Only a handful of long noncoding RNAs have been functionally characterized. They are known to modulate regulation through interacting with other biomolecules in the cell: DNA, RNA and protein. Though there have been detailed investigations on lncRNA-miRNA and lncRNA-protein interactions, the interaction of lncRNAs with DNA have not been studied extensively. In the present study, we explore whether lncRNAs could modulate genomic regulation by interacting with DNA through the formation of highly stable DNA:DNA:RNA triplexes.We computationally screened 23,898 lncRNA transcripts as annotated by GENCODE, across the human genome for potential triplex forming sequence stretches (PTS). The PTS frequencies were compared across 5′UTR, CDS, 3′UTR, introns, promoter and 1000 bases downstream of the transcription termination sites. These regions were annotated by mapping to experimental regulatory regions, classes of repeat regions and transcription factors. We validated few putative triplex mediated interactions where lncRNA-gene pair interaction is via pyrimidine triplex motif using biophysical methods.We identified 20,04,034 PTS sites to be enriched in promoter and intronic regions across human genome. Additional analysis of the association of PTS with core promoter elements revealed a systematic paucity of PTS in all regulatory regions, except TF binding sites. A total of 25 transcription factors were found to be associated with PTS. Using an interaction network, we showed that a subset of the triplex forming lncRNAs, have a positive association with gene promoters. We also demonstrated an in vitro interaction of one lncRNA candidate with its predicted gene target promoter regions.Our analysis shows that PTS are enriched in gene promoter and largely associated with simple repeats. The current study suggests a major role of a subset of lncRNAs in mediating chromatin organization modulation through CTCF and NSRF proteins.The online version of this article (doi:10.1186/s12967-017-1282-9) contains supplementary material, which is available to authorized users. Developments in the field of transcriptomics contributed to the discovery of vast number of RNAs, majority of which are non-coding RNAs . Long noSPHK1 (Sphingosine kinase 1) promoter by an antisense RNA khps1. This leads to up regulation of the SPHK1 gene via histone acetylation and ultimately cause increase in cell proliferation . The predictions were performed genome wide for each of the human chromosome independently keeping the parameters as -l 35 -e 10 -g 20 -m R/Y/M -fm 0 -of 1 -fr off . Additional file The potential DNA:DNA:RNA triplex sites were predicted using Triplexator  Chi-square test to check the mapping significance of UTA Transcription Factor Binding sites (UTA TFBS) with Potential Triplex Sites. [B] Chi-square test to check the mapping significance of HAIB Transcription Factor Binding sites (HAIB TFBS) with Potential Triplex Sites. [C] Chi-square test to check the mapping significance of SYDH Transcription Factor Binding sites (SYDH TFBS) with Potential Triplex Sites.Additional file 12. Distribution of Transcription factors across the Transcription Start Site (TSS) (A) Pol24h8 (B) NRSF (C) CTCF (D) CMYS (E) CTCFB.Additional file 13. Complete list for possible Hi-C (CCHiC-HeLaS3-G1mid-R2) interaction domains with PTS sites.Additional file 14. Complete list for possible Hi-C (CCHiC-HeLaS3-M-R1) interaction domains with PTS sites.Additional file 15. Complete list for possible Hi-C (CCHiC-HeLaS3-M-R2) interaction domains with PTS sites.Additional file 16. Complete list for possible Hi-C (Hi-C_PTS_CCHiC-HeLaS3CCL2p2-G1-0.25F) interaction domains with PTS sites.Additional file 17. Complete list for possible Hi-C (CCHiC-HeLaS3CCL2p2-G1-1FA) interaction domains with PTS sites.Additional file 18. Complete list for possible Hi-C (CCHiC-HeLaS3CCL2p2-M-0.25FA) interaction domains with PTS sites.Additional file 19. Complete list for possible Hi-C (CCHiC-HeLaS3CCL2p2-M-1FA) interaction domains with PTS sites.Additional file 20. Complete list for possible Hi-C (CCHiC-HeLaS3CCL2p2-M-98percent) interaction domains with PTS sites.Additional file 21. Complete list for possible Hi-C (CCHiC-K562-M-R1) interaction domains with PTS sites.Additional file 22. The results of Chi-square test for testing mapping significance of the Hi-C mapping to PTS. [A] Chi-square test to check the mapping significance of Hi-C interaction domain with Potential Triplex Sites forming purine motif (R). [B] Chi-square test to check the mapping significance of Hi-C interaction domain with Potential Triplex Sites forming pyrimidine motif (Y). [C] Chi-square test to check the mapping significance of Hi-C interaction domain with Potential Triplex Sites forming purine-pyrimidine motif (M).Additional file 23. Distribution of (A) Purine motif forming PTS and (B) Pyrimidine motif forming PTS across 98 DnaseI hypersensitivity datasets from ENCODE.Additional file 24. Correlation network constructed using Cytoscape for 23 lncRNAs and 51 Refseq genes. The Refseq genes are represented by blue node, lncRNAs by red node and the edge (grey) represents the correlation value. The thickness of the edge represents the value ranging from 0.08 to 0.91, thicker the line higher the correlation. [A] The network for lncRNAs forming triplex structure with promoters of Refseq gene predicted with these parameters (-l 35 -e 10 -g 20 -m R/Y/M -fm 0 -of 1 -fr off). [B] Interaction network for HOTAIR and MALAT1 lncRNAs, which are known to form triplex structure from previous literatures [14] and were predicted in our analysis with these parameters (-l 35/40 -e 20/20 -g 20 -m R/Y/M -fm 0 -of 1 -fr off).


The development and application of whole genome sequencing technology has greatly broadened our horizons on the capabilities of long non-coding RNAs (lncRNAs). LncRNAs are more than 200 nucleotides in length and lack protein-coding potential. Increasing evidence indicates that lncRNAs exert an irreplaceable role in tumor initiation, progression, as well as metastasis, and are novel molecular biomarkers for diagnosis and prognosis of cancer patients. Furthermore, lncRNAs and the pathways they influence might represent promising therapeutic targets for a number of tumors. Here, we discuss the recent advances in understanding of the specific regulatory mechanisms of lncRNAs. We focused on the signal, decoy, guide, and scaffold functions of lncRNAs at the epigenetic, transcription, and post-transcription levels in cancer cells. Additionally, we summarize the research strategies used to investigate the roles of lncRNAs in tumors, including lncRNAs screening, lncRNAs characteristic analyses, functional studies, and molecular mechanisms of lncRNAs. This review will provide a short but comprehensive description of the lncRNA functions in tumor development and progression, thus accelerating the clinical implementation of lncRNAs as tumor biomarkers and therapeutic targets. Cancer is a complex disease associated with multiple genetic mutations. Over the past few decades, oncogenes such as Src and Ras have been discovered, and the functions of their encoded proteins have been elucidated. While cancer awareness is gradually increasing worldwide, the current focus is still concentrated on the DNA sequence responsible for the abnormal protein synthesis. With the widespread application of next-generation sequencing (NGS) technology, a large number of non-coding genes have been identified and found to be strongly associated with tumor development and progression. Therefore, it is of paramount importance to understand the underlying mechanism through which non-coding genes influence the tumorigenic process.In 1961, the central position of RNA in the flow of genetic information was revealed , 3. ManyLncRNAs are structurally similar to mRNAs and are also generated through DNA transcription . Based oHere, we describe the specific regulatory mechanisms of lncRNAs, mainly focusing on their signal, decoy, guide, and scaffold functions in cancer cells. The lncRNAs in the nucleus are mainly involved in epigenetic and transcriptional regulation while the lncRNAs in the cytoplasm are often involved in post-transcriptional regulation, thus regulating the mRNA stability, protein translation and the competitive endogenous RNA (ceRNA) network. In addition, the research strategies used to identify the roles of lncRNAs in tumors are summarized, including lncRNA screening, lncRNA characteristic analyses, functional studies and molecular mechanisms of lncRNAs. The current review will provide a comprehensive description of the lncRNA functions and novel insights into their underlying molecular mechanisms.The lncRNA mechanisms of action can be divided into four categories: signal, decoy, guide, and scaffold , 27–29 Figure 1As signal molecules, lncRNAs are often considered to regulate the transcription of downstream genes. Previous studies have demonstrated that lncRNAs are specifically transcribed and they influence certain signaling pathways under different stimulation conditions. The transcribed lncRNAs act by themselves or in combination with certein proteins (such as transcription factors) to mediate the transcription of downstream genes. Huarte et al. showed that p53-induced lncRNA-p21 interacted with nuclear heterogeneous ribonucleoprotein-K to inhibit the expression of downstream genes in the p53 signaling pathway . LncRNA LncRNA commonly act as a decoy molecule by blocking a certain molecular pathway. After lncRNA is transcribed, it directly binds to some protein molecules such as chromosome folding proteins or transcription regulators and impairs the function of that protein. LncRNAs directly bind to transcription regulators to block the function of transcription factors, further suppressing the downstream gene transcription. For instance, DNA damage-induced lncRNA PANDA directly binds to the nuclear transcription factor NF-YA and impairs its function, thus preventing the expression of apoptosis-related genes and ultimately inhibiting the NF-YA-dependent apoptosis pathways . FurtherLncRNAs can also affect the expression of target genes by sponging miRNAs. In certain tumor cells and specific tissues, lncRNAs directly bind to the miRNA molecules and prevent them binding to the target mRNAs, thus upregulating of the expression of the target genes. In prostate cancer, lncRNA PCAT-1 acts as a sponge for absorbs miR-3667-3p and reduce its inhibitory effect on c-Myc mRNA, which promotes the proliferation and migration of the tumor .LncRNAs can act as guiding molecules by aiding specific protein in reaching their target location and exerting their biological functions. As guiding molecules, lncRNAs are often interacting with transcription factors, which are located on a specific DNA sequence and regulate gene transcription. Numerous studies showed that lncRNAs can regulate gene transcription through cis regulation, thus regulating the transcription of adjacent mRNAs. Wang et al. found that lncTCF7 can recruit the SWI/SNF complex to the TCF7 promoter and regulate the