Cloning And Functional Analysis Of Cadmium Tolerance Related Gene CsNRSP In Cucumber

Cucumber(Cucumis sativus L.)is one of the indispensable cash crops in facility cultivation.In recent years,with the rapid development of modern industry and agriculture,and the use of fertilizers or pesticides containing heavy metals in greenhouse,cucumber and other vegetable crops are vulnerable to heavy metal pollution.Cadmium(Cd)is one of the most serious elements in heavy metal pollution,which directly threaten people's health after entering the ecosystem through the food chain.Therefore,it is of great significance for food safety to study the mechanism of Cd tolerance in cucumber,strengthen the breeding of Cd tolerant varieties and improve their resistance to stress.To study the molecular mechanism of Cd tolerance in cucumber,a SDR family gene CsNRSP was obtained based on bioinformatics analysis.CsNRSP was cloned and its expression was analyzed under Cd stress.Combined with transgenic technology,the function of CsNRSP was analyzed from the perspective of physiology and molecular biology,and mechanism of Cd tolerance in cucumber were preliminarily expounded,which provided an experimental basis for further application to the improvement and breeding of Cd-tolerant cucumber varieties.The main results are as follows:(1)In this study,we reviewed the related literatures of Arabidopsis thaliana and other model crops,and analyzed genes in Cd stress related transcriptome.Based on the gene expression patterns in the literature and the results of comparison with cucumber homologous genes,the potential Cd tolerance genes were screened in cucumber by bioinformatics,the function of which in heavy metals was unknown yet.The homologous gene of AtNRSP in cucumber was preliminarily identified as the primary research object.BLAST analysis revealed that CsNRSP had 72% homology with AtNRSP in the amino acid sequence by DNAMAN software.The similarity of sequences can be inferred that their functions have some similarity,qRT-PCR analysis revealed that the expression of CsNRSP was significantly increased(4.45 folds)compared with the control under Cd stress.Finally,the homologous gene CsNRSP in cucumber of AtNRSP was identified as the experimental object,which was a new Cd tolerance induced gene.(2)The length of CsNRSP was 822 bp,encoded 273 amino acids,the molecular weight was 29.02 kD,the theoretical isoelectric point was 5.86,the instability coefficient was 91.79,and the average hydrophilic coefficient was 0.016.Phylogenetic analysis showed that CsNRSP had 73%-93% homology by comparing with 12 kinds of NRSP protein from cucurbita maxima,peach and sweet orange in protein homology,melon was the closest one showed 93%.(3)Constructed the overexpression vector,then transformed it into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation.Three positive transgenes were obtained by screening,detected and identified the relevant indicators under no Cd and 50 ?M Cd.The results revealed that the root length of wild type was reduced under Cd stress,but transgenic plants were not significantly affected by Cd,and were larger than those of control,which were 1.25,1.59 and 1.58 times of the control,respectively.The root lengths of transgenic plants and wild type had no significant difference under no Cd and 50 ?M Cd.In addition,.the fresh weight of wild type decreased significantly under Cd stress,but over-expressed lines changed little.(4)To investigate the response of CsNRSP in photosynthesis under Cd stress,chlorophyll content was detected in this study.Results showed that the chlorophyll content of wild type and transgenic strains were basically the same on MS medium without Cd,and was significantly reduced on MS medium containing 50 ?M Cd.But the chlorophyll content of three transgenic lines was significantly higher than that of wild type.Then antioxidant enzyme activity were investigated in transgenic and wild type lines and the related indexes were detected.The results showed that the CAT and POD contents of wild type and transgenic Arabidopsis were basically the same on MS medium without Cd,but the contents of three transgenic lines were significantly higher than that of wild type.(5)In addition,the expression levels of Cd related genes AtHMA4,AtMGT1,AtATM3 and AtDWF4 in transgenic lines were significantly higher than wild type under Cd stress,suggesting that the Cd tolerance of CsNRSP transgenic lines increased due to the cooperative work of these genes.The above results revealed that overexpression of CsNRSP might enhance the capability for Cd tolerance in Arabidopsis.(6)Agrobacterium-mediated transformation of cucumber was completed by cotyledon node infection,and transgenic cucumber plants were successfully obtained,which laid a foundation for the cultivation of new Cd resistant cucumber varieties by transgenic technology.