Fusarium Wilt Resistance Related-gene Cloning And Functional Analysis Of Gossypium Barbadense L.

Cotton is the main cash crops,which has a pivotal position in the national economy.Xinjiangcotton district,as the main raw cotton production base,which has an important role in the wholecountry.Fusarium Wilt is one of the main diseases of serious harm to the cotton production,breeding and planting new disease-resistant varieties of cotton is the most cost-effective only wayto solve this problem.Using traditional breeding methods to cultivate the new varieties needs toolong cycle,and it is unable to meet the urgent need of the production,so cloning of resistance geneand studying of the gene expression have great significance in breeding for disease resistance.In this study,resistance to Fusarium wilt resistance to sea-island cotton tightly linkedmolecular markers sites,obtaining the sequence of resistance related genes;And then using theprocess of Fusarium Wilt pathogen infection of cotton resulting in the onset,in order to clear theabove gene expression level,further to explain the role of this gene in resistance to Fusarium Wilt.The results are as follows:1. Resistance gene sequence published in the GenBank database,designed107pairs the RGAprimers,the use of high resistance to Fusarium wilt Island Cotton “06-146”highly susceptible toFusarium wilt Island Cotton Xinhai14andmiscellaneous home F2groups randomly select180plant materials,use of RGA primer material between parent screening out12pairs of primersmatter screening anti,sense gene pool, preliminary to determine Gbrga14island cotton wilt diseaseclosely linked to themolecular markers,molecular markers and the Sea Island cotton wilt diseaseresistance exchange values were15.5%, and Sea Island cotton resistance to Fusarium wilt genegenetic distance to15.92cm.Finding the source of the linked markers found,according to theprimers of the Arabidopsis thaliana the RPP5gene design,4(33.33%). This12pairs ofpolymorphic RGA markers mainly composed of three classes, which TIR-NBS-LRR class of sixpairs, accounting for50%; NBS-LRR class of five pairs, accounting for41.67%; NBS class1pair,accounting for8.33%.2. The through on for recycling Gbrga14loci amplification between the two parentalsequencing,the sequencing fragment as a probe,by means of silico cloning method,obtaining twodisease-resistant gene, named into RGBCH and SGBCH,BLAST alignment analysis showed thatRGBCH、SGBCH possess a high genetic polymorphism.Bioinformatics analysis showed that theα-helix is the main part of the secondary structure of the two proteins.ProtFun forecasted the mainfunction of the RGBCH protein is participating energy metabolism and biosynthesizingcofactors;The main function of the SGBCH protein is participating energy conversion in theprocess of protein translation.3. Induced by Fusarium oxysporum five different materials in different tissues and fluorescence quantitative PCR using two primers,respectively,the results show, the use of thespecific primers GBCH-Q material quantitative PCR analysis shows:“06-146”strainsthe targetgene expression in different tissues4d peak;Xinhai14target genes in6d there is a maximumvalue, and then decreased to the original level;Zhongmiansuo35,Junmian1cotton and TM-1inthe induction ofbefore and after the induction of expression of the volume is very weak orrelatively consistent,remaining at a low level of expression, quantitative PCR analysis of thematerial shows through the use of primers StVe-Q:lines “06-146”,Xinhai14,Zhongmiansuo35,Junmian1cotton and TM-1target gene in the bacteria-induced conditions,there is no change withthe induction time showing expression status.Description the cloned RGBCH、SGBCH onlyexpressed in Sea Island cotton material,two genes associated with resistance to Fusarium wilt.4. RGBCH and SGBCH recombinating into pCAMBIA1301plant expression vector,constructing plant expression vector pCAMBIA1301-RGBCH and pCAMBIA1301-SGBCH,RGBCH and SGBCH gene integrated into the tobacco genome using of agrobacterium-mediatedmethod,obtaining T1generation seeds.T1transgenic and non-transgenic tobacco broth poured rootmethod(Each material of30planting),Turn RGBCH gene most of the leaves were yellowing, plantsurvival rate was78%,while the survival rate of to turn SGBCH transgenic plants to24%,most ofthe leaves dry death,non-transgenic plants almost all died.These results suggest that the genetransferred RGBCH tobacco resistance to Fusarium wilt bacteria.