Functional Characterization Of RNAi Fragments Derived From A Myo2 Gene From Fusarium Asiaticum And Construction Of Plant Expression Vectors

Wheat is an important food crop in the word,and its resistance to pathogen has always been an important research subject.With increasing incidence of wheat Fusarium Head Blight(FHB)in China caused by Fusarium asiaticum,the prevention and control of wheat FHB is more and more important.F.asiaticum not only can reduce wheat production,but also mycotoxin produces mycotoxins in field and during wheat storage,posing a threat to human and animal health.In the past three decades researches proved that the wheat varieties with high resistance to FHB can not be made by traditional breeding methods.Natural resources of wheat FHB resisatnce is very scarce,and thus breeders begin to consider genetic engineering to make wheat FHB breeding.RNA interference(RNAi)has high efficiency and may be used for FHB resistance breeding.In our study,Gateway was used to construct fungi expression vectors of different key growth genes of F.asiaticum.Small RNAi fragments were screened in F.asiaticum and wheat expression vectors for gene gun and grobacterium were generated.The results are as follows.1.Analysis and characterization of a small RNAi fragment of type ? Myosin synthetic gene(Myo 2)from F.asiaticum.The CDS region was divided into 15 segments.The target gene is amplified by PCR,and the attB1/attB2 site was added to both ends of the fragments.BP enzyme was used for recombination with intermediate vector pDON-201(containing attP1/attP2),generating BP entry vector.Then with pSXSH-RNAi vector(containing attR1/attR2 sites)using enzyme LR forconstruction.A fungal expression vector containing a stem loop structure was obtained,and a 177bp intron between sence and antisense chain was used.Protoplast transformation was deployed to integrate the vector into the specific PLS locus of F.asiaticum 5035,using the way of PEG mediated the protoplast transformation.RNAi fragments that have obvious effects on growth and virulence were selected.2.Using RNAi fragmanets from type ? Myosin gene and other RNAi fragments from other pathogenicity genes of F.asiaticum were used to construct two types of wheat expression vectors for the wheat transformation.