Cloningand Functional Analysis Of Transcription Factor GhWRKY31Gene Of Gossypium Hirsuturm L.

WRKY transcription factors in transcriptional regulation are one of the largestfamilies in plants, it is the main part of the information transmission network, andcontrols many growth processes of the plants. Recent findings that WRKY protein hasthe repressor, as activator, many of the WRKY families have repression and activationat the same time in the process of plant growth. In addition, a separate WRKYtranscription factor may participate in regulating the growth of several unrelatedprocesses. WRKY genes can perform adjustment of its own and the reverse adjustment,which can make the WRKY gene transcript in dynamic smoothly.In this study, ESTs related cotton WRKY31transcription factor gene as seedsequences were pieced together to form a complete GhWRKY31transcription factorgenes with open reading frame by electronic cloning method. Then, the ORF ofputative GhWRKY31transcription factor gene was cloned through specific primersdesigned according to electric cloning sequence, named GhWRKY31. WRKY31wasinserted respectively in silencing vector pYL156and binary plant expressing vectorpBin438and constructed interference expression vector pYL156-GhWRKY31andeukaryotic expression vector pBin438-GhWRKY31.pYL156-GhWRKY31andpBin438-GhWRKY31vectors were introduced respectively into Agrobacterium strainGV3101or LBA4404by electroporation. The transgenic tobacco plants wereobtained by Agrobacterium-mediated transformation via leaf disc. The molecularanalyses and resistance assay against pathogens of transgenic tobacco plants wereperformed. Main results in the present study show below:1. Design specific primers with enzyme sites according to the GhWRKY31transcription factor gene sequence which was pieced together by electronic cloning.Amplify target gene by cDNA from cotton cultivar “zhongmian35”. Determinationresults show that the full length of GhWRKY31gene is1904bp, open reading frame is1647bp, coding548amino acids, the prediction of protein molecular weight is59.195KDa, isoelectric point pI=4.932. With EcoRI and KpnI enzymes cut GhWRKY31gene fragment and interferenceexpression vector pYL156, and building interference expression vector pYL156-GhWRKY31, electric shock intoGV3101, injecting them into the sea island cottonuse blade injecting method, inoculated Verticillium lecaniiunder leaf1cm until the control gene PDS deficiency making cotton growing flowers white spot oralbefaction, making disease resistance and phenotypic observation to experiment andcontrol plant, the results showed that after the injection of Verticillium lecanii, controlgroup and experimental group of cotton plants are characterized by the etiolate, theexperimental group even worse, but ten days later, the control plant disease resistanceimproved significantly, and experimental group is more serious, and even the leavesall fall off.3. Use BamHI and SalI enzymes cut cloning vectorpGEM-T-GhWRKY31andexpression vector pBin438, construction pBin438-GhWRKY31vector, and electricshock it into agrobacterium LBA4404, through leaf dish transformation tansformGhWRKY31gene into tobacco, obtaining8strains of tobacco transformation plant, theRT-PCR detection shows that5strains of transformation plants having positive,resistance to the Pseudomonas syringae pv shows that the transgenic plants has highresistance, on the phenotype, the control group and experimental group′leaves areburned after vaccination Pst, eight days later tobacco leaf of experimental groupimproved significantly; Through test the Pst activity of inoculating leaf tobacco(including comparison and experiment group)showed that colony number in leaf inthe control group is more significantly the experimental group.4.The transgenic tobacco have resistance to salt (150mM NaCl), on one hand ofplant growth condition, without adding NaCl medium, transgenic tobacco and wildtype tobacco growth situation is similarity; And add the NaCl medium, wild typetobacco is very small, and three transgenic tobacco growing is same better than that ofwild type tobacco. In addition, the totalleaf chlorophyll content and electricconductivity detection show in the case of add and none add salt, total chlorophyllcontent of the control group and experimental group have significant differences.