Cloning And Functional Research Of A Cucumber Expansin Gene CsEXPb1

Cucumber(Cucumis sativus L.)is one of the larger demand vegetable crops.However,in production practice,expecially in multiseason production,low temperature,overhigh humidity,lack of male flowers,disease seriously or other factors affect cucumber growth and yield,seriously.Therefore,study on fruit developing mechanism,enhance cucumber growth potential and increase fruit setting rate are benefit to effectively solve the problems in production.Plant development is a complex process,a series of genes involve in,among which exists a class of genes,expansin,which present in plant cell walls.Expansins can make cell wall loosening,then induce cell division.Over the years,more and more researchs about expansin influence plant development were reported.On the study of cucumber fruit transcriptome,it was found that the target gene Csa5M561760.1 differentially expressed during fruit development and belongs toβ-expansin gene(GeneBank accession number:XP011655449).In this study,the gene was cloned and named CsEXPbl,subsequently,we carried out a research on CsEXPbl gene genetic transformation to cucumber.The main contents and results are as follows:1.Cloning and expression analysis of the expansin gene CsEXPbl in cucumberIn this study,RNA was extracted from female and male flowers and is reverse transcribed into cDNA.Then CsEXPbl was cloned using specific primers for PCR amplification.The result of sequencing showed that CsEXPb1 gene fragment full length was 942 bp,open reading frame(ORF)length was 549 bp,which was supposed to encode a protein of 182 amino acids.Homologous alignment showed that CsEXPb1 belongs to Calmodulin-Like protein and has high similarities with Solanaceae,Leguminosae sp.and Gramineae.Protein sequence analysis showed that CsEXPbl contains two conserved EF hand motifs,calcium-binding sites,which formed Ca2+ CaM complex,invoving in the regulation of metabolism by changing the interaction mechnisms with target enzymes.The ExPasy analysis showed that CsEXPbl was hydrophilic and the subcellular localization also demonstrated that CsEXPbl existed in plasma membrane,cytoplasm and nucleus.Tissue expression analysis showed that there were high expression in young leaves and female flowers,in which had rapid cell division.While only little expression in roots,stems,mature leaves and male flowers.Expression analysis of CsEXPblgene in fruit development showed that expression levels were increased continually during 0～6 d in ’EC 1’ovaries and’8419’ pollinated ovaries,but was decreased since 4 d in the ’8419’ unpollinated ovaries and far lower than the same period of the ’EC1’ ovaries and ’8419’pollination ovaries.The expression of CsEXPb1gene is induced by exogenous hormones GA,6-BA and NAAs.2.Construction of sense and antisense plant expression vectors of CsEXPblgeneThe clone vector PMD19-T and the expression vector PLP35S-100 were digested with enzymes PstⅠ and XbaI.Fragments of CsEXPb1 gene were inserted and reverse inserted between CaMV3 5 S promoter and the NOS terminator region of the expression vector PLP35S-100,then was transformed into Agrobacterium tumefaciens strain C58 by by freeze-thaw method.So the sense and antisense plant expression vectors PLP100-CsEXPb1 were constructed,carring CaMV35S promoter,NOS terminator,a reporter gene GUS,a Kanamycin sensitivity gene NPT Ⅱ.3.Genetic transformation and functional research of CsEXPblgeneThe sense-CsEXPb1 and antisense-CsEXPb1 were transiently expressed in non-parthenocarpic cucumber ’8419’ and parthenocarpic cucumber ’EC1’,respectivly.The results showed that overexpressed CsEXPbl gene could promote the development of ovaries,inhibited CsEXPbl gene expression could slow down growth speed.So,it was supposed that CsEXPb1 gene influenced fruit development.Then CsEXPbl gene was transformed into cucumber cultivars’Changchunmici’by Agrobacterium-mediated cotyledon node method and genetic transformation system was studied.lt was show that highest differentiation rate reached 96.2%.One To transgenic plants was obtained after phenotype observation and molecular identification to resistant buds,numbering T0-M-1.Using pollens from T0-M-1 pollinated non-transgenic ’Changchunmici’,500 T1 generation seeds were havested.After Kan screening to T1 generation seeds,242 T1 plants were obtained.After PCR detection,dot blotting and expression analysis to the 242 T1 plants,3 T1 transgenic plants were obtained,numbering T1-79,T1-173 and T1-177.The results of phenotypes observed on T0-M-1 and T2 transgenic plants showed that transgenic plants grow faster than non-transgenic plants,the differentiation of the flower buds were early than that of ’Changchunmici’ and the blunt with blossom happened.So it was speculated that the CsEXPb1 gene can promote young tissues development,accelerate flower buds differentiation and influence apical meristem differentiation.