Transformation Of Ralstoia Solanacearum Effector Poppl Into Tobacco Plant And Its Contribution To Resistance To Bacterial Wilt

Ralstonia solanacearum PopP1 is an effector protein delivered into host plant cells through the type three secretion system.It,together with AvrA,plays a role in determining the incompatible interaction of R.solanacearum with tobacco plants.Transient expression of PopPl induced cell death in Nicotiana benthemiana,suggesting that R.solanacearum PopP1 is an Avr protein.In this study,PopPl gene was transformed into tobacco plants by Agrobacterium-mediated transformation to determine whether it could increase the resistance to bacterial wilt disease.This research will provide new idea and clue to plant breeding for disease resistance.The main contents of this thesis include several items as follows:(1)The constitutive 35S promoter and the pathogen-inducible PRla promoter were separately fused with PopPl gene by the overlapping PCR method.After digested with Hind?-EcoRI,two fused DNA sequences were inserted into binary vector pCAMBIA2300,generating p2300-35P1and p2300-PRP1;(2)To confirm whether the recombinant constructs could be used for expressing PopPl gene,p2300-PRP1 was transiently expressed in N.benthemiana leaves.At 2 days post inoculation,clear cell death was observed the inoculation sites;(3)The p2300-35P1 and p2300-PRP1 were transformed into tobacco NC89 variety by using Agrobacterium tumefaciens EHA105-mediated transformation.In total,fifty-five TO generations of p2300-35P1 transgenic lines,and fifty p2300-PRP1 transgenic lines were obtained.The gDNA of 105 transgenic lines was extracted and used as a template for PCR amplification to by using PopPl gene specific primers.For the p2300-35P1 transformation plants,50 transgenic lines showed positive results,and 43 positive lines were identified from p2300-PRP1 transgenic lines.The test positive efficiencies were 90.9%and 86%,respectively.(4)The RNAs were extracted from TO generation,including thirteen p2300-35P1 and six p2300-PRP1 transgenic lines.The expression level of PopPl gene was evaluated by using Real-time PCR experiment.Four lines from each kind of transgenic plants were chosen to make T1 generation plants for testing the resistance to bacterial wilt disease.After inoculation with irrigation method,the transgenic plants showed no distinct difference in disease incidence,but the disease index was reduced.