Abstract:

【Objective】 This research aimed to screen resistance genes from cycled Chinese wild grape Vitis pseudoreticulata cv. Baihe35-1 cDNA library which was constructed from leaves induced by grape powdery mildew (Uncinular necator) inoculation. 【Method】Specific oligonucleotide primers were designed according to P-loop region of conservative NBS and LRR sequence of resistance genes and the universal sequence primers on cloning vector. According to the method of three dimensional library, colony PCR was employed to screen the circled cDNA library. 【Result】 Twenty-four super pools, each contains 768 colonies, were built, obtaining 18 432 colonies, and 585 positive colonies were obtained, in which 330 by NBS primers and 255 by LRR primers. The sequencing results were assembled into 422 contig sequences that 250 and 172 sequences from NBS and LRR primer screening respectively, including 25 sequences from the both. 【Conclusion】 There were 8 sequences (GW787758, GW787739—GW787742, GW787746, GW787747, GW787749) belong to P-loop superfamily and 5 sequences (GW787743, GW787744, GW787751, GW787753, GW787750) with LRR and a specific gene in Vitis pseudoreticulata (GW787754). Ten sequences which have different resistance domains were chosen for real-time quantitative PCR to check the expression profile in the process of white powdery mildew invasion. Five showed significant variation during Uncinular necator induction and supposed to be a pathogenesis related gene.

Key words: Vitis pseudoreticulata, cDNA library, library screening, disease resistance gene, real-time quantitative PCR