Construction and ESTs Analysis of a Normalized Full-Length cDNA Library of Brown Prunus salicina
Total RNA was extracted from the fruit of " Oil Nai" with beginning of browning.The full-length normalized cDNA library was constructed by the combination of long distance PCR,DSN(Duplex-Specific Nuclease) treatment and directed cloning.The titer of primary library was about 2.0×106pfu/mL and 99% clones were recombinant,and the length of insert cDNAs was about 1.8 kb.The cDNA library was well normalized with 8.4% redundancy.A total of 720 random clones in this cDNA library were then sequenced by 5'EST.A total of valid 684 ESTs were attained by fragment assembly,removing the contaminated sequences and frameshift mutation.Then 647 unigenes were attained,which included 21 contigs and 626 singlets.Annotation by the online Blast 2 go search against Genbank database on NCBI web server revealed that most of the genes with predicted function were related to energy metabolism,protein synthesis,degradation of secondary metabolites,cell wall metabolism and transcription factors.These results would lead to further research of mechanism of browning fruit in molecular level and develop good foundation of cloning related important genes of browning from "Oil Nai".