Cloningofthemelon’ CmHSP83 Gene And Genetic Transformation

Melon is widely cultivated and extremely moisture intolerance melon crop. Due to the characteristic of the seasonal rainfall in China, especially Huanghuai plain and the middle and lower reaches of the Yangtze river region, water-logging stress has become a important stress factor of impacting on the growth of melon and simultaneously limiting melon yield and quality. In order to elucidate the mechanism of water-logging resistant melon, the researchers applied molecular biology techniques to study of the response to water-logging stress genes in melon, which could provide the theoretical basis for melon breeding and cultivation in pluvial region. Some water-logging tolerance genes were screened out from the result of the previously conducted RNA-seq, including the gene Cm HSP83. The key work of this study was to identify function of the water-logging tolerance gene Cm HSP83. To date,the gene Cm HSP83 from melon M19 was successfully cloned and its nucleotide sequence was analyzed. Subsequently, the over-expression vector,PTCK303-Cm HSP83, was successfully constructed. Ultimately, the gene was transformed into the melon M63 by Agrobacterium mediated transformation. Results are summarized as follows:1.The Melon ’ Cm HSP83 gene expression analysis under Submergence Stress.The expression pattern of the gene Cm HSP83 upon water-logging treatment were detected by q RT-PCR.Cm HSP83 expression in melon M19 was accumulated induced and subsequently decreased in 0-12 h stage of water-logging treatment.However, the expression in melon M63 showed an upward trend and finally changed to be steady. The two expression patterns indicated that Cm HSP83 expression was much higher in melon M19 of water-logging tolerance than in melon M63 of water-logging sensitivity.2. The gene Cm HSP83 from melon M19,related to water-logging resistant, was cloned, and the sequence is consistent with the published in NCBI database. And it constructed over expression vector PTCK303-Cm HSP83 by PTCK303 vector. The gene Cm HSP83 was imported melon M63 by agrobacterium tumefaciens-mediated.3.The Biological function of the melon Cm HSP83 gene was analysised. The size of the Cm HSP83 c DNA was found to be approximately 2.17 kb,CDS region have fulllength of 2097 bp, encoding an non-membrane protein consisted of 698 amino acids in the endoplasmic. Conserved domains of the protein are similar to conserved domain of HATPase_c protein family and HSP90 protein subfamily. Through Homology search and phylogenetic analysis, the gene Cm HSP83 belongs to HSP90 subfamily, suggesting that the gene might be involved in response to water-logging resistance.4. We constructed the melon regeneration system: first, the best condition of the higher rate of inducing adventitious buds was that cotyledon blocks near the axial end of 3d seedlings were selected as explants, and 6-BA concentration was 1.0mg/L;secondly, the kindest elongation medium was MS within 0.2mg/L KT, the most moderate rooting medium was 1/2MS, contained 0.4mg/L IBA; finally, the regenerated seedlings were domesticated, effectively improving transplant survival.5. Based on the Melon regeneration system, the melon genetic transformation system was established. Melon M63 is very sensitive to Kan, then 50 mg/L Kan, as a selection pressure in medium, can be better to screen resistant buds. Additionally, the medium, contained 400mg/L Carb and 400mg/L Cef, can effectively inhibit agrobacterium. The optimum conditions of the melon genetic transformation system included pre-incubation for 2-3d, co-cultivation for 3d, infection for 15-20 min, and agrobacterium bacterial concentration of 0.5 at OD600. Consequentially, the rate of transformation was 4% by statistication and calculation in my experiments.6. The over-expression vector, p TCK303-Cm HSP83, was constructed. The gene Cm HSP83, which is related to water-logging tolerance, was transformed into melon M63 by agrobacterium-mediated transformation, finding that 6 melon strains were positive, we identified the all of successfully obtained resistant seedlings by PCR.