| As an economically important fruit, grape (Vitis L.) is very popular among peole. Anth- racnose (Spheceloma ampelinum de Bary) is one of the fungal diseases which affect fruit qua- lity and can cause severe yield loss. Researching the interaction mechanisms between grapes and Elsinoe ampelina is very important to further studies of resistance mechanisms of grape to anthracnose and molecular breeding for disease resistance. In this study, a suppression sub- tractive hybridization (SSH) cDNA library was constructed with V.quinquangularis Shang-24 leaves inoculated by Elsinoe ampelina and bioinformatics were employed to analyze the exp- ression of defence relating cDNA fragment. Main conclusions of this paper are as follows:1. A suppression subtractive hybridization cDNA library was constructed with artificially sprayed leaves with Elsinoe ampelina and V. quinquangularis Shang-24 which is highly resistant to anthracnose,and the un-inoculated leaves as driver on the day of inoculation, the leaves induced for 12 h, 24 h, 48 h, 72 h and 120 h as tester. 1024 cDNA library clones were selected. 670 high-quality ESTs were obtained after clearing pollution, redundant, low quality(﹤100 bp)sequences. 461 Unigenes, including 85 Contigs and 376 Singletons,were obtained after clustering.2. Similarity analysis based on BLAST X softwares in GenBank was performed for obtained Unigenes. The results indicated that 370 (80.3%) of the ESTs could find encoding putative proteins, 86(18.7%)was hypothesis protein with unknown function, 5(1.0%)of the ESTs were found with no significant similarity which may represent new genes or high variant non-coding regions of cDNAs. The function of those best matched protein mainly involves resistance signals transduction, transcriptional regulation, elementary metabolic, ubiquitin pathway, active oxygen metabolism, hypersensitive response, defense response et al. Furthermore, those best matched protein were from many plant species, such as grape (Vitis vinifera L.), Arabidopsis thaliana (Arabidopsis thaliana), maize (Maize), ricinus communis (Ricinus communis), populus trichocarpa(Populus trichocarpa), tobacco(Nicotiana tabacum), clover (Medicago)and rice (Oryza sativa L.) .3. All of the acquired ESTs were analyzed with Gene Ontology (Go) in biological process and molecular function. Results showed that genes relating to cell metabolic process (enery metabolic, lipid metabolic and protein metabolic) and response to stress process which were 116 (11.1%) and 79 (7.6%), respectively, existed high expression in biological process, what'more, binding (protein binding, nucleic acid binding and DNA binding), catalytic activity and hydrolase activity which were 108 (17.0%), 94 (14.8%) and 62 (9.7%), respectively, existed high expression in molecular function. The results indicated that after V. quinquangularis Shang-24 leaves inoculation with Elsinoe ampelina, many cell metabolic process related genes and responsnse to stress process related genes involved in plant defense process, the function of those gene related to which primarily focused on binding, catalytic activity and hydrolase activity.4. According to functional classification, the expression of 22 different expressed ESTs were validated by Real time PCR, which inculded three resistance signals transduction related genes, three ubiquitin pathyway related genes, three active oxygen metabolism related genes, a hypersensitive response related gene, eight defense genes (six ESTs were secondary metabolic related genes), three transcriptional factors and a transporter protein. These results accord with results of suppression subtractive hybridization. The results shows that 22 ESTs were involved in the resistance to anthracnose and were up-regulated genes.5. PR-1 gene cDNA sequence was obtained from SSH library, which was involved in plant defense reactions. The full length cDNA sequence of PR-1 was 529 bp, had an complete open reading frame (ORF), and encoded 160 amino acids. The non-translation of 5′and 3′-terminal region were 13 bp and 33 bp, respectively. The results of phylogenetic tree of PR-1 gene shows that V.quinquangularis Shang-24 PR-1(VqPR-1)was most closely to V. vinifera (VvPR-1), V. shuttleworthii(VsPR-1), was more closely to Populus trichocarpa (PtPR-1), and was farthest to Brassica napus (BnPR-1). By semi-quantitative PCR test, PR-1 was constitutive expressed in flower, leaves and stems of V. quinquangularis Shang-24 while almost not expressed in tendril and pericarp. The result of Real time PCR shows that PR-1 was expressed at high level at 8 h and 120 h after anthracnose inoculation, which was 8.7- and 4.3-fold over-expressed respectively compared with control; What't more, it futher shows that PR-1 play an important role in defense response, and its expression may reflect the trend of SAR(systematic acquired resistance) reaction; Based on analysis of PR-1 expression by pathogens inoculated and hormone induced, we deduced that salicylic acid (SA) and ethylene (Eth) cooperatively regulated the expression of PR-1 during early stage of infection(0~3 h), while the expression of jasmonic acid(JA)was repressed. SA, JA and Eth cooperatively regulated the expression of PR-1 during later stage of infection(3~48 h). |
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