Screening And Identification Of Wheat Plants Co-transformed With Fusarium Graminearum RNAi And Disease Resistance-related Genes Peptide Genes

Wheat Fusarium Head Blight（FHB）,caused by Fusarium graminearum,is one of the major fungal diseases in wheat production in China.In this study,wheat varieites Xiangmai 76,Yangmai 158 and Zhengmai 9023 were used as experimental wheat materials.Clean cassettes（minimal expression cassettes without vector frame sequneces）containing disease resistance-related genes such as that enclding antimicrobial peptides and antibody fusion proteins,and RNAi fragments derived from F.graminearum were co-transformed into wheat embryogenic callus by gene gun,and forty-nine transgenic lines were obtained through differentiation and screening.In greenhouse conditions,single-floret inoculation experiments were carried out to assay the resistance of stable transgenic plants to FHB.The main research results are as follows:1.Screening and identification of wheat transformed with RNAi fragments and antibody fusions/antifungal peptide genes（1）RNAi fragments,Chs3b-As5*2,from F.graminearum regulated by different promoter Cmps,Lem1 and 35S s were introduced into embryogenic callus of Xiangmai76 and Yangmai 158 by gene gun.Eleven positive plants containing Chs3b-As5 gene were screened by PMI/mannose system,which were numbered as ch A and ch C,and now in T₄ generation.（2）RNAi fragments Cmps-t-chs3b-PKC5-chs7-4-gls6-myo II-14 and a gene encoding antibody fusion Lem2-ech42-D2 were co-introduced into embryogenic callus of Yangmai 158 by gene gun.Three positive plants were obtained.They all contained the RNAi fragment and antibody fusion Lem2-ech42,which are now in T₄ generation.（3）RNAi fragment d35s N-CYP51 and genes encoding antifungal peptides[Cmps-D4E-Ocs]-[ubi-Opt-Nos]-[lem1-AFP-Mas]were used to co-transform Yangmai158.Seven positive plants were obtained.Six of them contained the marker gene Pmi and antifungal peptides and one contained RNAi fragment and antifungal peptide.These plants are now in T₃ generation.（4）RNAi fragments Ubi N-As5-t RNA-As2c-NOS were introduced into Zhengmai9023 by gene gun.Six positive plants were obtained.These plants contained RNAi fragments and had no marker gene.They are now in T₁ generation.（5）Genes encoding antifungal peptides Ubi-AFP2,[Cmps-D4E-Ocs]-[ubi-Opt-Nos]-[lem1-AFP-Mas]and lem2-Exhvrip were co-transformed into Zhengmai 9023.A total of 16 positive plants were obtained.Eight of them contained PMI,Opt1,D4E and Exhvrip,while the other eight only contained Pmi and D4E.These plants are now in T0generation.（6）RNAi fragments Ubi N-Tri5AS-（R-intron）,Ubi N-Tri6AS-（R-intron）-t RNA-Tri10As-（PDK）were co-transformed into Zhengmai 9023 by gene gun,generating two positive plants.These plants contained Pmi and Tri10 RNAi fragments and are now in T₀generation.2.Screening and identification of wheat transformed with genes encoding mycotoxin-detoxification enzymesTwo mycotoxin-detoxification genes Ubi N-AKR13B2 and Ubi N-PQQ-ADH were transformed into Zhengmai 9023 by gene gun.Four positive plants were screened out.Two of them contained the marker gene Pmi,and AKR13B2 and ADH-Nos,while the other two contained only ADH-Nos.These plants are now in T₀ generation.3.Propagation and identification of transgenic wheat lines with higher generation（1）Seven lines（EA,XVII-E and 3Ba）containing RNAi fragments Chs3b-As5*2 in the background of the wheat variety Xiangmai 76 were propogated and identified.Single floret inoculation with F.graminearum showed that XVII-E line（T3）had significantly enhanced resistance compared to that of non-transgenix control Xiangmai 76.（2）Propogation and identification of SC line containing RNAi Gls gragment in the background of Xiangmai 76.Single floret inoculation with F.graminearum（T3）showed that the transgenic line had significantly higher resistance than non-transgenic Xiangmai76.（3）Propogation and identification of transgenic lines containing micro RNA 8m R7.The line 7A2 was generated with wheat variety Xiangmai 76 for transformation while lines 8A,11A2 and 11A3 were generated with Yangmai 158 for transformation.The four lines have been propogated for two generations.