CsCBS Cloning Of Cucumber And Functional Verification Of Resistant To Target Spot And Downy Mildew

Cucumber(Cucumis sativus L.)can be a fruit or a vegetable,which loved by everyone becaue of its refreshing taste.Cucumbers belong to herbaceous plants,it has a long history of cultivation in China and it is one of the main vegetable crops in China.It is can be planted in open field and spread all over the country.Cucumber target spot and downy mildew are dangerous leaf diseases in cucumber production,resulting a serious decline in cucumber yield and economic losses.A large number of chemical agents will cause environmental pollution.Excessive exposure can lead to the occurrence of pathogen resistance.At the same time,pesticide residues can also harm human health.Therefore,breeding and using resistant varieties is a green,economical and effective way to control diseases.At present,the disease resistant varieties of cucumber in the market are very short.On the basis of the sequencing of the transcriptome,we screened a gene which has the CBS domain(Csa6M496430.1,named CsCBS).CBS domain proteins play an active role in wheat disease resistance,but it has not been reported in cucumber.In this study,CsCBS gene was studied by molecular biology.(1)the expression pattern of CsCBS under the stress of pathogens was analyzed by qRT-PCR technology.The results showed that under the treatment of single inoculation of downy mildew,target spot and inoculation of two pathogens,the expression of CsCBS in the early stage of inoculation was Significantly up-regulated and there was no significant change in the expression of CsCBS in the susceptible strain D0401,indicating that CsCBS could actively respond to the stress of the pathogen of frost mycophenal fungus and the leaf spot pathogen.(2)the full length of CsCBS sequence was cloned by PCR technology.CsCBS contains a 1263 bp open reading frame,which encodes 420 amino acids.Contain a CBS domain and a DUF domain,belonging to the CBSDUF family.(3)analysis of the physicochemical properties of amino acid sequences encoded by CsCBS,the molecular weight of the protein encoded by CsCBS is 103711.6,the theoretical isoelectric point is 6.07,the average hydrophilic coefficient is 0.107,the hydrophobic protein,and the non signal peptide,it is a transmembrane protein.(4)the protein structure of the amino acid sequences encoded by CsCBS was analyzed,and 192 alpha helices accounted for 45.71% of the whole peptide chain in the CsCBS encoded protein;75 extended main chains accounted for 17.86% of the whole peptide chain;31 beta folds accounted for 7.38% of the whole peptide chain;122 random curls were 29.05% of the whole peptide chain.(5)CsCBS and green fluorescent protein(GFP)were fused and transferred into the protoplast of arabidopsis thaliana.Under the fluorescence microscope,it was found that the CsCBS protein was located in the cytoplasm.(6)After that,the plant overexpression vector pCXSN-CsCBS was successfully constructed.The CsCBS gene was transferred into the susceptible cultivar D0401 by Agrobacterium tumefaciens mediated method.The transgenic plants were inoculated in vitro.The results showed that overexpression of CsCBS could enhance cucumber resistance to downy mildew and target spot.(7)In this study,the content of malondialdehyde in overexpressing plants was decreased,and the content of soluble sugar increased.