Study On Agrobacterium-mediated Transformation Of Wheat Wiht Genes Against Fusarium Head Blight

Wheat is one of the major sources of food.Wheat Fusarium head blight(FHB)is caused by Fusarium graminearum.FHB not only reduces wheat production,but also affects quality of wheat flour.Traditional chemical control of FHB has certain effects on FHB,but it causes environmental pollution and its controlling efficiency t decreases over years of use.Genetic engineering technology provides a new way to improve wheat FHB resistance.In this study,commercial wheat varieties in of the Middle and lower reaches of the Yangtze River were used for transformation.Suitable sizes of young grains after flowering for 12?14 days were selected,from which young embryos were used for inducing calli.Bar or Pmi gene was used as a selection marker.Vectors with double borders for Agrobacterium-mediated transformation were constructed and used for wheat transformation.Positive T0 transgenic plants were selected through screening.PCR was applied to identify the transgenes in transformed wheat plants.RT-PCR analyses was used to analyze gene expression.Transgenic wheat plants without maker gene were selected.To test the FHB resistance response,the high generations of the transgenic plants were inoculated in the field with F.graminearum.The main results are as follows:1)Transformation and identification of wheat transgenic plants with wheat varieties Yangmail58(Y158),Zhengmai9023(Z9023),Xiangmai76(X76)and Xiangmail3(X13)as receiver wheat materials and a barley Hvglu gene.Barley glucanase Hvglu gene may break down the glucan of fungal cell wall,thus delaying fungal growth in plant tissues.Vector containing a Hvglu gene was introduced into Agrobacterium cells that were then used to transform wheat.After screening and PCR identification,nine transgenic wheat plants were generated,four plants derived from wheat variety Y158,three from Z9023,one from X76 and one from X13,respectively,through the Pmi/mannose screening system.T6 generations from Y158 and Z9023 as wheat receivers for transformation were obtained and some transgenes have segregation.The transgenes in X76 as receiver were stable in in T2?T5 generations.RT-PCR analyses detected Hvglu gene expression.To test wheat FHB resistance response,high generations of the transgenic plants with the Hvglu gene were assayed in the field after inoculation with F.graminearum.The results showed that T6 generations from Y158 had increased resistance by 13%and T6 generations from Z9023 had increased resistance by 12%and an increased resistance of 11%was observed for T6 generations from X76.2)Wheat variety Y158 was transformed with OAPAR gene and transgenic plants were screened and identified.The OAPAR gene contains five different genes,Omega/AG/Pep3/ACE/PsD2.The multiple genes were transformed into wheat cells at the same time by using Agrobacterium-mediated transformation.One positive To plant was generated,and selection marker gene was Bar gene.T4 generation of the transgenic plants was obtained and the transgene was in segregation.3)Wheat variety Y158 was transformed with CHS7 RNAi gene and transgenic plants were screened and identified.The CHS7 gene is RNA interference gene,including CHS7As3 and CHS7As4.One positive plant in T0 generation was generated,and its selection marker gene was Pmi gene.T4 generation of transgenic plants was obtained.These results showed that the four wheat varieties can be used for Agrobacterium-mediated transformation and Y158 appeared to be most suitable wheat genetype.