expression of TCF7. This process can trigger the activation of the Wnt signaling pathway, and ultimately promote the self-renewal of liver cancer stem cells and the proliferation of cancer cells . NeverthLncRNA can act as a “central platform” and facilitate the interaction of numerous molecules and protein. Furthermore, the scaffold properties of lncRNAs enable the assembly of different types of macromolecular complexes, thus promoting the convergence and integration of information among different signaling pathways , 36. OneAccumulating evidence shows that lncRNAs play different roles in the nucleus and cytoplasm. In the nucleus, lncRNAs regulate the epigenome by recruiting chromatin remodeling complexes as well as chromatin modification complexes. Furthermore, lncRNAs act as transcriptional regulators by themselves or by recruiting transcription factors and are involved in the process of pre-mRNA alternative splicing. Nevertheless, numerous studies indicated that there are a large number of lncRNAs in the cytoplasm, which are involved in post-transcriptional regulation processes such as the mRNAs stability, mRNAs translation, protein stability, and “ceRNA” network , 45 , the 5-methylcytosine on the TCF21 promoter (tumor suppressor) was demethylated, thereby mediating the transcriptional activation of TCF21 .In eukaryotic cells, transcription factors bind to DNA and control the transcription, localization, and stability of the RNA transcribed. Some lncRNAs that act as ligands often interact with transcription factors to form complexes and control gene transcription . LncRNAsIn addition to the two regulatory mechanisms aforementioned, lncRNAs are involved in post-transcriptional regulation, including alternative splicing of pre-mRNA, stabilization of mRNA, and translation and stabilization of proteins –59. MostLncRNAs often affect the expression of their target genes by interacting with miRNAs, which are the main post-transcriptional regulation factors. In some tumor cells and specific tissues, some lncRNAs carrying “seed sequences” of certain miRNAs bind to miRNAs and act like sponges, thereby preventing miRNAs from binding to their target mRNAs –67. Qu eIn summary, it has been found that lncRNAs regulate gene expression at following three levels by interfering with the epigenome and by regulating the transcriptional as well as post-transcriptional processes of the targeted genes.With the development of high-throughput sequencing technology, an increasing number of lncRNAs are annotated; however, the function of most lncRNAs in tumors remains unclear. Since the field of lncRNAs remains broadly unknown, its exploration is of paramount importance. Previous studies showed that the function of lncRNAs in tumors can be investigated using lncRNA screening, lncRNA characteristic analyses, functional studies and lncRNA molecular mechanisms analyses Figure 3Under the premise of guaranteeing at least three samples, differentially expressed lncRNAs can be screened using high-throughput sequencing technologies such as NGS . VariousThe lncRNAs of interest were also obtained from databases such as the TCGA database, a multi-omics database related to tumors, which includes DNA-Sep, RNA-Sep, protein-Sep, and other omics data .Following lncRNA screening, qRT-PCR/northern blot were used to reduce the number of candidate lncRNAs and verify their expression in clinical specimens and cell lines. Furthermore, the correlation between the lncRNA expression and clinical indicators would be determined, thus identifying the importance of the candidate lncRNAs in clinical diagnosis and treatment.The mostly explored characteristics of lncRNAs include coding potential, location information on the genome, secondary structure, correlation with disease, full-length analysis, and cell localization, which can be determined through bioinformatical analysis. NONCODE, LncBook, LncRNAdb v2.0, and LncRNADisease are some softwares that can be used to obtain a provide comprehensive annotations of lncRNAs –77.Rapid-amplification of cDNA ends (RACE) is a method that can provide the full length of the lncRNAs by extending and amplifying the two ends of a known cDNA fragment based on PCR technology –80. Befoin situ hybridization (FISH) is a method often used to identify the cellular localization of the candidate lncRNAs. The principle is that the foreign nucleic acid containing radioactive labels  or non-radioactive labels  is complementary paired with the DNA or RNA to be tested on tissues, cells or chromosomes to form a specific nucleic acid hybrid molecule  Use of bioinformatics to predict the possible “ceRNA” networks. There are many databases that can predict possible “ceRNA” networks, such as Starbase , LncACTdIn conclusion, the use of bioinformatics to predict the functions of lncRNAs is favored over the traditional, time-consuming, and expensive experimental methods. For the lncRNA field, it is essential to establish a variety of databases meant to help researchers identify and name their newly discovered lncRNAs. The already established databases for lncRNA are listed in Table 1.The role of lncRNAs has become one ofmain focus for fundamental and clinical tumor studies. Given the increasing pool of evidence regarding the role of lncRNAs in tumor development and progression, here we summarized the four main models of action through which lncRNAs influence tumor progression . The lncRNAs in the nucleus are often involved in chromatin remodeling and modification, transcriptional regulation, and alternative splicing of pre-mRNA, while the lncRNAs in the cytoplasm are often involved in the stability of mRNAs, protein translation, and the ceRNA network . Based ovia exosomes can promote sunitinib resistance as a competitive endogenous RNA in renal cancer .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PLIN1 gene is regulated remain unknown. In this study, we investigated the role of retinoid X receptor α (RXRα) in transcription of the chicken PLIN1 gene. Notably, reporter gene and expression assays showed that RXRα activates transcription of the chicken PLIN1 gene in a PPARγ-independent manner. Furthermore, promoter deletion and electrophoretic mobility shift assay (EMSA) analysis revealed that the chicken PLIN1 gene promoter region (-774/-785) contains an RXRα-binding site. Further study demonstrated that RXRα overexpression promotes differentiation of an immortalized chicken preadipocyte cell line (ICP1), causing a concomitant increase in PLIN1 transcripts. Taken together, our results show for the first time that RXRα activates transcription of the chicken PLIN1 gene in a PPARγ-independent manner, which might be at least in part responsible for RXRα-induced adipogenesis.Perilipin1 (PLIN1), the most abundant lipid droplet (LD)-associated protein, plays a vital role in regulating lipid storage and breakdown in adipocytes. Recently, we found that the overexpression of PLIN1 promotes chicken preadipocyte lipid accumulation. However, the mechanisms by which transcription of the chicken  Obesity is a major risk factor for the development of various diseases such as type 2 diabetes, cardiovascular disease and cancer . ObesityPerilipin1 (PLIN1) is the most abundant LD-associated protein in adipocytes and plays dual roles in controlling both basal and β-adrenergic receptor agonist-stimulated lipolysis in adipocytes . ConsistPLIN1 gene is transcriptionally regulated by numerous factors including peroxisome proliferator-activated receptor γ (PPARγ)  , estroge (PPARγ) , liver X (PPARγ) , constit (PPARγ) , tribble (PPARγ) , tumor n (PPARγ) , RAR-rel (PPARγ) , docosah (PPARγ) , 17 β-es (PPARγ) , acylati (PPARγ) , serum a (PPARγ) , eicosap (PPARγ)  and estr (PPARγ) . HoweverAll animal work was conducted in accordance with the guidelines for the care and use of experimental animals established by the Ministry of Science and Technology of the China  and approved by the Institutional Biosafety Committee of Northeast Agricultural University . Plasmid construction and transfection were performed according to the directions of the Regulation on Safety Administration of Agricultural Genetically Modified Organisms (RSAGMO) established by the China (revised version 2017).g for 10 min at room temperature (22°C) to separate the stromal-vascular fractions from undigested tissue debris and mature adipocytes. Stromal-vascular cells (including preadipocytes) or ICP1 cells were seeded at a density of 1 × 106 cells/cm2 in Dulbecco’s modified Eagle’s medium/F12 medium (Invitrogen) with 5% fetal bovine serum  and maintained at 37°C in a humidified atmosphere of 5% CO2 until confluency (day 4). The cells were then trypsinized (0.25% trypsin + 0.04% EDTA) and passaged. DF-1 chicken fibroblast cells  were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 5% FBS at 37°C in a humidified atmosphere of 5% CO2.Abdominal adipose tissue was excised from 12-day-old Arbor Acres birds and digested. Primary chicken preadipocytes and an immortalized chicken preadipocyte cell line (ICP1) were cultured and differentiated according to the methods of our laboratory . BrieflyOne day after propagation (day 5), when the cells had reached 50% confluence, primary chicken preadipocytes and ICP1 cells were induced by growth in complete medium containing 160 μM sodium oleate  for differentiation. Subsequently, the medium was removed every 24 h and replaced with fresh medium containing DMEM/F12 supplemented with 10% FBS and 160 μM sodium oleate. Preadipocytes and ICP1 cells were differentiated for a total of 72 h.