Construction Of Suppression Subtractive Hybridization Library From Cucurbita Induced By Phytophthora Capsici And Analysis Of Expressed Sequence Tags

Pumpkin is annual economic crop. And it is a plant genus of the family cucurbitaceae. It has very important economic value and medicine value. As the foreign trade and the serial products about pumpkin developed, cultivated area of pumpkin keeps expanding. And because of that, farmers gained more economic returns. But pumpkin blight caused huge loss in some years and some areas. So pumpkin loemia is one of the limiting factors about pumpkin production. And it is a troublesome question need to be deal with. At first ,we need to know the resistance mechanism and the mechanism of action between pumpkin and pathogenic bacterium. In this study, a suppression subtractive hybridization (SSH) cDNA library was first constructed. Then using expression sequence tags (ESTs) and bioinformatics to analyze the expression of related gene resisted pumpkin loemia. To study the resistance mechanism about pumpkin at molecule level. And the study may help disease resistance breeding.In this study, SSHcDNA library was detected by reserve northern blot, PCR, double digestion on plasmid. And then 60 cDNA positive clones were randomly selected and sequenced. 55 ESTs with high quality were obtained. Similarity analysis was based on BLASTx and BLASTn softwares in GenBank. 52 of the ESTs were identified encoding putative proteins. 3 of the ESTs were found no significant similarity and they may represent new genes or high variant non-coding regions of cDNAs. The best matched protein sequences with those encoded by the 52 ESTs were from so many plant species, such as Cucumis sativus, Citrullus lanatus, Arabidopsis thaliana, Oryza sativa, Brassica napus, Brassica oleracea, Solanum tuberosum, Vitis vinifera, Zea mays, Nicotiana tobacum, Sorghum vulgare, Beta vulgaris, Phaseolus vulgaris and so on.After EST analysis, we supposed that calcium-mediated signal transduction system and jasminate acid signal transduction system play roles in pumpkin's resistance to blight. But the interactions among various signal transduction pathways are not clear. Transcription factor related resistance contains zinc finger protein, MYC transcription factor, G-box binding protein. They may play important roles in expression and regulation of gene related pumpkin's resistance.In this study, we found two resistance gene analog (RGA). They should be to participate pumpkin's resistance, and play a very important part in it. So this two genes need to be studied to know the resistance mechanism. In addition, in the ESTs obtained, there are some ESTs for abiotic stress-induced protein, such as heat shock protein, salt induced protein and so on. This result suggests that a possible common mechanism exist during the stress reaction of plants towards different environments.These ESTs contained other aspects, such as RNA processing, protein interpretation and processing, photosynthesis, energy metabolism. These genes should be related to pumpkin resistance mechanism.