Cloning And Functional Analysis Of CsLecRK Related To Copper Tolerance In Cucumber

Cucumber（Cucumis sativus L）,one of the largest consumable and popular vegetables around the world,it is also a sort of vegetable crops that is extremely sensitive to copper（Cu）stress.In recent years,Cu pollution of soil is getting worse as well as lower yield and quality of vegetable crops with many unreasonable applications such as the high copper livestock manure,fungicides and insecticides.Moreover,it is also possibly harmful for the health of animals and humans through the transmission of the food chain.Therefore,it is of great significance to effectively improve the Cu stress tolerance of cucumber and to cultivate Cu tolerance cucumber varieties for the creation of green agriculture and modern agriculture.To study of the molecular mechanism of Cu tolerance and identification of related genes will provide theoretical reference for pollution-free vegetable breeding.To further increase our understanding of the adaption for Cu tolerance in Cucumber,‘649’was used as experimental materials in the present study.The mechanism of Cu tolerance in cucumber was investigated based on expression pattern and conservative of genes response to Cu,and techniques such as transgenic technology.The function of Cu tolerance related gene was also analyzed.Discussion of the molecular and physiological mechanism of Cu tolerance in Cucumber.The main results are as follows:（1）Based on the analysis of gene expression patterns of Arabidopsis thaliana under Cu stress,the gene sequences were then homologously compared with the cucumber genome database,and the potential Cu-related genes in cucumber were further screened using a combination of bioinformatics analysis methods.And to ensure that the function of the gene in related heavy metals has not been reported,the Arabidopsis LecRK gene was initially set as the object of study.The amino acid homology of this gene and CsLecRK was compared by DNAMAN software,and the homology between them was 46%.Further qRT-PCR analysis showed that the expression of CsLecRK under Cu stress was significantly down-regulated by 4.39 times.The results of this study indicate that CsLecRK is specifically expressed in cucumber induced by Cu stress,and this gene may be related to Cu tolerance of cucumber.It is also suggested that CsLecRK is a new gene that is resistant to Cu stress in cucumber.（2）The full-length sequence of CsLecRK CDS was amplified by PCR,which is 2121 bp,encoding 706 amino acids with predicted molecular mass of 78.36 kD and a pI of 7.76.Both the results of NCBI Blastp and phylogenetic tree based on LecRK proteins showed that CsLecRK protein has highest homology with muskmelon（95%）,the evolutionary distance was shortest.It belongs to a member of LecRK superfamily.（3）Consruct over-expression vector 1250-3301::CsLecRK,transformed into Columbia wild type Arabidopsis thaliana.The results showed that,under 50 Cu treatments the wild-type root length was significantly inhibited and reduced,0.8443±0.0628 cm.However,there is no difference between transgenic plant and wild-type under control conditions,but under 50 Cu treatments,the transgenic plants root length was higher than that of the control,respectively.In addition,under 50Cu treatments,the fresh weight and dry weight of the wild type and the three transgenic plants were reduced,the fresh weight of shoot were reduced by 59.16%,33.92%,29.36%,43.65%,the fresh weight of root were reduced by 68.46%,51.39%,51.42%,49.49%.The dry weight of shoot were reduced by 66.19%,40.72%,32.06%and 24.47%respectively,compared to the normal condition.（4）Determination of H₂O₂ and MDAunder control conditions and 50 Cutreatment on wild type and transgenic Arabidopsis.Under normal conditions,the transgenic plants of shoot H₂O₂content was significantly higher than that of the wild type.There is no difference between transgenic plant and wild type uneder 50 Cu treatments,while the content of H₂O₂ of transgenic plant of root is lower than wild type with 50 Cu.In addition,There is no difference between transgenic plant of shoot and wild type uneder normal conditions,but under 50 Cu stress,whether or not the content of MDA in root tissues of transgenic plants was significantly lower than that of wild type.While the activity level of SOD,POD and CAT in the shoot and root of the transgenic lines was significantly higher than that in the wild type under Cu stress.The germination rate of the results showed that:The germination rate of three transgenic Arabidopsis seeds was significantly higher than those in wild type with 0.3μM ABA.The results showed that the overexpression of CsLecRK gene may be involved in ABA signal transduction,the CsLecRK could further improve the antioxidase the level of activity of the plant by decreasing the content of H₂O₂and MDA in plant,which could enhance the Cu tolerance to maintain the normal growth and development.In addition,the expression levels of the Cu regulatory gene AtSPL7 and ABA response gene AtRAB18 and AtABI5 in the transgenic plants under Cu stress were significantly higher than those in wild type.This indicates that the transgenic plants exhibit copper tolerance probably due to the cooperative work of the proteins encoded by these genes.These results indicated that overexpression of CsLecRK could enhance the Cu tolerance in Arabidopsis thaliana.