® Reagent (Invitrogen) following the supplier’s protocol. Total RNA was treated with DNase I , and RNA quality was assessed by visualization of the 18S and 28S ribosomal RNA bands on a denaturing formaldehyde agarose gel. Only RNA with a 28S:18S ratio between 1.8 and 2.1 was used for reverse transcription. Reverse transcription was performed according to the directions of the ImProm-IITM Reverse Transcription System .Total RNA was extracted from chicken abdominal fat tissue and cells with TRIzol-actin and the TATA-box binding protein (TBP) gene were used as internal references. Quantitative RT-PCR was performed using FastStart Universal SYBR Green Master (ROX)  on a 7500 Real-Time PCR System . From each 10-μL reaction, 1 μL of product was amplified. The following PCR conditions were used: incubation for 1 cycle at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Dissociation curves for each PCR were analyzed using Dissociation Curve 1.0 software (Applied Biosystems) to detect and eliminate possible primer-dimer artifacts. The relative level of target gene expression, as determined with ABI software, was calculated using the comparative 2–ΔΔCt method for relative quantification. The sequences of the primers used to analyze gene expression levels are shown in Quantitative real-time RT-PCR was used to analyze gene expression levels. Expression levels of βPLIN1 promoter and its subsequent 5’ truncation construct were generated by PCR from chicken genomic DNA using different forward primers and the same reverse primer as shown in RXRα gene (GenBank Accession No. XP_003642339.1) was constructed by RT-PCR from chicken abdominal fat tissue total RNA and cloned into the pCMV-Myc vector . All primers used are shown in The chicken To prepare nuclear extracts, the pCMV-Myc-RXRα expression vector was transfected into DF-1 cells. After 48 h of transfection, nuclear extracts were collected using NE-PER extraction reagents . Part of the nuclear extracts was used for Western blotting. After being mixed with 6× denaturing loading buffer and boiled for 5 min, nuclear proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an Immun-Blot polyvinylidene fluoride membrane . Western blotting was performed using anti-Myc bodies with ECL (Beyotime Institute of Biotechnology).PLIN1 promoter DNA probe (5′-3′ Biotin) for 20 min at room temperature and then separated by electrophoresis on a 5% non-denaturing polyacrylamide gel with 0.5 × TBE running buffer. DNA-protein complexes were transferred onto nylon membranes (Pierce) and then crosslinked for 1 min with a UV crosslinker. The signal was detected with a Chemiluminescent EMSA Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. For the competition assay, nuclear extracts were incubated with unlabeled probes (Invitrogen) for 10 min at room temperature before the addition of biotin-labeled oligonucleotide. For the supershift assay, protein-DNA complexes were incubated with 1 μg of antibody specific to the Myc tag  for 30 min at room temperature before electrophoresis. Sequences of the probes used for EMSA are shown in Nuclear extracts were incubated with a biotin-labeled DF-1 cells at 70–80% confluence were washed with PBS and transiently transfected using Lipofectamine 2000 reagent (Invitrogen). Forty-eight hours later, luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions on a FB12 luminometer . Firefly luciferase (Fluc) activity was normalized to Renilla luciferase (Rluc) activity.2O and analyzed under a microscope .The differentiated ICP1 cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min at 4°C. After being washed with PBS and distilled water twice, the cells were stained with oil red O working solution (oil red O dye in 60% isopropanol) at room temperature for 15 min. Cells were then washed immediately with ddHTo quantitatively measure lipid accumulation, an oil red O extraction assay was performed. Briefly, after removing the staining solution, oil red O was extracted by the addition of 1 mL of 100% (v/v) isopropyl alcohol, and the absorbance at 510 nm was measured with a spectrophotometer .t-tests conducted with GraphPad Prism 5. Statistical significance was indicated when ∗P < 0.05, ∗∗P < 0.01.All data are presented as the mean ± SEM. Differences between groups were analyzed using unpaired Student’s PLIN1 and RXRα genes during the adipogenesis of primary chicken preadipocytes. During preadipocyte differentiation, the mRNA expression levels of chicken PLIN1 gene were gradually elevated; the expression level of chicken RXRα gene increased rapidly after preadipocyte induction and maintained high expression throughout the differentiation process  and pCMV-Myc-RXRα, and luciferase activity was measured. DF-1 cells transfected with the pCMV-Myc vector were used in the control group. As shown in PLIN1 promoter activity was higher in the RXRα overexpression group than in the control (P < 0.01).To determine whether RXRα regulates transcription of the chicken  plasmid . Then, DPLIN1 gene in the form of only a PPARγ2/RXRα heterodimer  (PLIN1 gene (P < 0.01)  . Similar < 0.01) . HoweverIN1 gene . These rPLIN1 gene is regulated by RXRα, we investigated the effect of PPARγ knockdown on RXRα-induced transcriptional activation of the chicken PLIN1 gene using RNAi. The sh-PPARγ and control (nc-PPARγ) vectors, whose construction and confirmation were reported in our previous study  vector. As shown in PLIN1 promoters, even the promoter truncated to -123/-11, could be activated by the expression of RXRα compared with promoter activity in the control (P < 0.01). The promoter construct from -838/-11 had the highest promoter activity, while promoter activity decreased significantly as the PLIN1 promoter was truncated from −838 bp to −680 bp  in the -838/-680 region. To define the function of this predicted RXRα-binding site, a pGL3-PLIN1-MUT-838/-11 promoter reporter construct was generated by site-directed mutagenesis using DNA synthesis (GCC to AAG) caused a 69.86% decline in chicken PLIN1 promoter activity with the expression of RXRα compared to that observed with the wild-type reporter (P < 0.01)  , suggestPLIN1 gene promoter, -785/-774 and -123/-11, are directly recognized by RXRα, an electrophoretic mobility shift assay (EMSA) was performed with recombinant nuclear RXRα protein. First, to assess the -123/-11 region, three labeled probes  were designed. Two adjacent probes overlapped fragments of at least 10 bp , farnesoid X receptor (FXR), Nurr1, Nur77, retinoic acid receptors (RARs), vitamin D receptor (VDR), and thyroid receptors (TRs) . Studieseceptors . Interesγ ligand . In conto groups . Thus, waffected , which iaffected . TherefoPLIN1 gene promoter from -1992 to -123 bp, the RXRα-mediated positive regulation of chicken PLIN1 gene transcription was maintained, suggesting that there are RXRα-binding sites in the -123/-11 region of the PLIN1 gene promoter  in this region caused an abrupt decline in chicken PLIN1 promoter activity with the expression of RXRα, which indicates that the -785/-774 site is the crux involved in RXRα-mediated positive activation of the chicken PLIN1 gene  and that the amino acid compositions and domains of mammalian and chicken RXR are relatively conserved . In this study, RXRα positively activated the promoter activity of the chicken PLIN1 gene in a PPARγ-independent manner. Therefore, we predict that RXRα regulates transcription of the chicken PLIN1 gene by forming a homodimer through binding to the -785/-774 region.The structures and functions of steroids, retinoic acids, vitamin D and thyroid hormone nuclear receptors encoded by a single gene are evolutionarily conserved. As transcription factors, these nuclear receptors can efficiently identify target genes through a conserved DNA-binding domain and regulate the transcription of these genes. RXRα also has a conserved DNA-binding domain and interacts with a hexanucleotide motif (5’-(A/G)G(G/T)TCA) . The RXRPLIN1 gene promoter region, we could not determine the specific regulatory parameters in this region, such as the oligomeric form of RXRα (homodimer or heterodimer) or the presence of specific binding sites.Activation of a specific target gene by RXR homodimers depends on not only the local abundance of RXR, other NRs and related cofactors but also the specificity of the transcriptional response, which is achieved by cooperation between different nuclear receptors or a given NR coupled to other transcription factors . TherefoPLIN1 gene can enhance lipid accumulation in chicken preadipocytes  the role of RXRα in adipogenesis, which may allow us to identify novel therapeutic strategies to protect against obesity.Taken together, our results revealed (i) a novel RXRα-mediated mechanism by which transcription of the chicken The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the Institutional Biosafety Committee of the Northeast Agricultural University .YW conceived and supervised the study. YW, YS, and WZ designed the experiments. YS, GZ, and RL performed the experiments. YL and ZC contributed the reagents and materials. YS analyzed the data and wrote the manuscript. All authors made manuscript revisions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Western blotting and immunostaining results revealed that SAHA pretreatment could induce global hyperacetylation of histone H3 at lysine 9 (H3K9) and histone H4 at lysine 5 (H4K5) under 250 mM NaCl stress, indicating that SAHA could act as the HDACi in cotton. Chromatin immunoprecipitation and chromatin accessibility coupled with real time quantitative PCR analyses showed that the upregulation of the ion homeostasis-related genes was associated with the elevated acetylation levels of H3K9 and H4K5 and increased chromatin accessibility on the promoter regions of these genes. Our results could provide a theoretical basis for analyzing the mechanism of HDACi application on salt tolerance in plants.Histone acetylation plays an important role in regulation of chromatin structure and gene expression in terms of responding to abiotic stresses. Histone acetylation is modulated by histone deacetylases (HDACs) and histone acetyltransferases. Recently, the effectiveness of HDAC inhibitors (HDACis) for conferring plant salt tolerance has been reported. However, the role of HDACis in cotton has not been elucidated. In the present study, we assessed the effects of the HDACi suberoylanilide hydroxamic acid (SAHA) during high salinity stress in cotton. We demonstrated that 10 μM SAHA pretreatment could rescue of cotton from 250 mM NaCl stress, accompanied with reduced Na Na+ exclusion and vacuolar Na+ sequestration, mediated by Na+/H+ antiporters, are two important ways to reduce Na+ concentration in the cytoplasm  [Total RNA was isolated using an RNAprep pure Plant Kit , cDNA was synthesized from 1 μg of total RNA with HiScript II 1st Strand cDNA Sythesis Kit , and qPCR was performed with ChamQ Universal SYBR qPCR Master Mix  with a LightCycler480 instrument , using the following profile: 95 °C for 5 min, and then 45 cycles at 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. The following antibodies were applied for Western blotting, immunostaining, and ChIP experiments. Anti-H3K9ac (07–352), anti-H4K5ac (07-327), and FITC-conjugated goat anti-rabbit IgG (12-507) were purchased from Merck Millipore . Anti-H3 (ab1791) was obtained from Abcam  and HRP-conjugated goat anti-rabbit IgG (A21020-1) was obtained from Abbkine .https://imagej.nih.gov/ij/).Extraction of plant proteins was carried out using an Animal-Plant Total Protein Miniprep Kit . Western blotting was performed as previously described . The proChIP assay was performed according to Zhang et al. . BrieflyCHART-PCR assay was carried out to determine the conformational change of chromatin according to Zhang et al. . This pr+ content, leading to relief of the damage caused by salt stress in cotton seedlings. This study provides the preliminary results for the application HDACi on the development of salt-tolerant varieties of cotton.Overall, our results suggested that exogenous application of SAHA with relative low concentration (10 μM) elevated the acetylation levels of H3K9 and H4K5 and chromatin accessibility on the promoter regions of ion homeostasis-related genes, and then upregulated the expression of these genes, moreover reducing the Na


