Construction Of CDNA Library And Analysis Of EST Sequence Of Chinese Wild Vitis Pseudoreticulata And Its Hybrid Inoculated With Uncinula Necator

Powdery mildew caused by(Uncinula necator(schw.) Burr.) is one of the most serious diseases which influence the grape production in the world.Breeding resistant cultivars has been proved the most effective way to control this disease.China wild vitis not only possess the resistance to the powdery mildew but alse inhere the defence genes to the offsprings.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,We constructed cDNA library with Vitis pseudoreticulata W T Wang native to china and its hybride inoculated with Uncinula necator by using SMART technology..After sequencing,bioinformatics.method.was.used to analyze the EST sequences characteristics and functions,with the aim to obtain defence-related EST sequences and genes.The results are as follows:1.The grape materials of Chinese wild V.pseudoreticulata clone Baihe-35-1 highly resistant to powdery mildew in the grape germplasm resources orchard,Northwest A&F University,Yangling Shaanxi,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 powdery mildew from 8:00 am to 10:00 am on July 7,2006.The inoculated leaves were immediately covered with plastic bags to prevent them from being infected by other pathogens.At 1d,2d,3 d,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(1.5μg) from V.pseudoreticulata clone Baihe-35-1 was used to construct the cDNA library.The titer of primary library was 3.0×10~6pfu/ml.The recombinant percent accounted for 98%.The titer of amplified library was 1×10~9 pfu/m.The size of insert fragments basically ranged from 0.5 kb to 3.0 kb with average of 1.0 kb.2.We constructed cDNA library with Chinese wild V.pseudoreticulata clone 6-12-4 inoculated with Uncinula necator.The methods of inoculation,collection,RNA extraction were the same to baihe-35-1.The titer of primary library was 2.0×10~6pfu/ml.The recombinant percent accounted for 98%.The titer of amplified library was 8×10~8 pfu/m.The size of insert fragments basically ranged from 0.5 kb to 3.0 kb with average of 1.0 kb.3.181 sequences with high quality were obtained;and these sequences have submitted to GenBank database,The GenBank accession numbers are FG579859-FG580039;114 sequences were sent to GenBank for homologue search using Blastx programs and were further classified based on their functions.The results are as follows:78 EST sequences showed high homologue to 13 clusters of genes with known function;35 EST sequences were matched to the genes whose protein function were unclassified;1 EST sequences hit no any sequence in GenBank.Among the 78 EST sequences had strong homology to previously identified genes,28 EST sequences showed homologue to putative genes related to disease resistance,which are involved in the whole course of disease resistance response These genes included similar to SGT1,Calmodulin,MYB transcription factor,His-Asp phosphotransfer protein,participating in signal transduction;proteins such as Glycosyl hydrolases family 38 protein,PR14,LRR protein,glutaredoxin family protein,cyclophilin and cytochrome P450 involving in defense response; proteins metallothionein,cysteine protease,heat shock protein 90,chalcone-flavanone isomerase family protein indispensable for basal defense and non-host resistance;proteins.of.proline-rich protein,abscisic stress ripening protein,ultraviolet-B-repressible protein;for Stress induced protein.cysteine proteinase inhibitor,fiber protein related to cell self protection in disease-resistance response and host factor interacting with pathogen.4.After randomly sequencing,1019 unieqs were obtained,in which,785 were singletons, 234 were contigs.The nanlysis of bioinformatics were ounder going.5.A compete CAM gene was obtained from the cDNA library of Chinese wild vitis.