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Grape is an important fruit worldwide, loved by the people. Grapes black pox disease (Spheceloma ampelinum de Bary) against the European grape fungal diseases, and serious harm to the yield and quality of grapes. Study grape anthracnose pathogen interaction mechanisms, has important significance to reveal of Grape Anthracnose Resistant Mechanism and grape disease resistance molecular breeding. In this study, the use of suppression subtractive hybridization technique to build a grape anthracnose the black pox germs induced high anti wild hair Grape (Vitis quinquangularis) in supplier -24 suppression subtractive library, bioinformatics functional annotation of the resulting differences cDNA fragment and classification, and expression analysis, made the following main results: 1 black grapes with high anti-pox disease the Vitis supplier -24 blade for the material, using spray inoculated black pox germs, building the unvaccinated control for test driven (Driver), after inoculation black pox germs 12 h, 24-h, 48 h, 72 h and 120 h five points of the mixed samples for the test side (Tester) suppression subtractive hybridization cDNA library, 1024 clones picked After sequencing decontamination repeated sequence of low quality (<100 bp), a total of 670 high-quality EST sequences to login GenBank, serial number for HS102358 HS103027; After sequence assembly and assembly 461 Unigenes, including 85 of Contigs and a 376 Singletons. 2. The resulting Unigenes GenBank database BLAST X comparative analysis of the results showed that 370 EST sequences (80.3%) with known functional genes of high homology, 86 EST sequences (18.7%) function is yet to be determined the hypothetical proteins, five EST sequences (1.0%) no homology in GenBank speculated that these do not have any homology to genes may be a new disease resistance gene, or a high degree of variability in the cDNA non-coding region . EST sequences with known function, and its function is mainly related to disease resistance signal transduction, transcriptional regulation, the ubiquitin pathway, reactive oxygen metabolism, allergic reactions, defense reaction, primary metabolites, secondary metabolites, systemic acquired resistance . BLAST comparison to see the source of the gene of the highest homology, mostly derived from grape (Vitis vinifera L.), Arabidopsis (Arabidopsis thaliana), Corn (Maize), castor (Ricinus communis), trichocarpa poplar (Populus trichocarpa), tobacco (Nicotiana tabacum), alfalfa (Medicago) and rice (Oryza sativa). 3. Classification of ESTs using Gene Ontology (Go), respectively, from the two aspects of the biological process and molecular function functional annotation. In biological processes, cellular metabolic processes (including energy metabolism, lipid metabolism, protein metabolism) and stress response related to the number of EST up, 116 (11.1%) and 79 (7.6%); molecular function classification, bind to molecules (including protein binding, nucleic acid binding and carbohydrate-binding), catalytic activity and hydrolase activity EST large number of, respectively, 108 (17.0%), 94 (14.8%) and 62 (9.7%) a. That black pox pathogen infection Vitis -24 cell metabolism related biological processes involved in the process of plant disease resistance and stress response and gene function with these metabolic processes are mainly concentrated in the molecular binding, catalytic function hydrolase activity of these three aspects. On this basis, according to the functional classification, screened from the library of 22 differentially expressed genes, three of which were related to signal transduction, three related to the ubiquitin pathway, three related to the active oxygen metabolism, a associated with allergic reactions, eight defense genes (six of them involved in secondary metabolism) 3 is a transcription factor, a secondary metabolite transporter protein 1 genes of unknown function, Real time PCR technology vaccination black pox germs 0 h, 4 h time points for 8 h for 12 h, 24 h, 48 h, 72 h and 120 h 8 expression analysis. Proven results with suppression subtractive hybridization experiments consistent 22 differentially expressed genes are the black pox germs inducible gene, belonging to the up-regulated genes. PR-1 gene from Chinese wild grapes Vitis -24 anti the black pox disease SSH library cloned into a participation in the plant defense responses full-length cDNA sequences of the gene is 529 bp, with a complete open reading frame , 5 'end non-coding region of 13 bp, 3' end of the non-coding region of 33 bp, and encodes 160 amino acids. Phylogenetic analysis showed that: Vitis supplier -24 PR-1 (VqPR-1) protein encoded Vitis European grape (VvPR-1), the Saudi grape (VsPR-1) kinship with trichocarpa Young (PtPR-1), Castor (RcPR-1) closer relationship with the European rapeseed (BnPR-1) kinship far. The semi-quantitative PCR showed that PR-1 gene was constitutively expressed in the flowers, leaves and stems of Vitis -24, and the highest expression in stem tendrils and peel almost undetectable expression. Real-time PCR results showed that the PR-1 expression in the the black pox pathogen infection at 8 h and 120 h volume reached the peak of its expression level of the expression level of 8.7 times and 4.3 times respectively as background, indicating that the PR-1 in anti-black pox disease defense responses play an important role in the expression change to some extent, reflect trends in the SAR response; expression analysis and hormone-induced expression analysis by pathogen inoculation, suggesting that infection early in black pox germs (0 to 3 h) role of salicylic acid (SA) and ethylene (Eth) collaboration, jasmonic acid (JA) expression is suppressed; late pathogen infection (3 to 48 h), SA, JA, the Eth three of PR-1 regulation The effect is synergistic.
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