TECHNICAL FIELD

The present invention relates to plant genetic engineering, particularly, a gene and a vector containing the gene or a homologous gene thereof and the application for regulating leaf erectness of plants in agricultural production.

BACKGROUND OF INVENTION

The architecture of crops is a key characteristic for high-yield breeding. Leaf erectness (or the leaf-stem angle) is one of the important characteristics for plant architecture development. The field planting density of crops may be increased with a proper leaf-stem angle to further promote the utilization ratio of light energy and increase the yield of crops. The leaf pulvinus plays a key role in leaf erectness, and its development directly affects the leaf erectness. Therefore, finding and utilizing the key gene affecting the leaf erectness (or the leaf-stem angle) is the key to improve the rice architecture for promoting the breeding via molecular design and increasing the rice yield per unit area.

The leaf pulvinus of monocotyledonous plants is an intermediate hub connecting the leaf blade and leaf sheath and serves as a basic structure for forming the leaf-stem angle. As a mechanical mechanism, the leaf pulvinus promotes the leaf blade to bend towards an abaxial direction when the leaf blade and the leaf sheath undergo complete elongation, therefore, the development of leaf pulvinus closely relates to the leaf erectness. Both brassinosteroids (BR) synthesis and signaling pathways play an important and unique role in regulating the leaf erectness in monocotyledons, however, the underlying cytology and molecular mechanisms are unknown. It is reported that mutants in BR synthesis pathway, d2, dwarf4, d11, and brd2, are all characterized by erect leaves. A positive-regulator-deficient mutant in the BR signaling pathway is also characterized by a small leaf-stem angle and erecting leaf, such as receptor mutants d61, dlt, d1, and tud1. The transgenic plants of OsBAK1 and OsGSK2, which are obtained by homologous cloning, exhibit changes in the leaf-stem angle. The leaf erectness may also be promoted by suppressing the gene expression of the positive regulator OsBZR1 at the downstream of the rice BR signaling pathway via RNAi technology. Moreover, a gain-of-function mutant lic-1, a negative regulator of the BR signaling pathway, is also characterized by a small leaf-stem angle and erecting leaf. A gain-of-function mutant ili1-D is characterized by a large leaf-stem angle due to the amplification of a BR signal. In addition, a target gene OsIBH1 of OsBZR1 is directly involved in the leaf erectness regulation in rice; and the external application of BR enlarged leaf angles of wild type and BR-synthesis mutants, and the BR signaling mutant, such as a receptor mutant d61, is almost unaffected.

In addition to the brassinosteroids pathways, other signaling pathways are also involved in the regulation of the leaf-stem angle. Although the genes of the BR-related pathways play a significant role in regulating the rice leaf angles, other independent pathways are also involved in the regulation of such biological characters. When decreasing the expression level of SPINDLY (SPY), a negative regulated gibberellin signal, the leaf-stem angle can be increased. However, the leaf-stem angle of the rice may be increased due to the over-expression of a family member OsIAA1, a negative regulator in auxin signaling pathway. LC1, encoding a gene for regulating the auxin homeostasis, determines the rice leaf-stem angle by regulating the cell elongation in the leaf pulvinus. Rice leaf inclination2 (LC2), a VIN3-analogous protein, may also enlarge the leaf-stem angle after mutation. A gene ILA1, a member in Group C of a MAPKKK family, has a high expression in a vascular bundle of the leaf pulvinus, and the insertional mutation of ILA1 thereof leads to the decrease of the contents of major components including fibrin and xylan in the cell wall of the leaf pulvinus, resulting in the reduction of the mechanical strength of the leaf pulvinus and the increase of the leaf angle. A mutant of LPA1, a Loose Plant Architecture1 in the rice, is characterized as a loose architecture with both the tiller angle and leaf-stem angle increased.

Leaf erectness has been demonstrated to be closely related with the crop yields. There is a broad application prospect in the crop yields by using the gene in the BR-related pathway as a target gene for improving the rice architecture. For example, the expression of endogenous OsBRI1 of the rice is partially suppressed by introducing an intracellular region (OsACTIN::OsBRI1-KD) of OsBRI1 as driven by a rice actin promoter with a co-suppression strategy, thereby an erect leaf transgenic line is isolated from the offspring; and the yield is estimated to increase by about 35% and 26% through close planting. Through field experiments, it has been demonstrated that the yield may be increased by up to 32% by the reasonable close planting of the mutant based on a BR synthesis pathway OsDWARF4 of the rice. 244 RIL groups from Zhenshan 97B and Miyan 46 are genetically analyzed in terms of the flag leaf angle and the main ear output. Five QTLs for controlling the flag leaf angle have been detected in total on chromosomes 1, 3, 5 and 11, where the QTL for controlling the flag leaf angle (FLA) and seed setting percentage (SF) have been detected simultaneously at an interval RM24-RM294A of the chromosome 1, with opposite additive effect directions. It demonstrates that the flag leaf angle is in negative correlation with the yield, and it is favorable to increase the seed settling percentage by properly reducing the flag leaf angle of the rice, and to finally increase the yield.

