Analysis Of Defense-related Genes In Cucumber Induced By Pseudoperonospora Cubensis

Cucumber,a kind of vegetable which plays an important role in agriculture production,is cultivated in large areas and planted quite long time in China.Downy mildew,the most severe oomycetes ePIdemic disease in cucumber cultivation,spreads fast and does harm seriously.Once downy mildew becomes prevalent,there would cause huge losses to growers.With FQ-PCR(Fluorescent Quantitative PCR)and prokaryotic expression to fuse,prepare and purify proteins,14-3-3 protein family genes and other 10 primary anti-disease genes of the two varieties,""and "Changchunmici",was studied to obtain polyclonal antibodies of CsPI?CsTLP and CsHirp proteins.What's more,Western blotting was used to detect the difference of protein expression between "PI08 8"and `Changchunmici",which lay the foundation for cucumber molecular mechanism of resistance to downy mildew.Main results for this study are as follows:(1)Gene expression of the two varieties at the early stage when cucumber leaves were infected by pseudoperonospora cubensis was analyzed using the technique of real-time fluorescent quantitation PCR.The results showed that:in the 14-3-3 protein family of cucumber,both 14-3-3-1 and 14-3-3-8 are connected with disease-resistant stress at the early stage before infection by this peronospora,14-3-7 and 14-3-3-11 are concerned with the disease-resistant stress at the late stage after infection.There are also the relationship of CsCupi1?CsNtp27?CsPI?CsPI-F4?CsTLP?CsGS?CsHirpwith disease-resistant stress at the early stage before infection(2)The prokaryotic expression vector of pET28a-CsPI?pET28a-CsTLP?pET28a-CsHirp was constructed successfully.The vector was transformed and shifted into BL21(DE3),correct recombinant protein,induced by IPTG and expressed,was detected by SDS-PAGE.CsTLP,CsHirp proteins are inclusion bodies including most CsPI proteins and a small part of CsPI proteins are soluble expressed.The induced temperature,duration and the concentration of IPTG were optimized and the optimum condition was confirmed,a great quantity of proteins were also made.To the recombinant protein,pET28a-CsPI,the optimum shaking bacteria temperature is 37?,the optimum concentration of IPTG is 0.2mM,the optimum induced duration is 2h.To pET28a-CsTLP,the optimum shaking bacteria temperature is 37?,the optimum concentration of IPTG is 0.8mM,the optimum induced duration is 8h;To pET28a-CsHirp,the optimum shaking bacteria temperature is 37?,the optimum concentration of IPTG is 0.5mM,the optimum induced duration is 4h.Both of pET28a-CsPI and pET28a-CsHirp recombinant proteins were purified through Ni-chelating affinity chromatography.The objective straps were cut after SDS-PAGE gel electrophoresis.However,the recombinant protein,pET28a-CsTLP couldn't be purified through Ni-chelating affinity chromatography because of insolubilization.So the objective straps were cut directly after SDS-PAGE gel electrophoresis and these gel straps were used to make polyclonal antibodyWestern blotting detection showed that idiosyncratic reaction between proteins extracted from cucumber leaves and positive serum of CsHirp explains successful preparation of polyclonal antibody.Expression detected through Western blotting showed that the very low content of CsHirp in CCMC made CsHirp not be detected.However,the initial content Hirp in is higher than that in CCMC and hardly changes with the difference of induced duration.