SOX17 containing two independent signals. SOX17 is an important transcription factor in embryonic development and in the homeostasis of pulmonary artery endothelial cells (hPAEC) in the adult. Rare pathogenic mutations in SOX17 cause heritable PAH. We hypothesised that PAH risk alleles in an enhancer region impair TF-binding upstream of SOX17, which in turn reduces SOX17 expression and contributes to disturbed endothelial cell function and PAH development.Pulmonary arterial hypertension (PAH) is a rare disease characterised by remodelling of the pulmonary arteries, increased vascular resistance and right heart failure. Genome-wide association studies (GWAS) of idiopathic/heritable PAH established novel genetic risk variants including conserved enhancers upstream of transcription factor (TF) SOX17 expression. Electromobility shift assays (EMSA) were used to confirm in-silico-predicted TF differential binding to the SOX17 variants. Functional assays in hPAEC were used to establish the biological consequences of SOX17 loss. In-silico analysis using the connectivity map (CMap) were used to predict compounds that rescue disturbed SOX17 signalling. Mice with deletion of the SOX17 signal 1 enhancer region (SOX17-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia.CRISPR manipulation and small interfering RNA were used to modulate SOX17-signal 2 and deletion of SOX17-signal 1 specifically decreased SOX17 expression. EMSA demonstrated differential binding of hPAEC nuclear proteins to the risk and non-risk alleles from both SOX17 signals. Candidate TFs HOXA5 and ROR-α were identified through in silico analysis and antibody EMSA. Analysis of the hPAEC transcriptomes revealed alteration of PAH-relevant pathways upon SOX17 silencing, including extracellular matrix regulation. SOX17 silencing in hPAEC resulted in increased apoptosis, proliferation, and disturbance of barrier function. Using CMap, compounds were identified that reversed the SOX17-dysfunction transcriptomic signatures in hPAECs. SOX17 enhancer knockout in mice reduced lung SOX17 expression, resulting in more severe pulmonary vascular leak and hypoxia or SU5416/hypoxia-induced pulmonary hypertension.CRISPR-Inhibition of SOX17 promoter reduce endothelial SOX17 expression, at least in part, through differential binding of HOXA5 and ROR-α. Reduced SOX17 expression results in disturbed hPAEC function and PAH. Existing drug compounds can reverse the disturbed SOX17 pulmonary endothelial transcriptomic signature.Common PAH risk variants upstream of the  BMPR2 variants), environmental factors like hypoxia, change in shear stress, inflammation, drugs or toxins can directly injure the endothelial barrier, leading to apoptosis, loss of barrier integrity and vascular remodelling of the pulmonary artery wall  is a rare but lethal disease. With no intervention, the mean survival is 2.8 years  and withery wall .BMPR2, are associated with PAH  in a region located 106-200kb upstream of the SOX17 gene promoter. The risk alleles are common in the populations tested and enriched in PAH, with 59% of patients homozygous for the risk allele of both signals compared to 46% of controls  binding at the two signals which affects SOX17 expression, hPAEC function and PAH development.The ntiation  and haventiation . SOX17 intiation ,11. EC dntiation . Conditimyopathy . Thus faFor full methods and materials please see the supplemental section. We have deposited the RNAseq data to GEO with accession number GSE214742. All other data are available upon reasonable request.Individuals with a diagnosis of idiopathic PAH (n=11) diagnosed according to international guidelines  and healSOX17-signal 2 and -signal 1, CRISPR−Inhibition (CRISPR-I) was used as previously described  was used. Two guides -T2A-HygR vector (#118153) using a one-step dual CRISPR/Cas9 guide RNA cloning protocol as previously described  EMSA kit according to manufacturer’s instructions. To investigate which transcription factor binds to the SOX17 variants, supershift assays with anti-HOXa5/RORa antibodies were performed. Five hPAEC donors were also used for chromatin immunoprecipitation (ChIP) qPCR assays with the same antibodies.To investigate transcription factors (TF) whose binding may be affected due to the SOX17 in hPAECs, siRNA experiments were performed using silencer select siRNA (ThermoFisher) targeting SOX17 (#s34626). Comparable control siRNA was also used and included two scrambled-siRNA  and a control targeting GAPDH (#ASO2FLIC).To assess the role of SOX17 by siRNA and CRISPR-I SOX17-signal 2 and -signal 1, RNA sequencing (RNAseq) was carried out by the Imperial College BRC Genomics Facility and analysis of the dataset was performed in RStudio. To assess gene ontology changes resulting from either siRNA-SOX17, CRISPR-I SOX17-signal 2 or signal 1, over-representation analysis was performed using the WEB-based Gene SeTAnaLysis Toolkit .To assess whole transcriptomic effects of knockdown of -deltaCt).To investigate the change in expression of target genes, reverse transcription-PCR (RTPCR) and qPCR was performed with actin beta (ACTβ) used as a reference gene  supplemented with protease and phosphatase inhibitor cocktail (ThermoFisher) and immunoblotted with anti-SOX17 .SomaLogic SomaScan measurements were available from a recent proteomics study by Rhodes et al  Patient SOX17 signal 1 (rs13266183) were obtained from a whole genome sequencing study from the UK National Institute for Health Research BioResource  method. All analyses were completed in R using RStudio v1.4.1106 and the volcano plots were designed using the package “EnhancedVolcano”.The SNP genotypes for SOX17 on hPAEC function, assays investigating proliferation, apoptosis, cell viability, adhesion and barrier function were used and are discussed in full in the supplemental section.To assess the effect of siRNA-SOX17 expression is manipulated, RNA-sequencing of SOX17-signal 1 CRISPR-I hPAECs was performed as previously stated. To discover compounds that could be repurposed for the treatment of SOX17 dysfunction, the CMap was used  is unknown. To test whether it is an enhancer that targets SOX17, we first targeted the region using CRISPR-inhibition in hPAECs. Guide RNAs targeting either rs765727 or the SOX17 promoter led to a significant decrease in SOX17 expression  using CRISPR-deletion guides resulted in a significant decrease in SOX17 but had no effect on nearby genes TMEM68 or MRLP15  derived from healthy controls or PAH patients after stimulation with the hypoxia mimic DMOG , the inflammatory stimulus LPS (lipopolysaccharide) and the BMPR2 ligand BMP9 (bone morphogenetic protein-9). ECFC from IPAH patients have reduced barrier function versus those from control subjects and are more susceptible to LPS-induced permeability (SOX17 expression in both control (n=5) and PAH patient (n=11) ECFCs (To determine whether eability . LPS treeability . Expresseability . Stimula1) ECFCs .SOX17 expression. To determine the downstream effects of SOX17 depletion we performed RNAseq analysis of hPAEC following modulation of SOX17 by siRNA-mediated silencing or by CRISPR inhibition of SOX17-signal 1 and -signal 2 . Consistent with the siRNA analysis, gene ontology analysis of these differentially expressed genes shows enrichment for pathways linked to the extracellular matrix organization and cell adhesion . There were 26 genes in common between SOX17-signal 1 proteomics and CRISPRI or siRNA analyses (23)((23) in response to either TNF-α or LPS as compared to siRNA controls . Aminopurvalanol-a is also negatively connected to SOX17-signal 1 repression (-99.65). YK4279 is predicted to mimic SOX17 promoter activation . Comparisons of each compound’s signature from the Cmap and our RNAseq signature resulted in the gene lists shown in SOX17 effects we measured SOX17 levels in the treated hPAEC and found that both YK-4279 and sirolimus significantly increased SOX17 relative expression compared to vehicle . Using the standard protocol of 20 mg of SUGEN and 3 weeks of hypoxia, there were no significant differences in the severity of PH that developed between, SOX17-enhKO and WT mice. However, SOX17-enKO were more susceptible to developing SUGEN/hypoxia-PH as evidenced by the development of severe PH at lower levels of SUGEN-hypoxia (5 mg/kg and 12% O2) which did not produce PH in WT littermates  in which SOX17 is the only gene, making it the most likely target for these two enhancers  demonstrated that HOXa5 or ROR-alpha bind to the non-risk alleles present at SOX17-signal 1 and signal 2 respectively. Thus, an individual homozygous for one or both risk alleles would be more resistant to HOXa5 and/or ROR-alpha induced SOX17 expression than an individual hosting non-risk alleles. While it is not a requirement that these transcription factors are themselves associated with PAH, HOX transcription factors expression have been shown to differ in PAH lung tissue  associated with loss of SOX17. This was supported by our plasma proteomic analysis between patients with risk or non-risk SOX17 enhancer genotypes, where we found an enrichment of adhesion- and ECM-associated proteins. The basement membrane is thicker in the lungs of iPAH patients, and regulation of the ECM is established as an important factor in PAH  in future trials would be valuable.That rare pathogenic variants in nger age , emphasicy trial . Severalcy trial  and an acy trial . It woulSOX17 silencing in arterial endothelium  is a CDK1/cyclin B inhibitor that arrests proliferating cells in the G2/M stage of the cell cycle and prevents proliferation. It has been investigated in human microvascular endothelial cells as an anti-angiogenic drug and was shown to inhibit proliferation, increase apoptosis and prevent tube formation . Some CDothelium . In bothothelium . Both CDothelium .SOX17 gene in HUVEC, suggesting a possible role for ERG in the regulation of SOX17 in this cell type  was sufficient to lead to a worse PH phenotype in two independent laboratories, under hypoxia or lower levels of SUGEN-hypoxia than drove PH in wildtype animals. The concept that a ‘second hit’ may be required to exhibit the PH phenotype is well understood and in patients harbouring a SOX17 enhancer risk allele, this may comprise inflammation or drug toxicity as well as hypoxia.ed veins . Endotheed veins ,45. SOX1ed veins . These slar beds ,48, the SOX17 enhancer variants. In our EMSA experiments, use of a ROR-α antibody lead to loss of signal rather than a clear supershift, which is most likely due to the antibody preventing formation of the ROR-α-probe complex. SOX17 is not included on the proteomics platform used in this study. The numbers of homozygotes for rarer variant alleles are small so variability in these measurements is high. Validation of key findings in hPAECs with  variants in SOX17-signal 1 or 2 in addition to the complete deletion of the enhancer would have even more robustly supported functional conclusions. The effects would likely be more subtle than deletion or inhibition of the enhancer requiring larger n numbers. Aminopurvalanol did not affect SOX17 expression but was identified as a drug which can alter the downstream transcriptomic signature produced by SOX17 loss.It remains possible that other transcription factors also bind differentially to PAH-associated SOX17 and can reduce susceptibility to PH. Loss of SOX17 leads to downstream alterations in extracellular matrix regulation and hPAEC function. SOX17 is a priority for therapeutic rescue and predicted compounds which restore endothelial gene expression offer candidates for future investigation.In summary, we provide comprehensive insight into how common variation influences the binding of HOXa5 and RORα to enhancers upstream of Supplementary MaterialUnedited gels


