Cloning And Functional Characterization Of Responsing To Low Nitrogen Stress Related Gene CsAOC3 In Cucumber

Cucumber(Cucumis sativus L.),one of the important horticultural crops in China,a typical nitrogenous crop.Nitrogen deficiency will affect the growth of plants seriously,and lead to the decreasing of the yield and quality.There will be excessive application of nitrogen fertilizer in cucumber for higher yield,when the nitrogen fertilizer is applied excessively,not only the utilization rate of nitrogen of crops will be lower but also be harmful to the agricultural and ecological environment.using rationally of nitrogen fertilizer,identifying related gene and cultivating low nitrogen cucumber varieties are effective ways to solve the above problems.In this study,cucumber D0328 was used as the experimental material,cloned of the nitrogen stress-related gene CsAOC3 and the expression of CsAOC3 was detected by qRT-PCR,to further analyze the function by transgenic technology.The main findings are as follows:(1)The gene CsAOC3 was screened out combined with previous expression profile data results which may be involved in the response of nitrogen stress.The results by PCR with template from cDNA of cucumber leaves and primers designed by using the gene sequence information of cucumber genome showed a about 750bp bright band,the total length of the gene was 753 bp,encoded 251 amino acids,the molecular weight of the protein was 27.17 kD,the instability coefficient was 51.92,the theoretical isoelectric point was 8.93,and the average hydrophilic coefficient was-0.155.The CsAOC3 showed 65%-94%homology by comparing with 16 kinds of AOC protein from Pear,Camptotheca acuminata and Mung bean in protein homology,melon were the closest one showed 94%.Subcellular localization results showed that the protein is most likely to be located in the chloroplast.(2)The results of fluorescence quantitative analysis showed that the expression of CsAOC3 in D0328 was significantly increased by 11 times under low nitrogen stress comparing with normal nitrogen treatment.However,for D0422,the expression of CsAOC3 did not change significantly nomatter with low nitrogen treatment or normal nitrogen treatment.(3)In order to study the function of CsAOC3 in response to nitrogen,firstly,constructed the overexpression vector of CsAOC3 gene,then transformed it into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation.Three positive transgenes were obtained by screening,detected and identified the relevant indicators after treated them with low nitrogen and normal nitrogen.Compared with that of the normal nitrogen treatment,he root length of the wild type and the transgenic plant was significantly decreased under the low nitrogen treatment condition,but the root length of the transgenic plant was longer than that of the control Un’ler normal nitrogen treatment.the root length of transgenic plants was longer than that of wild type,but the root length growth rate was significantly lower than that of low nitrogen treatment.Under normal nitrogen conditions,there was no significant difference in transgenic and wild plants.The fresh weight of transgenic Arabidopsis and wild type plants was significantly lower than that of normal nitrogen treatments,but significantly higher than wild type plants in overexpressional lines.The results indicated that overexpression of CsAOC3 could improve the ability of Arabidopsis thaliana to respond to low nitrogen stress.