Screening And Identification Of Wheat Plants Transformed With RNAi Fragments Derived From Fusarium Head Blight Pathogens

Fusarium head blight(FHB)in wheat is a worldwide fungal disease which mainly infects the wheat spikes causing lots of yield losses.Breeding for FHB resistance has been rather slow because of limited genetic resources and tools via traditional approaches.However,transgenic technology is a powerful approach that could introduce any resisting genes from various origins,which may substantially facilitate the progress of FHB breeding.Hence,introducing of resistance genes from different resources into wheat varieties and analysis of the genetic behavior of these genes could provide new germplasm and information for breeding durable and stable resistance materials against FHB.In this study,by using particle bombardment and agrobacterium-mediated transformation,along with selection marker gene Pmi,different RNAi fragments targeting chitin synthetase(Chs),membrane protease(Gls),cell division gene(Myo)and toxin synthetase gene(miRNA)were transformed into commercial wheat varieties Xiangmai 76,Yangmai 158 and Zhengmai 9023 which were cultivated in middle and lower reaches of Yangtse River valley.After a series of work including selection,regeneration and PCR analysis,transgenic plants with various generations were obtained.The results are as the follows:1.Transformation and identification of RNAi fragments targeting pathogenicity genes from FHB pathogens(1)With particle bombardment strategy,co-transformation of five different combinations of membrane protease RNAi fragments(gls2-glsAs6,gls3-glsAs6 and glsAs6-i-glsAs6)and chitin synthetase RNAi fragments(Chs3b-As5-chs3b-As1 and Chs3b-As3-As3)into Xiangmai 76 resulted in 18 positive plants in T0 generation.Analysis of their progenis showed the results listed below: A,one line of ZR containing glsAs2-glsAs6,glsAs3-glsAs6 and chs3b-As5-chs3b-As1 in T4 generation;B,two lines of CA and CB which contain glsAs2-glsAs6 and glsAs3-glsAs6 in T3 generation;C,one line of 03 gls only containing glsAs2-glsAs6 in T3 generation;D,twelve lines of Ch contains glsAs2-glsAs6 and chs3b-As3-chs3b-As3 in T1 generation;E,one line of 07 gls containing glsAs2-glsAs6 and glsAs6-i-glsAs6.(2)With particle bombardment,co-transformation of RNAi fragment Myo2-As6-i-As6 with marker gene Pmi,we obtained 6 positive transgenic plants from 4different calli which contain both Myo2-As6 and Pmi,and named MY2 ～ MY7.(3)With agrobacterium-mediated transformation,transformation of RNAi fragment Chs7-As3-Chs7-As4 trageting chitin synthetase into immature embryo calli of Yangmai158,and under the PMI/mannose selection system generated,we obtained one line06Chs7 in T1 generation which was confirmed contain the transgene with PCR analysis.2.Transformation and identification of RNAi fragments targeting toxin synthetase gene(miRNA)from FHB pathogensBy co-transformation of different combinations of RNAi fragments(ubi-8mR7、35SN-8mR7 、 Lem1-8mR7 、 Lem2-8mR7 、 cmps-8mR7 、 cmps-8mR12-47 、ubi-8mRFD10787、cmps-8mRFD10787 and ubi-8mR12-47)targeting toxin synthetase gene(8mR7)into Xiangmai 76,we obtained ten positive transgenic plants in T0 generation.Analysis of the progenies showed the results listed below: A,eight lines of mR in T1 generation containing ubi-8mR7 and 35SN-8mR7;B,one line of 07 mR containing Lem1-8mR7,Lem2-8mR7 and cmps-8mR7;C,one line of 08 mR containing35SN-8mR7 and cmps-8mR7.D,three lines of Z11 mR in T0 generation containing Lem2-8mR7,cmps-8mR12-47 and ubi-8mRFD10787;E,three lines of C11 mR in T0 generation containing Lem2-8mR7,cmps-8mRFD10787 and ubi-8mR12-47.3.PCR identification of high generation materialsThe transgenic lines,obtained by Lab preliminary study and using Yangmai 158 and Xiangmai 76 as receptor,were generated and PCR identified.As the follows: A,The generation of one transgenic line having cmps-Chs3b As2-Chs3bAs3.PCR identification of T3 and T4 lines found transgenes were co-segregation.B,The generation of two transgenic lines carrying Chs7,Pkc and Blf genes.Trough PCR identification of T6 and T7 lines,the transgenes were segregated.C,The generation of transgenic line with OAPAR gene.T5 and T6 lines,analyzing by PCR identification,were linkage inherited.All of these transgenic wheat materials that were confirmed by PCR analysis in more than one generations display similar phenotypes as their non-transgenic control plants.These results suggest that RNAi fragments derived from fungal pathogens were stably integrated into genome of wheat cultivars and heritable.These transgenic lines provided new materials and resources for further selection and breeding of FHB resistant wheat varieties.