| As the first largest nut tree specie in China, Chinese jujube(zizyphus jujube Mill.) played an important role in nut industry. Jujube fruit shrink disease caused by Alternaria alternate, was one of the most serious diseases of jujube. The complexity of pathogen infection and the diversity of pathogens had brought difficulties to control this disease. Conventional actericide controls not only polluted the environment, but also palliatives. Pollution difficulty and abortion were common phenomenons in artificial hybrid breeding pollination. Therefore, looking for resistance genes was an effective method to prevent jujube fruit shrink disease. In our study, a suppression subtractive hybridization(SSH) c DNA library was constructed with jujube fruit induced by Alternaria alternate in order to understand the gene expression after the interaction between Chinese jujube fruit and the Alternaria alternate and analyze the genetic characteristics related to defense response. The study of the mechanism of resistance of the molecular level laid a theoretical foundation for breeding. Main conclusions of this paper are as follows.1. A suppression subtractive hybridization c DNA library was constructed with the fruit of Ziziphus jujube var. Fengmiguan during white-ripening stage. Picked the fruit after inoculated with Alternaria alternate 12 h, 24 h, 48 h, 72 h, 96 h. Mixed the 5 samples’ RNA and transcripted them into c DNA used as tester, and fruit inoculated with water were used as driver. At last 1000 c DNA library clones were conserved and 200 positive clones were selected and sequenced, and 182 high quality ESTs were obtained. After clustering we get 118 Unigenes, including 7 Contigs and 111 Singlets.2. Similarity analysis based on BLASTx softwares in Gene Bank was performed. 84(71.19%) ESTs could find encoding putative proteins. 34(28.81%) of the ESTs were found with no significant similarity. Then analyzed the 34 sequences by BLASTn. 18 ESTs with the significant similarity. Function of 95 ESTs was known, 7 were unknown. The known function of ESTs was mainly involved in protein degradation, protein synthesis, protein modification and transport, disease defense response, cell structure, signal transduction and transcriptional regulation, energy and metabolism, etc. Defense genes(27.17%) were the first major categories. Energy and metabolism related genes(20.65%) were the second major categories. Protein synthesis and processing and decomposition related genes(16.30%) were the third major categories. The best matched protein sequences were from many plant species, such as Ziziphus jujuba, Morus notabilis, Theobroma cacao, Cajanus cajan and so on.3. Based on the analysis of the genes with known function, we speculate that the genes code for basic leucine zipper, serine/threonine protein kinase, WRKY31 were supposed to function as signal transduction and transcription regulating genes in jujube after inoculated with Alternaria alternate. Pathogenesis-related protein10, Thaumantin-like protein, protein DETOXIFICATION, hypersensitive-induced response protein and E3 ubiquitin ligase might be involved in the main resistant course of jujube.4. All of the acquired ESTs were analyzed with INTERPROSAN and WEGO in cellular component, molecular function and biological process. Results showed that sort and number of resistance relating genes induced by Alternaria alternate were significantly different. Genes relating to metabolic process and cellular process existed high expression, binding and catalytic molecules increased. And these led biological regulation and response stimulus enhanced; cell, cell part and organelle relating genes also expressed extensively. And these differencially expressed genes might be closely related to jujube resistance to jujube shrink diease. |
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