The juxtaposed blue eggshell allele A-A exhibited higher promoter activity and stronger nuclear protein binding capacity than the white eggshell allele G-G. Transcription factor analysis suggested differential binding capability of CTCF between blue eggshell and white eggshell alleles. Knockdown of CTCF expression significantly decreased the promoter activity of the blue eggshell but not the white eggshell allele. DNA methylation analysis revealed similar high methylation of the region upstream of the CTCF binding sites in both blue-eggshelled and white-eggshelled ducks. However, DNA methylation levels downstream of the binding sites were decreased and 35% lower in blue-eggshelled ducks than in white-eggshelled ducks. Consistent with the in vitro regulatory pattern of causative sites, ABCG2 exhibited higher expression in uteruses of blue-eggshelled ducks and also showed polarized distribution in their endometrial epithelial cells, distributing at the apical surface of endometrial epithelial cells and with orientation toward the uterine cavity, where the eggshell is pigmented.Avian eggshell color is an interesting genetic trait. Here, we report that the blue eggshell color of the domestic duck is caused by two cis-regulatory SNPs upstream of ABCG2 are the causative mutations for blue eggshells in ducks. The blue eggshell variant up-regulated ABCG2 expression through recruiting CTCF binding, which may function as a barrier element to shield the downstream region from high methylation levels present upstream. ABCG2 was identified as the only candidate causative gene for blue eggshells; it may function as an efflux transporter of biliverdin to the uterine cavity.In conclusion, our results suggest that two  cis-regulatory variations that synergistically up-regulate ABCG2 expression in the uterus. ABCG2 was identified as the only candidate causative gene for blue eggshell; it may function as an efflux transporter of biliverdin to the uterine cavity, where the eggshell is pigmented. Our study provides useful molecular markers for breeding of blue-eggshelled ducks.Avian eggshell color is an interesting genetic trait that has been related to numerous interesting biological functions, such as crypsis, mimicry, and protection from ultraviolet radiation. In ducks, blue eggshells are a dominant Mendelian trait. The color is preferred by customers and has become one of the main breeding targets in laying ducks in China. In this study, we identified that duck blue eggshells are likely caused by two  Avian eggshell color is very diverse and has gained sustained scientific attention. This attractive trait has been proposed to account for numerous interesting biological functions, such as crypsis, mimicry, and protection from ultraviolet radiation protection , 2. BlueSLCO1B3 is considered the candidate causative gene for blue eggshells in the chicken, being exclusively expressed in the uteruses of chickens laying blue eggs and a potential contributor in the transportation of biliverdin to the eggshell. However, SLCO1B3 is not expressed in the uteruses of ducks [cis-regulatory SNPs that together up-regulate the expression of ATP-binding cassette sub-family G member 2 (ABCG2) in the uterus.In contract with the diversity of eggshell colors, eggshell pigments are monotonous, with only two main pigments accounting for the diverse eggshell colors: biliverdin, which gives a blue-green color, and protoporphyrin IX, which gives a rusty-brown color , 12. A sof ducks . Althougof ducks . In the S1 Table), and variant calling (SNPs and Indels) using these data identified 22,844,207 SNPs and 2,404,821 Indels. Of the identified SNPs, 15,086,311  were used for the genome-wide association study (GWAS). A 283 kb region between nucleotide positions 3,343,841 and 3,627,116 bp in scaffold KB742619.1 showed a significant association with the blue eggshell phenotype (S2 Table). Structural variations and assembly gaps were not found in this region. Genotyping screen of all SNPs and Indels (including 7413 SNPs and 1011 Indels) from this region revealed that six variations displayed perfect genotype-segregation across the three genotypes of resequenced individuals (S3 Table). That is, these six variations were fixed for homozygous reference genotype in all resequenced white-eggshelled ducks, fixed for homozygous mutant genotype in all resequenced homozygous blue-eggshelled ducks, and heterozygous in resequenced heterozygous blue-eggshelled ducks. In addition, these six variations showed perfect association with eggshell color in an extended genotyping screen of 1328 additional individuals from a divergent set of breeds/species (Table 1). Linkage analysis revealed that they were in complete linkage disequilibrium . These results prompted us to speculate that the causative mutation(s) should be among the six variations.Positional candidate loci were identified by whole-genome resequencing of 25 blue-eggshelled ducks as well as 24 white-eggshelled ducks. The genotypes of 25 blue-eggshelled ducks were determined with crossing tests, which determined eight birds to be homozygous and 17 heterozygous. In the genome resequencing, an average of eight-fold coverage of the duck genome was generated (PRKG2) and ABCG2. To determine the exact distance between candidate variations and the two nearby genes, we performed 3’RACE analysis for PRKG2 and 5’ RACE analysis for ABCG2 using uteruses from white-eggshelled ducks and blue-eggshelled ducks. We found that neither the transcription end site of PRKG2 nor the transcription start site of ABCG2 showed difference between the two genotypes. The six candidate variations were determined to be sited 676 bp downstream from PRKG2 and 2.6 kb upstream from the transcription start site of ABCG2 . Considering positions of these candidate variations, we analyzed their enhancer and promoter activity on the downstream gene and enhancer activity on the upstream gene using luciferase assays. For these assays, two fragments each containing three candidate variations were synthesized: Fragment 1 containing variations M1, M2, and M3, and Fragment 2 containing variations M4, M5, and M6. We found that Fragment 1 showed neither enhancer nor promoter activity, while Fragment 2 exhibited promoter activity on the downstream gene . Relative to the promoterless vector (pGL3-Basic), the blue eggshell haplotype of Fragment 2 increased luciferase activity 56-fold, whereas the white eggshell haplotype of Fragment 2 increased the activity only 4-fold, suggesting that while both haplotypes had promoter activity, the blue eggshell haplotype exhibited a much stronger effect than the white eggshell haplotype . Thus, further promoter activity assays were focused on the three candidate variations in Fragment 2, which we assayed separately or in pairs. Results showed that constructs containing the paired variations M5 and M6 exhibited results consistent with those from constructs containing the whole of Fragment 2. Specifically, relative to the pGL3-Basic vector, the blue eggshell construct increased luciferase activity 43-fold, while the white eggshell construct only increased luciferase activity 3-fold ; this suggested that the blue eggshell alleles of variations M5 and M6 synergistically increased promoter activity. Although weak promoter activities were also found either for the three separate variations or for the paired variations M4 and M5, in these cases, the blue eggshell and white eggshell alleles showed no difference in luciferase activity . Electrophoretic mobility shift assays (EMSAs) further revealed that both variation M5  and variation M6  exhibited differences in nuclear protein binding capacity between blue eggshell and white eggshell alleles . These results led us to speculate that variations M5 and M6 are the causative mutations responsible for the blue eggshell phenotype, and that they may induce differential expression of the downstream gene by altering protein binding.Annotation of the duck reference genome suggested that the six candidate variations are located between Protein kinase, cGMP-dependent, type II . ChIP-qPCR confirmed this endogenous binding. As shown in Fig 4B, CTCF binding fragments were significantly enriched in homozygous blue-eggshelled ducks compared to white-eggshelled ducks. Given that CTCF has been reported to mediate transcriptional activation [CTCF expression in duck embryonic fibroblast (DEF) cells cotransfected with a luciferase reporter containing either blue eggshell or white eggshell alleles. As expected, reducing CTCF expression led to a 2.5-fold loss of luciferase activity of the blue eggshell vector (p = 0.0004), whereas the activity of the white eggshell vector was unaffected , revealing a strong correlation between CTCF expression and promoter activation of the blue eggshell alleles.To identify proteins exhibiting different binding capacity between blue eggshell and white eggshell alleles, we scanned the surrounding sequences of variations M5 and M6 for predicted transcription factor binding sites. At variation M5, CCCTC-binding factor (CTCF) was predicted to bind to the blue eggshell allele but not the white eggshell allele. To experimentally verify this prediction, we conducted a supershift EMSA using CTCF antibody. We observed that CTCF specifically bound to the blue eggshell but not the white eggshell allele at variation M5, and a supershift was evident upon incubation with CTCF antibody . Bisulfite conversion and sequencing analysis of uteruses from eight blue-eggshelled and eight white-eggshelled ducks revealed similar methylation levels between phenotypes in the upstream region, with 88.5% and 87% methylation in white-eggshelled and blue-eggshelled ducks, respectively. However, methylation of the downstream region was 35% lower in blue-eggshelled ducks than in white-eggshelled ducks , leading us to speculate that CTCF binding may decrease the methylation level of the region downstream of its binding site, and it may function as a barrier to shield the downstream gene region from the high methylation level upstream.CTCF can affect DNA methylation , 18. To in vivo should be consistent with the regulatory pattern of causative sites in vitro and should fit the dominant single-gene inheritance pattern. Four genes were found adjacent to the causative sites, with RasGEF domain family member 1B (RASGEF1B) and PRKG2 being upstream and ABCG2 and Polycystin 2, transient receptor potential cation channel (PKD2) being downstream . Expression analysis of these four genes in uteruses from three genotypes of Shaoxing ducks, a Chinese local duck breed, revealed ABGC2 