CDNA Library Construct Of Vitis Amurensis Skins And The Cloning And Expression Of Related Genes

Vitis amurensis is a kind of fruit tree resources with very high economic value, Which berries contain anthocyanins, protein and minerals and other nutrients. The study successfully constructed cDNA library of V.amurensis before skins coloring; The full-length cDNA sequences of CHS and CHI genes was cloned by the RACE technology and the bioinformatics was analyzed; The gene expression in seven stage of V.amurensis skins maturation was analyzed by the real-time fluorescent quantitative PCR technology, the main results obtained are as follows:The period before coloring skins of V.amurensis was used as to construct cDNA library by the technology of In-Fusion(?) SMARTer(?) Directional cDNA Library Construction. The result showed:the titer of primary and amplified cDNA library was 1.15 ×106 pfu.mL"1 and 2.86×109 pfu.mL"1 respectively. The recombination rate was about 100%, the insert fragment length was between 0.5-2 kb which the average size was about 0.9 kb. It showed that the cDNA library of V.amurensis before skins coloring was constructed successfully. This study laid a foundation for gene cloning and functional genomic research of V.amurensis.The full-length cDNA sequences of CHS and CHI gene in V.amurensis were cloned by the technique of RT-PCR and SMART RACE. The results of bioinformatics analysis showed that the two genes have the complete open reading frame, the full-length of CHS cDNA was 1461 bp with open reading frame of 1182 bp, which encodes 393 bp amino acids and the full-length of CHI cDNA was 955 bp with open reading frame of 705 bp, which encodes 234 amino acids. The common feature were that they have the stable protein,the polypeptide chain performancing hydrophilic, they were not containing transmembrane domain and signal peptide. Phylogenetic analysis showed that they all have the highest homology with Vitis vinifera.Using seven stage skins of V.amurensis as experimental material, ACTIN for internal genes to analysis the expression pattern of CHS and CHI genes by the real-time fluorescent quantitative PCR technology. The result showed:they were expressed in seven stage, in addition to the expression quantity of before fruit coloring and 10% turn colored were similar, the expressed quantity of CHS were significantly higher than the CHI in the other five stage. The expression of CHS in seven stage has obvious regularity, the expression quantity increase in the trend form the period of before coloring to 100% turn colored and the full ripeness period decreased expression. The expression quantity had not obvious regularity, the expression quantity was the lowest in before coloring period and had the highest in the full ripeness period.