Construction Of Rnai Vector Of ATP/ADP Translocase-Encoded Gene And RNA Interference Of Gene Encoding Haplotype Gentisate 1,2-Dioxygenase Of Gaeumannomyces Graminis Var.Tritici

Gaeumannomyces graminis is a soil inhabiting wheat root disease,which has a serious effect on wheat yield.So far,no high or immune-resistant varieties have been found in common wheat.Previous study in our lab found RNAi of genes encoding the cell division control protein(Cdc),haplotype gentisate 1,2-dioxygenase(Gdo),and ATP /ADP translocase(At1)can affect the growth and development of Gaeumannomyces graminis.In this study the RNAi transformants of Gdo were obtained by using Agrobacterium tumefaciens-mediated transformation,and growth and pathogenicity of some transformants were further analyzed.The main aspects are summarized as followings:1.RNAi vector of gene At1 was successfully constructed by inserting two opposite-direction segments of the same ATP/ADP transposase-encoded gene into up-stream and down-stream regions of the chalcone synthetase gene intron sequence(CHSA Intron)in the p BHt2-CHSA-Intron vector by seamless cloning and double enzyme digestion and ligation.2.Some factors which affecting transformation efficiency were optimized: Mycelial mechanical crushing and lyticase treatment prior to Agrobacterium tumefaciens co-culture showed little effect on the transformation efficiency;The transformation rate of the solid co-culture system was 43.01% and 35.77% for the liquid co-cultivation system,so solid co-culture system is more conducive to improve the transformation rate;The optimal concentration of hygromycin B inhibiting growth of Gaeumannomyces graminis on PDA plates was 200?g/m L.The concentration can be used for the screening and identification of transformants.3.Progenies of Agrobacterium tumefaciens-mediated genetic transformation were sub-cultured three generations on media containing 200?g/m L hygromycin B,then 30 transformant candidates were randomly selected and inoculated on hygromycin B-free plates and cultured at 25°C in darkness.After 7 days,the colony diameters of Gaeumannomyces graminis were measured and compared to wild types.The results indicated that the growth rates of transformants were statistically significantly reduced.Hyg gene sequence-specific PCR amplification of four candidate transformants showed that T-DNA were inserted into transformants genome.4.The pathogenicity of four transformants were evaluated by infection of wheat roots,the results showed two transformants has obvious weakened pathogenicity.