Construction Of Cdna Library And Cloning Of Resistance Gene From Gossypium Barbadense Induced By Verticillium Dahliae

Verticillium wilt is one of the most serious diseases which influence the cottonproduction in the world. Breeding for resistant cultivars has been proved the mosteffeetive way to control this disease. Previous researches indicated that，Pima90–53possessing resistance to Verticillium wilt genes. Cloning the resistance genes or therelated genes is important to understand disease resistance mechanism and molecularbreeding. In this study, a cDNA expression library of Pima90-53(G. barbadense)was constructed after inoculation with defoliating strain of Verticillium dahliae.cDNAlibrary screening and RACE technique were used, structure and expression of thetarget sequences were analysed in order to obtain disease-resistant related gene.Theresults are as follows:1. Construction and characterization of full-length cDNA expression libraryfrom Pima90-53root induced by Verticillium dahliaeThe SMART technology was used to construct a full-length cDNA Library ofPima90-53root tissue after inoculation with defoliating strain of Verticillium dahliae.The capacity of the primary cDNA library was1.28×106PFU, the titer of theamplified cDNA library was more than1010PFU·mL-1and the recombination ratiowas94%. The average insert size of the cDNA library was about1.1kb. Fourty-fivesequences were obtained. Blastx was used to homologue searching for thesesequences. Four ESTs sequences showed homologue to putative genes related todisease resistance, and three EST failed to show significant homology to any proteinsin the public databases, suggesting that they represent novel sequences.2. A novel GbWRKY1gene was isolated by cDNA library screening and RACEtechniqueGbWRKY1gene was isolated by cDNA library screening and RACE (GenBankaccession No. JF831361). The1971bp GbWRKY1cDNA sequence contained a271bp5′-UTR, a230bp3′-UTR and a polyA polyadenylation signal，as well as an openread frame of1470bp encoding a489amino acids. The deduced GbWRKY1proteincontains two characteristic WRKY domains and two zinc finger motifs. It couldclearly be assigned to GroupⅠ. Alignment of the putative GbWRKY1protein withrelated sequences indicated that it had the highest sequence homology with theVvWRKY2(amino acid identity62%), AtWRKY4(61%) and AtWRKY3(59%). The putative GbWRKY1protein contained N-glycosylation site, Protein kinase Cphosphorylation site, N-myristoylation site, and so on. It was soluble protein and nosignal peptide and transmembrane domains.In GbWRKY1, there were three introns which contained conservative GT-AGcleavage site with size of599bp,81bp and325bp, respectively. A1762bp fragmentof the GbWRKY1promoter was obtained using PCR from Pima90-53genome. Weidentified pathogen/elicitor-related elements including RAV1AAT, Box-W1, ARE,W-box, CGTCA-motif, ERE and TGACG, abiotic stress responsive element includingHSE, TC-rich repeats, WRKY71OS, WBOXNTERF3and MYCATERD1. It suggeststhat GbWRKY1may play a role in the response to environmental stress.We constructed an expression vector containing the GbWRKY1-GFP fusiongene driven by the CaMV35S promoter. Afterwards, the35S::GbWRKY1-GFPvector and the control35S::GFP vector were directly introduced into onion epidermiscells by particle bombardment. Those transiently expressed GbWRKY1-GFP fusionproteins were found exclusively in the nucleus, whereas GFP occurred in both thenucleus and cytoplasm. Therefore, these results indicated that the GbWRKY1proteinis indeed localized to the nucleus.3. Understanding expression patterns of GbWRKY1by inoculation andhormone treatment, the plant expression vector pCamE-GbWRKY1wasconstructed and successfully transferred into Arabidopsis thalianaQuantitative real-time PCR showed that GbWRKY1expressed in all tissues ofroot, stem and leaf. The GbWRKY1gene expression pattern was different afterinoculation and hormone treatment. GbWRKY1was induced and reached to a peak at8h after inoculation with Verticillium dahliae and maintained at high level within12h. After SA treatment, the expression levels declined at8h, dropped to the nadir at12h, and then increased normal lever at24h. MeJA treatment induced the GbWRKY1expression quickly accumulated and reached maximum level at12h, the expressionlevels declined at24h. For ACC treatment, GbWRKY1expression increasedobviously at12h, and maintained at high level within24h.The plant overexpression and antisense vector pCamE-GbWRKY1andpCamE-iGbWRKY1were constructed and then transferred into Arabidopsis thalianavia Agrobacterium-madiated method. Seven transgenic plants were verified byeffective selection for hygromycin B resistance and PCR.