as the only differentially expressed gene for blue eggshells. Consistent with the regulatory pattern of causative sites, ABCG2 exhibited significantly higher expression in blue-eggshelled ducks than in white-eggshelled ducks, but no difference between homozygous and heterozygous blue-eggshelled ducks . This phenotype-associated expression was also demonstrated in another two Chinese local duck breeds, Shanma and Youxian ducks . The other three genes were excluded from candidate genes, as they showed similar expression levels between phenotypes, or their expression patterns were inconsistent with the regulatory pattern of causative sites and the dominant single-gene inheritance pattern  upstream of ABCG2 together cause the blue eggshell phenotype. These variant alleles up-regulated ABCG2 expression through recruiting CTCF, which may function as a barrier element to shield the downstream gene region from high methylation levels upstream. ABCG2 was identified as the only candidate causative gene for blue eggshell and may function as an exporter of biliverdin.Blue eggshell color is a dominant Mendelian trait in the domestic duck. This bright eggshell color has been considered to have advantages in terms of antioxidant ability and eggshell strength. In this study, our results suggested that two CTCF significantly decreased the promoter activity of blue eggshell alleles, suggesting that CTCF is crucial to the promoter effect of blue eggshell variant of M5. CTCF usually influences DNA methylation status around its binding sites [Fig 5C), suggesting that CTCF binding may reduce methylation in the region downstream of its binding site. Acting as a chromatin insulator, CTCF could block the transmission of either active or repressive signals along the chromatin [ABCG2 expression in blue-eggshelled ducks. We identified that the causative sites were 2.6 kb from ABCG2, which position seems like it should locate in an enhancer element rather than a promoter element; however, a clear promoter effect was observed for the causative alleles. We speculate that this may be because of CTCF binding, with CTCF being one of the most important elements in the regulation of chromatin folding [ABCG2, thus allowing the causative sites to act as a promoter element.CTCF is a versatile transcription regulator, with functions relating to gene activation, repression, and chromatin insulation. In our study, CTCF was found to specifically bind to the blue eggshell allele of variation M5. Knockdown of ng sites . Our DNAhromatin . Based o folding . Its binFECH) and heme oxygenase-1 (HMOX1), revealed similar expression levels between uteruses from blue-eggshelled ducks and those from white-eggshelled ducks ; along with our previous transcriptional analysis [Fig 7C and 7D). We speculate that most biliverdin in the uteruses of white-eggshelled ducks may be reduced to bilirubin, because our previous transcriptome data [BLVRA), which can reduce biliverdin to bilirubin, was significantly higher in uteruses from white-eggshelled ducks. The very small amount of biliverdin remaining is not sufficient to pigment the eggshell, thus eventually a white eggshell is formed. Consistent with the abundant biliverdin distribution observed in uteruses of blue-eggshelled ducks, ABCG2 was highly expressed in uteruses of blue-eggshelled ducks. ABCG2 is an efflux transporter that can transport a wide variety of structurally unrelated compounds. In our study, ABCG2 was identified as the only candidate causative gene for blue eggshells. We speculate that it may function as an exporter of biliverdin, which is a linear tetrapyrrole. Protoporphyrin IX (PPIX), a cyclic tetrapyrrole, has been demonstrated to be a direct substance transported by ABCG2 [ABCG2 in uteruses of blue-eggshelled ducks could result in abundant biliverdin export to the uterine fluid, pigmenting the eggshell, and eventually a blue eggshell is formed. Consistent with the exporter hypothesis, ABCG2 was found to have a polarized distribution in endometrial epithelial cells: distributed at the apical surface and oriented toward the uterine cavity .In this study, the 25 blue-eggshelled ducks used in whole-genome resequencing were male. Their genotypes were determined with crossing tests in which each male was mated with 20 white-eggshelled females, and the genotype of the male was inferred from the eggshell colors laid by its 20 daughters over a 20-day period during the egg-laying peak. According to the principle that blue eggshell color is a dominant Mendelian trait in ducks, if all 20 daughters laid blue eggs, the male was genotyped as a homozygous blue-eggshelled duck; if about half of the 20 daughters laid blue eggs, the male was genotyped as a heterozygous blue-eggshelled duck. The 24 white-eggshelled ducks used in whole-genome resequencing were female, and were observed laying white eggs for 20 consecutive days during their egg-laying peaks.An additional 1328 individuals from six duck breeds/species were used for extended genotyping of the six candidate variations. Of the six breeds/species, five are Chinese indigenous breeds originating from Shaoxing, Huzhou, Jinyun, and Wuhan. The wild species, mallard, was captured and then bred in captivity separately by Aoji Duck Farm, which has captured permission granted by the local government. Eggshell colors of all indigenous individuals were recorded over at least 20 days during their egg-laying peaks to determine their phenotypes. Eggshell colors of each mallard were recorded over a reproduction season to determine their phenotypes.Whole-genome resequencing was performed for 24 white-eggshelled ducks, eight homozygous blue-eggshelled ducks, and 17 heterozygous blue-eggshelled ducks. For each sample, a paired-end sequencing library with an insert size of 400 bp was generated and then sequenced on a HiSeq X-TEN platform according to the manufacturer’s protocol. The resequencing data have been submitted to the SRA database in NCBI with a BioProject accession number PRJNA668048.http://picard.sourceforge.net). To enhance alignment around Indels, local re-alignments of sequences around Indels were performed with the IndelRealigner tool in the Genome Analysis Toolkit (GATK) (version: 3.8) [Filtered reads were aligned to the duck reference genome (BGI_duck_1.0) using BWA-MEM (version 0.7.12) with default parameters . After aon: 3.8) .Variations (SNPs and Indels) were called using the Unified Genotyper implemented in GATK (version: 3.8) and subsequently filtered using the hard filtering process recommended by GATK . For eacGWAS was performed using the Efficient Mixed Model Association eXpedited (EMMAX) method . The matp-value  < 0.05 was considered to be a genome-wide significant association.A Bonferroni corrected An extended genotyping screen for the six candidate variations was conducted in 1328 individuals from six duck breeds. Primers and methods used for genotyping of the six candidate variations are listed in Linkage disequilibrium (LD) for candidate variations (pairwise r2 statistic) was calculated using SHEsis  based onNheI and XhoI sites to assay its enhancer or promoter activity on the downstream gene. The fragment was also cloned into the pGL3-Promoter vector using SaII and BamHI sites to assay its enhancer activity on the upstream gene. To further analyze the promoter activity of the three variations in fragment 2 in pairs or separately, appropriate sequences  were cloned into the pGL3-Basic vector using NheI and XhoI sites.To analyze promoter and enhancer activity of the six candidate variations by luciferase assay, two 250bp fragments each containing three candidate variations were synthesized: fragment 1  contained variations M1 , M2  and M3 ; fragment 2  contained variations M4 , M5  and M6 . Each fragment containing either reference or variant alleles was cloned into the pGL3-Promoter vector  or pGL3-Basic vector  using DEF cells in 24-well plates were transiently transfected with 720 ng Firefly luciferase vector (pGL3) and 80 ng pRL-TK Renilla luciferase vector  using 1.5 uL Lipofectamine 3000 . Twenty-four hours after transfection, luciferase activities were measured with a dual-luciferase reporter assay kit  on a SpectraMax M5 . Renilla luciferase activity was used as the transfection control for normalizing Firefly luciferase activity. Empty pGL3-Basic vector and pGL3-Promoter vector were used as the reference for promoter activity and enhancer activity assays, respectively. Empty pGL3-Control vector (Control) was used as the positive control for enhancer activity. All tests were conducted in triplicate with independent readings in triplicate (n = 3×3 = 9). The data are presented as means with their standard deviation.S4B Table. Two microliters of nuclear proteins (5μg/μl) were incubated with 40 pmol of biotin-labeled probes containing blue eggshell allele or white eggshell allele. Simultaneously, cold competition controls were conducted by adding 200-fold excess of unlabeled competitors containing the blue eggshell allele or white eggshell allele. For supershift analysis, the anti-duck CTCF polyclonal antibody made in rabbit using a partial CTCF peptide (S4C Table)  was used. CTCF antibody (1μg) was incubated with 0.5 μg recombinant human CTCF protein  prior to addition of the binding buffer and probe. Complexes were subjected to electrophoresis on non-denaturing 5% polyacrylamide gels. Finally, a picture was obtained using a LightShift Chemiluminescent EMSA kit .Uterine nuclear proteins from a blue-eggshelled duck were extracted following the instructions described in the protein extraction kit . Sequences of oligonucleotides used in the EMSA are listed in S4D Table). ChIP with rabbit immunoglobulin G (IgG) and “input” were used as negative control and positive control, respectively.Briefly, tissues were fixed with 1% formaldehyde, and then chromatin was prepared with a Chromatin Extraction Kit . ChIP was conducted with the CTCF antibody using a ChIP kit  according to the manufacturer’s instructions. ChIP-enriched DNA was analyzed with qPCR using primers specific for variation M5  . siRNA1 exhibited higher knockdown efficiency and was used in subsequent experiments. To assay the influence of CTCF expression on the promoter activity of causative sites, 150nM CTCF siRNA1 or scramble siRNA was cotransfected with luciferase reporter vectors containing variation sites into DEF cells using Lipofectamine 3000 . Luciferase activities were measured as mentioned above.Two sets of siRNAs and one set of negative control siRNA (scramble siRNA) were designed for duck  The anticipated PCR bands were gel-purified using a Gel Extraction Kit , cloned into the pMD19-T Vector  and sequenced.Genomic DNA was isolated by phenol chloroform extraction, after which 2 μg genomic DNA was bisulfite treated with the EZ DNA methylation-Gold kit . To examine the methylation status of targeted genomic regions around the CTCF binding site, we carried out semi-nested PCR with the following conditions. The first round of amplification: 3 min at 94°C; 20 cycles of 94°C for 20 s, 50°C for 