Cloning And Stress Resistance Analysis Of A Metallothionein Gene CsMT4 In Cucumber

As an important crop with high economic value,cucumber is often subjected to various stresses in agricultural production,and its normal growth is hindered,thus reduce the yield greatly.It has very important effect and significance to increase the increment and economic benefit of cucumber to improve its resistance to stress.We analyzed the gene family of metallothionein in cucumber on the basis of whole genome sequencing,analyzed the expression level of metallothionein gene under stress by qRT-PCR,cloned the CsMT4 gene through RT-PCR,and analyzed the expression levels of the metallothionein genes in cucumber under stress by qRT-PCR.By restriction enzyme digestion,ligation and transformation,the pET32a-CsMT4 expression vector was successfully constructed.The resistance function of CsMT4 gene was analyzed by prokaryotic expression system.The main results are as follows:1.The metallothionein gene family members of cucumber were predicted.There are three metallothionein genes in cucumber.They are named as CsMT2,CsMT3,and CsMT4.according to their specific position on chromosomes.Phylogenetic analysis showed that cucumber CsMT3 was clustered with rice OsMT3 a,OsMT3b and Arabidopsis AtMT3,but the phylogenetic relationship was far away and the homology was not high.Cucumber CsMT4 was closely related to Arabidopsis AtMT2 a and AtMT4 b,and rice OsMT4,but its genetic relationship was not high,and CsMT2 was not highly homologous to rice OsMT1 d and OsMT2 c were clustered into one group,and the phylogenetic relationship was relatively close and the homology was high.2.The results showed that the expression of metallothionein genes in cucumber was tissue-specific: CsMT2 was expressed in all tissues,as well as in stems,leaves and flowers.CsMT3 was not expressed in root,but not very high in stem and high in other tissues,and CsMT4 was not expressed in stem and flower,but was lower in root and higher in leaf and fruit.3.The results of qRT-PCR expression analysis showed that,under abiotic stress(PEG,ABA and NaCl),the expression of CsMT4 was significantly increased.4.The cucumber metallothionein gene CsMT4 was cloned by RT-PCR to construct the pET32a-CsMT4 expression vector,which was highly expressed in BL21.5.The results of stress resistance analysis showed that the prokaryotic expression of cucumber metallothionein CsMT4 could effectively improve the resistance of E.coli to various stresses.