Construction Of CDNA Library Of Vitis Pseudoreticulata Native To China Inoculated With Uncinula Necator And Analysis Of Its EST Sequences

Powdery mildew caused by (Uncinula necator (schw.) Burr.) is one of the most serious diseases which influence the grape production in the world. Breeding for resistant cultivars has been proved the most effective way to control this disease. Previous researches indicated that Vitis pseudoreticulata W.T.Wang, native to china, possessing resistance to powdery mildew genes, was effective against all the current biotypes of U. necator in China. Cloning of resistance to powdery mildew genes or the related genes for powdery mildew resistance is of significance for understanding disease resistance mechanism and making use of disease resistance genes for breeding. In this study, bioinformatics method and PCR technique used to analyze the EST sequences characteristics and gene-expression profiles of specific stages of inoculation with U. necator besides sequencing strategy aim to obtain defence-related EST sequences and genes. The potential results are as follows:1. The grape materials of Chinese wild V. pseudoreticulata clone Baihe-35-1 highly resistant to powdery mildew and V. pseudoreticulata clone Hunan-1 highly susceptible to powdery mildew maintained in the grape germplasm resources orchard, Northwest A&F University, Yangling Shaanxi, People's Republic of China, were used in this study. The powdery mildew inoculation was carried out under natural field conditions. Before inoculation, the upper side of the uninfected young leaves of Baihe-35-1 pre-sprayed with sterile water, the upper side of the leaves was then inoculated by dry-pressing method with spores from infected leaves with U. necator of V. pseudoreticulata clone Hunan-1 from 8:00 am to 10:00 am on July 8, 2003. The inoculated leaves were immediately covered with paper bags to prevent them from infecting by other pathogens.At 1d, 2d, 3d, 4d, 5d, 6d and 7d post inoculation, leaves were collected from V. pseudoreticulata clone Baihe-35-1 and snap frozen in liquid nitrogen. Total RNA was isolated respectively from above leaf samples with SDS/phenol method modified partially. The pool of total RNA at various times (1 μg) from V. pseudoreticulata clone Baihe-35-1 was used to construct the cDNA library. The titer of primary library was 3.0×10~5pfu/ml. The recombinant percent accounted for 96.9%. The primary library was directly introduced into the E.coli BM 25.8 without amplification. The transformation efficiency exceeded over 90 %. The size of insert fragments basically ranged from 0.5 kb to 2.0 kb with average of 0.9 kb.