Study On Transformation Of Wheat With RNAi Fragments Derived From Pathogenicity Genes Of Fusarium Graminearum

Wheat is one of the main food crops for the survival of humans.Fusarium head blight(FHB)caused by Fusarium graminearum occurrs frequently in China and many other countries in recent years due to global climate change,which seriously affects the yield and quality of wheat.Cultivating new varieties that can resist to this disease is the key to solve this problem.Because of lacking of natural resistance germplasm against wheat FHB,progress of traditional breeding wheat varieties resistant to FHB is slow.With the help of genetic engineering technologies,the alien genes derived from other species can be used to improve wheat FHB resistance.In this study,the immature embryos of wheat varieties Yangmai 158 and Xiangmai 76 were used as receptor materials for genetic transformation by using the particle bombardment;several vectors that contain RNAi fragments from pathogenicity genes of F.graminearum were introduced into wheat;Pmi/mannose selection system was used to select positive transformants,and some transgenic wheat lines were assayed for their resistance to FHB.The main results are as follows:1.Generation of transgenic wheat transformed with two F.graminearum RNAi fragments and FHB resistance assay of the transgenic wheat lines.A vector that contains RNAi fragment of Fusarium protein kinase gene(pkcAs3)and RNAi fragment of chitin synthase gene 7(chs7As4),together with a screening marker gene Pmi,were transformed into Xiangmai 76.PCR analyses showed that a transgenic plant containing both RNAi fragments and a Pmi gene was generated through mannose screening agent.The Pmi and the transferred RNAi fragments were linked together.The results of FHB resistance assay of T4 transgenic plants inoculated with single-flower in the field showed that the transgenic plants displayed significantly improved resistance compared with that of non-transgenic control Xiangmai 76.2.Generation of transgenic wheat transformed with RNAi fragments regulated by two promoters and analyses of their resistance to FHB.A vector that contains two RNAi fragments,together with a screening marker gene Pmi,was transformed into Yangmai 158.Among these two fragments,one is the combination of husk specific promoter and RNAi fragments from a protein kinase gene Pkc(Lem2-pkcAs3-chs7As4)and a Chitin synthase gene Chs 7;another one contains constitutive promoter and RNAi fragments chitin synthase gene Chs7.PCR identifications showed that two transgenic lines,named Y1 and Y3,are generated through screening with mannose screening agent.Y1 and Y3 contained all the introduced gene based on PCR analyses from T1 to T4 generations.Transgenic plants of T4 generation were assayed for FHB resistance after single-floret inoculation with F.graminearum in the field.The Y1 transgenic lines showed significant resistance to FHB compared with that of control Yangmai 158,whereas the Y3 line had no significant difference relative to the control.3.Screening and identification of transgenic wheat transpformed with different RNAi fragments from F.graminearum.Different promoters were combined with small RNAi fragments that were derived from F.graminearum,including chitin synthase gene Chs3b,sterol 14a-demethylase gene CYP51,glucan synthase gene Gls;Six minimum expression cassettes were used that included Dubi35s-chs3bAs5Asl,Dubicmps-chs3bAs2As3,Dubi35ss-CYP51BAC-DNOS,Dubilem2-glsAs6-chs3bAsl,Dubi-ubi-gls3-gls6,Dubi-ubi-gls2-gls6.Four different gene combinations were used to transform immature embryos of Xiangmai 76.After the screening and identification,15 positive TO plants were obtained.Their gene combinations from TO and T1 generations are as follows.1)Generation of transgenic wheat transformed with minimum expression cassettes Dubi-ubi-gls3-gls6,Dubi-ubi-gls2-gls6 and Ubi-pmi.PCR analyses showed that five positive TO plants that were named as R1,R8,R12,R21 and R23 were obtained.R1,R8 and R12 contained all the genes used for transformation and screening marker gene Pmi.R21 only contained the screening marker gene Pmi.R23 contained the gene gls6,but did not contain Pmi.All the genes identified in R1,R8 and R23 plants were stably inherited to T1 generations.2)Generation of transgenic wheat transformed with minimum expression cassettes Dubi35ss-CYP51BAC-DNOS,Dubicmps-chs3b-As2As3,Dubi35s-chs3bAs5Asl and Ubi-pmi.PCR analyses showed that two positive TO plants that were named as R20 and R2 were obtained.R2 plant only contained screening marker genes.R20 contained all the four genes that were stably inherited to T1 generation.3)Generation of transgenic wheat transformed with minimum expression cassettes Dubi35s-chs3bAs5Asl,Dubicmps-chs3b-As2As3,Dubi-ubi-gls3-gls6 and Ubi-pmi.PCR analyses showed that two TO positive plants that were named as R14 and R7 were obtained.R14 plant contained all four genes used for transformation.R7 strain contained three genes except Dubi35s-chs3bAs5As1.In R7 and R14 lines all four genes in were stably inherited to T1 generation.4)Generation of transgenic wheat transformed with minimum expression cassettes Dubilem2-glsAs6-chs3bAsl,Dubicmps-chs3b-As2As3,Dubi35s-chs3bAs5Asl and Ubi-pmi.PCR analyses showed that six TO positive plants that were named as R19a1,R29al,R16a1,R16a2,R17al and R17a2 were obtained.PCR identification of TO plants showed that R19a1 and R29al contained all the four genes used for transformation.R16a1,R16a2 contained three genes except Dubi35s-chs3bAs5Asl.In T1 generations of R17al and R17a2,all genes identified in TO plants were lost.In T1 generation of R29al line,Dubicmps-chs3b-As2As3 gene was lost.All the genes identified in R19a1,R16al and R16a2 lines were stably inherited to T1 generation.In the R29al line,Dubicmps-chs3b-As2As3 gene was lost,while the other three genes in this line were stably inherited to T1 generation.4.Propagation and identification of genetically stable transgenic wheat.Two transgenic lines(Xiangmai 76 as receiver cultivar)were previously generated by others and further identified here.1)Transgenic line that was transformed with Blf,Hvchi and Bar genes.PCR analyses showed that all transgenes were inherited to T3 and T4 generations.2)Transgenic line that was transformed with Blf,Hvchi and Pmi genes.PCR analyses showed that all transgenes were linked together and inherited in T2 to T3 generations.