SUMMARY OF THE INVENTION

The present invention provides a gene for controlling rice leaf erectness development, in which with a reverse genetics method, a gene, specifically expressing in a leaf pulvinus, is screened through the specific gene expression chip analysis of a rice leaf and pulvinus, and the promoter of CYC U4;1 is proved to have a specific expression in the sclerenchyma cell of leaf pulvinus by utilizing a pCYCU4-GUS transgenic line and in situ hybridization; and by transgenically increasing the expression level of CYC U4;1 and decreasing the expression level of CYC U4;1 with RNAi technology, the gene CYC U4;1 is proved to be capable of controlling the rice leaf erectness development. The present invention provides a vector containing the gene or a homologous gene thereof and an application involving regulating the leaf erectness of plants by utilizing the gene or a functional analog thereof in agricultural production.

One object of the present invention is to provide a gene CYC U4;1 for controlling the leaf erectness in rice. The gene is identified from a specific expression chip of a rice leaf pulvinus and is isolated and cloned. The promoter region of CYC U4;1 as shown in SEQ ID NO: 1, the CDS nucleotide sequence of CYC U4;1 as shown in SEQ ID NO: 2 and the amino acid sequence of CYC U4;1 as shown in SEQ ID NO: 3.

Another object of the present invention is to provide an application of the gene CYC U4;1 to controlling of the rice leaf erectness. A leaf-stem angle may be reduced by over-expression of the CYC U4;1 driven by the native promoter (with the sequence as shown in SEQ ID NO: 1) of the gene CYC U4;1, thereby improving the architecture of rice varieties and increasing the plant density per unit area.

To achieve the objects as described above, a technical solution employed by the present invention is as follows.

Using a gene specific expression chip assay in rice leaf blades and pulvini the inventor created, a gene CYC U4;1 with a specific expression at the leaf pulvinus is selected from differentially expressed genes of the chip with a reverse genetics method, and the specific expression of CYC U4;1 at the leaf pulvinus is verified with real-time qRT-PCR. A gene CYC U4;1 for controlling erectness development of rice leaves is obtained with the following method.

The total volume of a reaction system is 50 μl, with a template including Nipponbare cDNA 1 ul (about 50 ng), 10×KOD enzyme reaction buffer (5 μl), 25 mM MgCL2(2 μl), 5 mM dNTP (5 μl), 5 uM primers (5 μl) (with a stepwise PCR means, primers CYCU4-U having SEQ ID NO: 4 and CYCU4-L-1 having SEQ ID NO: 5 are used the first time, and primers CYCU4-U having SEQ ID NO: 4 and CYCU4-L-2 having SEQ ID NO: 6 are used the second time, with each primer at 2.50), KOD enzyme (1 μl), and the balance of ddH2O (sterile deionized water) which is added till the total volume of 50 μl.

A reaction procedure is as follows: denaturing at 94° C. for 5 min, 94° C. for 30 sec, 55° C. for 1 min and 68° C. for 2 min for 35 cycles in total, and elongating at 68° C. for 10 min The primers are described as follows:

CYCU4-U:(SEQ ID NO: 4)ATATgagctcATGAGGACGGGGGAGGTGGCGGAGGCGGTG;CYCU4-L-1:(SEQ ID NO: 5)CGTCTTTGTAGTCGACGGCGAGCTGATGCTGCTGCTG;andCYCU4-L-2:(SEQ ID NO: 6)ATATtctagactaCTTGTCGTCATCGTCTTTGTAGTCGACGGCGAG.

A gene sequence containing the nucleotide as described in SEQ ID NO: 2 is finally obtained, and a protein sequence encoded by the gene is as shown in SEQ ID NO: 3.

The present invention also provides the promoter region (with the sequence as shown in SEQ ID NO: 1) of the gene CYC U4;1, a nucleotide sequence (with the sequence as shown in SEQ ID NO: 2) corresponding to an amino acid sequence as shown in SEQ ID NO: 3, as well as a protein having the same function, which is obtained by modifying the sequence as shown in SEQ ID NO: 3.

An application of the gene CYC U4;1 to controlling the erectness of the rice leaves comprises cloning a promoter and full-length CDS of CYC U4;1 to pCAMBIA1301 and transferring it into the Nipponbare, and transgenic lines obtained are characterized by having a smaller leaf-stem angle than those of wild rice (Nipponbare), therefore, the leaf-stem angle may be reduced with this method to increase the planting density.

Compared with the prior art, the present invention has the following advantages:

1. CYC U4;1 is a gene having a specific expression only in the leaf pulvinus of rice in a seedling stage, which makes it possible to only change the leaf-stem angle of the crops by using the gene without affecting other characters.

2. As an effective gene for changing the leaf-stem angle, CYC U4;1 may directly affect the specific cell division at the leaf pulvinus to further change the leaf-stem angle of the crops;

3. At present, the molecular mechanism of the leaf erectness regulated by brassinosteroids to further increase the yield of crops is still not clear, and the gene uncovered the cellular and molecular mechanism that brassinosteroids regulated the erectness of the leaves.