30 s and 72°C for 30 s; and a final extension at 72°C for 5 min. The second round of amplification: 3 min at 94°C; 35 cycles of 94°C for 30 s, appropriate Tm for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. Primers used in the semi-nested PCR are listed in PRKG2 and ABCG2 genes, 3’RACE of PRKG2 and 5’RACE of ABCG2 were respectively performed using the 3’ and 5’ RACE System for Rapid Amplification of cDNA Ends kits . RNA was extracted from duck uteruses using Trizol  and purified using a RNeasy MiniKit . Three micrograms of RNA were used for reverse transcription with SuperScript III  followed by a nested PCR for the first and second amplifications of the 5’and 3’UTRs. Both PCR amplifications were conducted as follows: 95°C for 3 min, followed by 33 cycles of 94°C for 30 sec, 58°C for 30 sec, 72°C for 60 sec, and finally a reconditioning step at 72°C for 7 min. RACE products were purified, sub-cloned into the pMD19-T vector , and sequenced. The primers used for RACE are presented in  The sequences of 3’ and 5’RACE products have been submitted to GenBank with accession numbers MF774072 and MF774071, respectively.To determine the exact distance between the causative sites and the S4D Table. Five birds were used for each group. Relative gene expression was normalized to the internal control gene GAPDH using the 2-ΔΔCt method [Uteruses were collected from white-eggshelled ducks, homozygous blue-eggshelled ducks, and heterozygous blue-eggshelled ducks. Total RNA was extracted from uteruses with Trizol  and quantified using a Nanodrop2000 . Following cDNA synthesis using M-MLV reverse transcriptase , relative gene expression was detected by quantitative real-time PCR conducted on a 7900 Sequence Detection System  under the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 30 sec and 60°C for 1 min. The primer sequences used for the real-time PCR are listed in t method .S4C Table) .In the immunohistochemical assay, the anti-duck ABCG2 monoclonal antibody was made in mouse using a partial ABCG2 peptide  for 30 minutes at room temperature and then incubated overnight at 4°C with ABCG2 antibody (diluted 1:400 in the blocking solution). After washing three times in PBS, the sections were incubated with HRP conjugated goat anti-mouse IgG  for one hour at room temperature and then developed with a DAB Horseradish Peroxidase Color Development Kit . The nuclei were counterstained with hematoxylin . Subsequently, the sections were visualized under an inverted microscope at 200× or 400× .Paraffin sections were prepared as mentioned above and dewaxed to water under the following conditions: washed with xylene twice for 20 mins, with anhydrous ethanol twice for 10 mins, then with 95%, 90%, 80%, 70% ethanol for 5 mins in turn, and finally with distilled water. Dewaxed sections were stained for biliverdin with Lugo's iodine solution for 6 to 12 hrs and stained for nuclei for 5 mins with nuclear fast red. Subsequently, the sections were visualized under an inverted microscope at 200× or 400× .S1 Table(XLSX)Click here for additional data file.S2 Table−7 are listed. Positions with Bonferroni adjustment < 0.05 were identified as significantly associated and highlighted in boldface.SNPs with a p-value <10(XLSX)Click here for additional data file.S3 TableVariations exhibiting perfect genotype segregation among the three genotypes of resequenced samples are highlighted in red.(XLSX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 FigGAPDH using the 2-ΔΔCt method. Data represent the mean±SD from at least five ducks per group.Relative mRNA expression was normalized to (DOCX)Click here for additional data file.S2 FigCTCF. CTCF expression was analyzed in DEF cells transfected without siRNA or with siRNAs or negative control siRNA (scramble siRNA). Expression in the without siRNA group (no siRNA) was set as control and normalized as 1, and expression in the siRNA groups is presented as the ratio to that of control. Data represent the mean±SD from three biological repeats per group. ** represents p<0.01.Two sets of siRNAs were designed for duck (DOCX)Click here for additional data file.


Poncirus trifoliata). PtAOS1 contains a putative chloroplast targeting sequence in N-terminal and shows relative to pistachio (Pistacia vera) AOS. A number of stress-, light- and hormone-related cis-elements were found in the PtAOS1 promoter which may be responsible for the up-regulation of PtAOS1 under drought and JA treatments. Transient expression in tobacco (Nicotiana benthamiana) demonstrated that the P−532 (−532 to +1) fragment conferring drive activity was a core region in the PtAOS1 promoter. Using yeast one-hybrid, three novel proteins, PtDUF886, PtDUF1685 and PtRAP2.4, binding to P−532 were identified. The dual luciferase assay in tobacco illustrated that all three transcription factors could enhance PtAOS1 promoter activity. Genes PtDUF1685 and PtRAP2.4 shared an expression pattern which was induced significantly by drought stress. These findings should be available evidence for trifoliate orange responding to drought through JA modulation.Jasmonic acid (JA) plays a crucial role in various biological processes including development, signal transduction and stress response. Allene oxide synthase (AOS) catalyzing (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT) to an unstable allene oxide is involved in the first step of JA biosynthesis. Here, we isolated the PtAOS1 gene and its promoter from trifoliate orange ( Poncirus trifoliata) is the main citrus rootstock in China because of its excellent tolerance or resistance to stresses including drought. However, its specific mechanism in drought tolerance is still not known well. Jasmonic acid (JA) is a kind of octadecanoid derivative synthesized from α-linolenic acid and released from galactolipids of chloroplast membranes. JA and its derivatives (JAs) serve as signaling molecules to regulate diverse aspects of plant life including leaf senescence [In the south of China, seasonal drought happening from July to September generally damages the citrus industry regularly. Trifoliate orange  when LeAOS expression was inhibited [StAOS2 in potato significantly reduced endogenous JA content and compromised disease resistance [BaAOS in Arabidopsis increased basal JA levels compared with wild type: with drought stress, transgenic lines exhibited a drastic increase in endogenous JA and higher tolerance [Allene oxide synthase (AOS) converting (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT) to an unstable allene oxide is involved in the first step of JA biosynthesis. AOS belongs to the superfamily of cytochrome P450. It is likely to be a key regulator in JA’s biosynthesis . Over-exild type . Silenci tobacco . Defensenhibited . Silencisistance . Furtherolerance .ClAOS promoter, indicating their possible roles in stress and hormone responses [AtAOS1 promoter activity but that significantly increased at 3 h after mechanical wounding, suggesting the AtAOS1 promoter could be an inducible dramatically by injury [GmAOS1 associated significantly with soybean defense against common cut worm (Spodoptera litura Fabricius) attack [Transcription factor interacts with cis-acting elements in a gene’s promoter region and activates transcription. In watermelon, lots of stress- and hormone-related cis-elements were found in the esponses . In Araby injury . Promote) attack .AOS1 in trifoliate orange (PtAOS1) were characterized. Gene expression and PtAOS1 promoter activity were detected here. Afterward, a core region contributing to the promoter activity of PtAOS1 was revealed through 5’ deletion assays. Using yeast one-hybrid and dual-fluorescence systems, three interacting proteins binding with the PtAOS1 promoter were identified and named PtDUF886, PtDUF1685 and PtRAP2.4. Responses of PtDUF886, PtDUF1685 and PtRAP2.4 to drought stress were investigated further.A large number of AOS genes were cloned and characterized in diverse plants, however, there was still limited information about how it worked ,25,26. OPtAOS1 was isolated from trifoliate orange. Sequence analysis showed that PtAOS1 encoded a protein of 499 amino acids with a predicted molecular mass of 59.5 kD. The PtAOS1 protein contains a chloroplast transit peptide in N-terminal that might function as a chloroplast localization signal. The phylogenetic tree showed that AOSs were divided into four groups in dicots (Citrus sinensis), PtAOS1 exhibited high similarity with pistachio (Pistacia vera) AOS. In addition, AOSs from walnuts (Juglans regia), bayberry (Morella rubra), California valley oak (Quercus lobata), cork oak (Quercus suber) and mulberry (Morus notabilis) were close to PtAOS1 in their evolutionary process.Full-length n dicots . Except PtAOS1 responding to drought stress and exogenous JA treatment were analyzed. The transcript of PtAOS1 was induced significantly by exogenous JA within 12 h. Peak expression happened at 24 h after JA treatment, about 30-folds higher relative to the control. At 72 h after treatment, PtAOS1 expression decreased sharply, still being about 3.2-folds higher relative to the control (PtAOS1 was induced soon after drought stress (PtAOS1 was found immediately at 70% soil relative water content (RWC) in which citrus plant grows well generally. From 70% to 50% RWC, its expression level was up-regulated, about 2-folds higher than the control. From 40% to 20% RWC, the PtAOS1 transcript decreased slightly.Expression patterns of  control A. Exprest stress B. Up-regPtAOS1 containing 1181 bp upstream of the start codon was isolated and analyzed. The promoter fragment typically comprised an element involved in MeJA responsiveness (CGTCA-motif), an abscisic acid responsiveness element (ABRE) and an MYB binding site (MBS) mentioned drought stress  and its truncated fragments P−1062 (−1062 to +1), P−922 (−922 to +1), P−668 (−668 to +1), P−532 (−532 to +1) and P−265 (−265 to +1) were assigned to drive a yellow fluorescent protein (YFP) in tobacco leaves. A YFP signal driven by P−1182 was detected successfully in tobacco epidermal cells which illustrated the transcription activation of the PtAOS1 promoter. A YFP driven by P−1062, P−922, P−668 and P−532 fragments exposed a fluorescence signal equally with that using P−1182. However, a YFP signal with a P−265 fragment drive decreased sharply, implying a core function lost (Transient expression assays were used to examine a core region in the ion lost A–C.PtAOS1 promoter to MeJA and abscisic acid (ABA), 50 µM MeJA and 25 µM ABA were applied on infiltrated tobacco leaves. Results showed YFP was stimulated significantly after MeJA and ABA treatments when most truncated promoters were used except only P−265  for the bait yeast strain was 300 ng/mL. After a screen in a cDNA library prepared using drought-treated trifoliate orange leaves, three clones exhibited AbA resistance on selective plates  catalyzes the first step in JA biosynthesis and the encoding gene works outstandingly after abiotic and biotic stresses ,23. Genemulation . In chicreatment . Transcrreatment . Expressnd LeHPL ,32. Overnd LeHPL . With Csl damage . Transgenfection . Tobacco2 stress . In thise orange B showingreatment A which wGmRD26 (a NAC transcription factor) promoter has been characterized as being involved in stress and ABA response; ABRE motifs and ABA responsive elements were thought to be the key factor influencing the stress-responsive gene expression regulated by GmRD26 under drought stress [napA promoter led to the complete abolishment of reporter gene expression, suggesting that E-box is a core motif conferring napA promoter activity [PtAOS1 promoter contained cis-acting elements engaging in defense and stress responsiveness like an MYB binding site [PtAOS1 promoter was enriched in some light-responsive elements such as AE-box, G-box and MNF1 motif, indicating possible involvement in light response [It is widely known that a gene expression induced by an external stimulus was modulated by a regulatory motif in the promoter region ,37,38. It stress . In tranactivity . Here, ting site  and two ing site . These iresponse ,44.VvβVPE promoter was demonstrated as seed-specific, whereas this specificity vanished with a deletion from −1306 to −1045 bp [OsHAK1 promoter; further, the 5’ deletion analysis demonstrated that the promoter region −3037 to −1821 bp was indispensable for osmotic stress response [PtAOS1 promoter  promoter region −221 to +40 bp was sufficient for JA-responsive transcription activation in tomato (Solanum lycopersicum) through a series of 5’ deletions [GGP promoter was deduced to a function of regulating GGP gene expression when subjected to JA [PtAOS1 promoter was known as a major cis-acting regulatory element in ABA-dependent drought tolerance. An ABRE element was required for stress-responsive gene expression induced by osmotic stress in an ABA-dependent pathway. Gene OSBZ8 was considered to regulate ABA-mediated transcription; the intensity of OSBZ8 binding to an ABRE element was high and constitutive in salt-tolerant rice cultivars compared with salt-sensitive cultivars [AREB1, AREB2 and ABF3 were induced by drought, ABA treatment and high-salinity in Arabidopsis: these three genes functioned as transcription activators in ABA-dependent response through directly binding to an ABRE sequence in the DREB2A promoter [PtAOS1 promoter activity but the inducibility was significantly reduced by a 268 bp deletion which contained a CGTCA-motif and an ABRE-element. These results demonstrated a CGTCA-motif and an ABRE-element located in −532 to −265 bp of PtAOS1 promoter could be responsible for the JA drought response in trifoliate orange.Our previous reports suggested that the JA  and ABA eletions . Similared to JA . ABRE exultivars . Expresspromoter . In thisAtAOS promoter in healthy Arabidopsis. However, the JJW complex would disintegrate and activate JA biosynthesis once the plant was injured by mechanical damage or insect attack [OsAOS1 promoter. A reduction in endogenous JA by over-expressed OsDOF24 affected multiple physiological processes including leaf senescence, plant architecture and grain yield [PtAOS1 promoter bait, and three interacting proteins PtDUF886, PtDUF1685 and PtRAP2.4 were obtained  and plant hormones  in soybean; over-expression of GmERF3 in tobacco improved its drought tolerance [TaERF3 in wheat also exhibited tolerance enhancement to salt and drought stresses [ORCA3 encoding an AP2/ERF transcription factor was involved in JA signaling via directly binding to the jasmonate- and elicitor-responsive element of the Strictosidine synthase gene promoter [ORA59 acted as the key regulator of JA and ET responsiveness [AaERF1 and AaERF2 from Artemisia annua were both strongly induced by JA and acted as positive regulators of artemisinin biosynthesis [PtRAP2.4 with an AP2 domain was an AP2/ERF transcription factor and its expression was induced by drought significantly  complex repressing JA biosynthesis through binding to the t attack . Similarin yield . Here, wobtained A–C. The obtained D. Domaintivities . Expresst stress . Expresseatments . In the e orange B,C. Our response ,60,61,62olerance . Similarstresses . Severalpromoter . ERF memsiveness . Genes Aynthesis . Here, Pficantly A. It couPtAOS1 from trifoliate orange which was induced by drought, ABA treatment and JA treatment. A region from −532 to −265 bp in the PtAOS1 promoter was indispensable for promoter activity and ABA and JA responses. An ABRE element and a CGTCA-motif in the PtAOS1 promoter were probably responsible for PtAOS1 responding to drought. Further, three novel proteins which were drought-inducible and worked through binding to the PtAOS1 promoter were identified. These findings may offer new insights into the drought tolerance of trifoliate orange through the JA pathway.In conclusion, we identified and analyzed Poncirus trifoliate) seedlings in this assay were planted in a green house. All tobacco (Nicotiana benthamiana) seedlings were grown in a plant growth chamber with 16 h light/8 h dark cycle at 28 °C.Six-month-old trifoliate orange . Control samples (CK) were collected from well-watered seedlings.One mM MeJA was sprayed on the leaves of trifoliate orange. Samples were collected at 0, 12, 24 and 72 h after treatment. All samples were stored at −80 °C until use.PtAOS1 open reading frame (ORF) was PCR-amplified from trifoliate orange cDNA with primers  and constructed into a pM18-T vector . The NCBI (http://www.ncbi.nl m.nih.gov/) database was used for a homologous sequence search through the BLAST program and protein sequences were aligned with the ClustalW 2.0 software. The phylogenetic tree for AOS proteins was constructed with the MEGA5.0 software using the neighbor-joining method. Chloroplast transit peptide was analyzed using TargetP (http://www.cb s.dtu.dk/services/TargetP) and ChloroP (http://www.cbs.dtu.dk/services/ChloroP/).The PtAOS1 , PtDUF668 , PtDUF1685  and PtRAP2.4 . The qPCR was performed with SsoAdvancedTM SYBR® Green Supermix  using the CFX96 system . The reaction mixture  comprised 1 µL cDNA template, 0.5 µL primers (0.25 µM forward and 0.25 µM reverse), 5 µL 2× SYBR Green Supermix and 3.5 µL RNase-free water. The cycling parameters were 30 s at 95 °C, followed by 40 cycles of 95 °C for 5 s, 58 °C for 15 s. The relative expression level was normalized against the β-actin gene. Relative gene expression was calculated using the CFX96 software. Three biological replicates were performed for each sample.Gene expression patterns were assayed using mature leaves. Total RNA was isolated using RNAprep Pure Plant Kit  according to the manufacturer’s protocol. The first strand of cDNA was synthesized from 1 µg of DNase treated total RNA using FastQuant RT Kit  according to the manufacturer’s instruction. The primers used in the real-time quantitative PCR (qPCR) were as follows: PtAOS1 promoter was amplified using primers  and cloned into a pMD18-T vector for sequencing. Core motif predicted by PlantCARE (http://bioinformatics.ps b.ugent.be/webtools/plantcare/html). The promoter of PtAOS1 and truncated fragments  were PCR-amplified using a specific forward primer  and the same reverse primer (5′-GGATCCTTCACAGCGATAATAATTGTTGT-3′). The PCR products were gel-purified and cloned into a modified pBI121-YFP vector with EcoRI and BamHI digestion. The corresponding vectors were named P−1181, P−1062, P−922, P−688, P−532 and P−265, respectively. All constructs were purified and transformed into agrobacterium (EHA105) for further transient expression.Total genome DNA was extracted from trifoliate orange leaves using Plant Genomic DNA Extraction Kit . The Nicotiana benthamiana, the transient expression assay was performed as according to Sparkes [600 reached 0.6. Then, it was centrifuged at 3000 rpm and resuspended with infiltration buffer containing 10 mM 2-Morpholino ethanesulfonic acid hydrate (MES), 10 mM MgCl2 and 100 µM acetosyringone to an OD600 of 0.8, followed by incubating at 28 °C for 2 h. Infiltration: tobaccos leaves were injected from the back with a syringe (needle removed) until the water spots spread all over them. After 3 days, the YFP fluorescence intensity was detected with the LSM700 Fluorescence microscope . Phytohormone treatment: tobacco leaves were treated with distilled water (CK), 50 µM MeJA or 25 µM abscisic acid (ABA) at 12 h after the infiltration, and the YFP signal was detected at 36 h after treatment.With  Sparkes  with min−532 fragment of the PtAOS promoter was cloned into a pAbAi vector with digestion by SacI and XhoI. The construct was then digested by SmaI and then transformed into yeast strain Y1HGold according to the Matchmaker™ Gold Yeast One-Hybrid System  protocol and selected on dropout medium lacking uracil (SD/-Ura). A positive yeast strain was then picked and resuspended in 0.9% NaCl to an OD600 of 0.002. Then, SD/-Ura with Aureobasidin A (AbA) medium was used to determine the minimal AbA inhibitory concentration. The cDNA library construction was performed following the manufacturer’s instruction for the MatchmakerTM Gold Yeast One-Hybrid Library Screening System . First, total RNA was extracted from trifoliate orange leaves using the method described before, and then mRNA was enriched and purified. Then, a cDNA for library construction was generated through first-strand SMART cDNA synthesis and long-distance PCR for double-strand cDNA (ds cDNA). The ds cDNA was purified with CHROMA SPIN+TE-400 columns. A one-hybrid cDNA library  screen was performed according to yeast maker transformation system two. Library transformation reactions were spread on SD/-Leu with 300 mg/mL AbA with incubation at 30 °C for 3–5 days. Positive interaction clones were rescued and prey plasmids were isolated from selective plates. To quickly identify the clones, the T7 primer was used for sequencing, and plasmids were retransformed into bait yeast to confirm the interaction.The PPtDUF886, PtDUF1685 and PtRAP2.4 were amplified from trifoliate orange cDNA with gene-specific primers and cloned into the effector vector pGreenII 62-SK. The clone primers used were as follows: PtDUF886 , PtDUF1685  and PtRAP2.4 . The P−532 fragment of the PtAOS1 promoter was amplified and ligated into the reporter vector pGreenII 0800-LUC. The corresponding constructs were transformed into GV3101 component cells. Effector and reporter strains were separately re-cultivated on an LB plate with 50 mg/L kanamycin at 28 °C for 48 h. We resuspended the colony in MES buffer to an OD600 of 0.8 and incubated it for 2 h at 28 °C. Resuspended effector: reporter = 5:1 before infiltration was mixed up. After infiltration, tobacco seedings were moved back to the growth chamber with no watering for 3 days. Activity assays for firefly luciferase (LUC) and renilla luciferase (REN) were performed via the Dual-luciferase Reporter Assay System  according to the manufacturer’s instruction.Full-length genes of p < 0.05 as statistically significant.All data were presented as mean ± SE with three independent replicates at least. The data were analyzed using the SPSS13.0 software running ANOVA according to Tukey’s multiple range test taking