CDNA Library Construction And Expressed Sequence Tags (EST) Analysis Of The Ripening Vitis Amurensis Cv. Shuang Hong Berry Skins

Vitis amurensis is the main material that makes wine in the northeast region, wine made by amur grape has special taste and deeply popular among the domestic and international consumer. For studying the related gene expression during the amur grape mature process, identifying related gene of maturity, anti-disease, production of flavor and biosynthesis of anthocyanins, carried on the following research:Total RNA was extracted by three methods and the CTAB method can separate high quality RNA, and can be used for the next step of molecular biology search. cDNA library from the amur grape was constructed by Creator SMARTM cDNA Library Construction Protocol. The library capacity was 1.001×106 with a recombinant rate of 94% and the inserted cDNA fragments ranged from 0.25 to 2.0 kb.935 available EST through random sequencing were obtained, and 636 unigenes which including 108 contigs and 508 singletons were generated after clustering. This study laid a foundation for gene cloning and functional genomic research of amur grape.The result of EST analysis shown that the percentage of function unknown gene was 17.92%, metabolism gene was 20.81%, cell rescue and defence gene was 5.15%, signal transduction gene was 4.68%, energy gene was 5.61%, protein synthesis was 9.59%, protein activity regulation gene was 15.43%, cell structure gene was 9.12%, transport gene was 6.55% and transcription gene was 5.15%.The most frequently found gene in the cDNA library was ribosomal protein (28 clones; 5.38%). Ribosomal protein was the most frequently found gene in the library indicated that the process of protein synthesis was actively in the V. amurensis berry skins. In the cDNA library we found some genes that correlated with the biosynthesis of anthocyanins such as chalcone synthase(CHS), flavonoid3',5'-hydroxylase (F3'5'H), phenylalanine ammonia-lyase (PAL), glutathione S-transferases (GST). These genes are shown to share significant sequence similarity (Evalue<10-15, Identity>80%) to a variety of known genes or gene families. In the cDNA library, there were 8 genes that correlated with production of flavor such as lipoxygenase, phenylalanine ammonia-lyase and alcohol dehydrogenase and so on. Related gene of maturity were polygalacturonase, ripening-related protein grip22, pectinesteras and lipoxygenase.According to the literature data, we chose 23 genes that related to anti-disease from the cDNA library. For better research the anti-disease mechanism provided foundation.