Construction And EST Analysis Of A Suppressive Subtraction CDNA Library Of Chinese Wild Vitis Quinquangularis Inoculated With Powdery Mildew

Grape powdery mildew, Uncinula necator (Schw.) Burr is a fungal diseases harming the cultivation and producing of grape growing around the world. Using of resistance to powdery mildew in Chinese wild grapes resources, understanding the interaction between grapes and powdery mildew, studies differentially expressed genes of grape powdery mildew fungus induced and screening powdery mildew resistance genes is an effective way to cultivate grape varieties of powdery mildew resistant. In this study, suppression subtractive hybridization was used to construct the suppressive subtractive hybridization (SSH) library of inoculated and non-vaccinated grape leaf. High resistance to host-specific gene fragment was screened after EST analysis. The main research results as fellows.Total RNA of inoculated and inoculated leaves of different periods of Chinese wild Vitis quinquangularis Shang-24 highly resistant to powdery mildew were extracted with modified SDS / phenol method, and mRNA were isolated successfully. The mRNA of non-vaccinated grape leaf as the control side (Driver), and the mRNA of vaccinated grape leaf as experiment side (Tester). Suppression subtractive hybridization library was constructed successfully with the high resistance to powdery mildew of Chinese wild grapes V. quinquangularis Shang-24 leaves.516 clones were selected randomly from the SSH library and then sequenced. 502 high quality ESTs were obtained. remove the vector sequence and the joint sequence, the size of fragment ranged between 250~800 bp, there are 19 sequences of of 3.7% total have no match after compared by Blastn and Blastx in GenBank, 483 sequences are highly homologous with known gene sequences, including 281 genes with known of the performance of a high degree of homology, 54.5% of the total sequence, 202 proteins is the unnamed or no specifically function, 39.1% of the total sequence.Analysised the 281 genes of known function from the library, 10 specific functions can be classified as follows: the active oxygen, known genes account for 5.0 of sequences; protein transport 12.1%, defense response 18.9%,signal transduction of 5.3%, 30% of energy metabolism, secondary metabolism of 9.3%, 7.1% in transcriptional regulation, protein co-metabolism of 9.6%, 2.1% in cell structure, osmotic adjustment of 0.7%. The resistance-related genes obtained as flows: flavonol synthase, pathogenesis-related proteins, chitinase, chalcone synthase, serine / threonine protein kinase, shaggy-related protein kinase (ASK), signal interaction kinase 02, a colorless anthocyanin reductase, cysteine protease, metal S protein, catalase enzyme, glutamine synthetase, glycolate oxidase, proline rich protein, calmodulin, DnaJ- like protein, F-box family protein, drought induced protein, senescence-related protein, lipid transfer protein et.al.