Analysis Of The EST Sequences And Construction Of SSH Library Of Chinese Wild Vitis Pseudoreticulata Induced By Plasmopara Viticola

Downy mildew caused by(Plasmopara viticola(Berk and Curtis) Bert.and de Toni) is a disease of all the world,and it is one of the most serious diseases which influence the grape production,breeding for resistant cultivars has been proved the most effective way to control this disease.The Vitis pseudoreticulata,native to china,possessing resistance to downy mildew genes,is one effective way to against downy mildew of grape.Cloning of resistance to downy mildew genes or the related genes for downy mildew resistance is of significance for understanding disease- resistance mechanism and making use of disease resistance genes for breeding.In this study,on the base of SSH library,besides sequencing Strategy aim. bioinformatics method were integrated to analyze the ESTs characteristics and gene-expression profile,aim to provide functional EST sequence for related resistance-genes.The grape materials of Chinese wild V.pseudoreticulata clone Baihe-35-1 highly resistant to downy mildew,downy mildew of grape was inoculated with spray method,at 1d,2d, 3d,4d,5d,6d and 7d post inculation,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 methed modified partially,and mixed them at the same mass as treatment,uninculating young leaves was used as control.Downy mildew of grape resistance gene SSH Up-regulated library and Down-regulated library was constructed with SSH.And antibiotics,blue and white spot,colony PCR cloning methods was used to identificate positive clone.Then sequencing,bioinformatics analysis were carried out.Colony PCR showed clips inserted in vertor in 150-900 bp,The Library was of good quality.Screening library and received 85 valid EST sequence of Up-regμlated library,29 valid EST sequence of Down-regμlated library,which were signed in GenBank database,The GenBank accassion numbers are:Up-regμlated library: FG106789 FG106790 FG106791 FG106792 FG106793 FG106794 FG106795 FG106796 FG106797 FG106798 FG106799 FG106800 FG106801 FG106802 FG106803 FG106804 FG106805 FG106806 FG106807 FG106808 FG106809 FG106810 FG106811 FG106812 FG106813 FG106814 FG106815 FG106816 FG106817 FG106818 FG106819 FG106820 FG106821 FG106822 FG106823 FG106824 FG106825 FG106826 FG106827 FG106828 FG106829 FG106830 FG106831 FG106832 FG106833 FG106834 FG106835 FG106836 FG106837 FG106838 FG106839 FG106840 FG106841 FG106842 FG106843 FG106844 FG106845 FG106846 FG106847 FG106848 FG106849 FG106850 FG106851 FG106852 FG106853 FG106854 FG106855 FG106856 FG106857 FG106858 FG106859 FG106860 FG106861 FG106862 FG106863 FG106864 FG106865 FG106866 FG106867 FG106868 FG106869 FG106870 FG106871 FG106872 FG106873Down-regμlated library: FG106874 FG106875 FG106876 FG106877 FG106878 FG106879 FG106880 FG106881 FG106882 FG106883 FG106884 FG106885 FG106886 FG106887 FG106888 FG106889 FG106890 FG106891 FG106892 FG106893 FG106894 FG106895 FG106896 FG106897 FG106898 FG106899 FG106900 FG106901 FG106902The 114 EST sequences were sent to GenBank for homologue search using Blastn and Blastx programs and were further classified based on their functions.The result is that 78 ESTs have high homology with known genes in GenBank,31 ESTs are unknown genes.the 31 ESTs are as follows: FG106797 FG106801 FG106802 FG106803 FG106805 FG106806 FG106807 FG106813 FG106814 FG106816 FG106817 FG106820 FG106822 FG106823 FG106824 FG106825 FG106827 FG106837 FG106838 FG106841 FG106860 FG106861 FG106864 FG106865 FG106866 FG106868 FG106869 FG106871 FG106873 FG106880 FG106894These sequences involving the function of signal transduction,energy metabolism, protein metabolism,nucleic acid metabolism and photosynthesis and membrane transport and metabolism et al.22 ESTs that was directly related to the resistance were received.The function of these disease-related genes includ anti-oxidation and defense-related genes such as the cytochrome P450 monooxygenase,a key enzyme glutathione metabolism-glutathione S-tran-sfer -ase(GSTs),Signal transduction genes such as ammonia-aminobutyric acid ammonialyase ubiquitin-binding protein and ubiquitin-binding enzymes associated with protein metabolism, receptor kinase et al.the study provide a good genetic resources for further studying of full length of genes related to resistance downy mildew.