Research On Application Of Pathogen Induced Promoter Based On TALE

Bacterial blight(BB)and bacterial leaf streak(BLS)of rice,are the representative diseases resulted in the most severe reduction of rice output.The most economic and effective measures to control diseases is to cultrue disease-resistant varieties.Now,there were 40 bacterialblight resistance genes have been found and nine clond,seldom genes with a broad spectrum resistance was observed in rice.Moreover,most of genes have characterd weak resistance andbeeding bacterial leaf streak resistanced very slowly.Currently,only found someBLS resistanced in cultivated and wild rice.It is too narrow spectrum to few with realproduction values.Therefore,cultivating broad-spectrum resistance to BBand BLS are the major probles.Due to the transgenic strains are mostly with selection markers,incapable to industrializations.In this study,we planed to breeding resistance rice with broad-spectrum toBB and BLS using inducible artificial expression promoter by Xoo and Xoc onthe basis of the unmarked system strategys,to constuct double right Boundary vectors with artificial promoter of Xa23 and Xa27,By genetically modified then through the strategy of selection of hybrid and the resistant test in field to breed the safe transgenic rice with marker-free resistance to both BB and BLS.Furthermore,though the molecular identification and resistance analysis of T1 transgenic generations,confirmed that the resistance is indeed caused by transgen,and the genes could stable genetic to future generations.The main results were as follows:1.The plant expression vectors pMNDRBBIN6-Xa23,pMNDRBBIN6-WXa23,pMNDRBBIN6-WXa23 and pMNDRBBIN6-WXa27 were successfully constructed using for rice transformation.2.The four expression vectors were transferred into TP309,respectively,byusing of agrobacterium transformation system,after selection,differentiation,21 lines and 33 lines of transgenic plants were obtained,respectively.Then afterdetection by PCR to T0 transgenic lines,20 lines of transgenic pMNDRBBIN6-Xa23 rice were posotive,and 27 lines of transgenic pMNDRBBIN6-WXa23 rice were posotive.3.T0 transgenic lines of pMNDRBBIN6-Xa23 and pMNDRBBIN6-WXa23 were further investigated by the five major agronomic characters,including Thousand seed weight,seedind rate,plant height,spike length,and tillering number.Results showed that the transgenic lines was no significant differences compared with TP309 controls;The seed setting rate compared with TP309 controls changed,but it was not caused by GM,may be tisse culture,planting environment,etc.4.Seed harvested from pMNDRBBIN6-Xa23 TO generation of PCR and resistance analysis of betters.Select 16 resistance strain for T1 generations,a total of 493 strains.With TaleXa23 primers and Hpt PCR identification.PCR results and statistics.From TaleXa23 primers:PMNDRBBIN6-Xa23 394 of 482 were Positive,Positive detection rate of 81.7%.There six strain are separation 3:1.Mendelian inheritance law.360 of 482 were Positive with Hpt primer.Positive detection rate of 74.7%.100 of 180 were with Hpt primer under the PMNDRBBIN6-WXa23 transgenic plants,Positive detection rate of 55.6%.The above show that pMNDRBBIN6-Xa23 and pMNDRBBIN6-WXa23 Genes can be stable inheritane.5.16 and 13 T1 transgenic lines were further verified by the identification of resistance to BB and BLS.respectively.Results showed that the T1 transgnic plants have abilities to improve the resistance and tolerance to BB and BLS in some extent.6.The Ti generation of GM pMNDRBBIN6-Xa23 plant with primers(Hpt)were negative,and primers(TaleXa23)were posotive(namely marker-free transgenic strains).Screened a total of 21 strains of unmarked transgenic plants.7.Choose phenotypic and molecular identification are good strain and unmarked resistance of plants,respectively,TP309 for comparison,The vaccination sterile water P6 and Xoc rspectively,then by the Real-time fluorescent quantitative PCR to detect the expression of foreign genes.Results show:In the vaccination P6 and Xoc leaves,Xa23 gene expression quantity rather than by sterile water,and the contrast TP309 with sterile water,P6 and Xoc,expression have extremely significant difference(p<0.05).shows Xa23 gene isinduced expresions by P6 and Xoc.Obtained with double resistance to BB and BLS of transgenic plants.