Transformation And DsRNA-mediated Gene Silence Of Gaeumannomyces Graminis Var.tritici

Take-all,which is one kind of wheat root diseases caused by Gaeumannomyces graminis var.tritici,is one of the most important wheat root diseases world-wide.There is no normal wheat that can immune from this disease.RNA interference(RNAi)is a phenomenon that double-stranded RNA-induced,specific degradation of homologous m RNA and highly conserved during evolution.In this study,we use ds RNA-mediated RNAi to find the growth and pathogenicity related genes of Gaeumannomyces,we also constructed a high efficiency transformation system of mycelium.this work make a foundation for gene function analysis and breed resistant wheat varieties using RNAi technology.The main contents and conclusions are as follows:1 In this study we attempt different methods for the mycelium quantitatively and different culture media.We find that when we broken the hyphae into small fragments for uniform quantification,the uniformity of hypha has been highly increased.The cell culture plate can offer a good interference microenvironment for mycelial growth when cultured with liquid medium.2 In this study,we used different concentration(50-500 ng/?L)of ds RNA to the culture experiment.The result showed that: when the concentration of ds RNA is reached at 100ng/?L,the inhibition of Gaeumannomyces mycelium was more pronounced,have statistics significant difference compared with control.By adding ds RNA of the 19 different candidate genes to liquid PDA media,the result show that different genes of ds RNA,the interference effect is different,the cell division control protein,haplotype gentisate1,2-dioxygenase,ADP-ATP translocase gene have a very significant inhibition in mycelium,the highest inhibition rate can reached to 8.5%,have statistics significant difference compared with control.Fluorescence quantitative PCR results showed that the three genes using ds RNA to interference during 1-5 days can make the gene expressing decreased,but they showed different trends.It was indicated that different genes can express different performance for vitro interference.This result show that adding ds RNA in vitro can interference gene expression of hypha.However,this interference has a relatively instability.3 This study use the plant RNA interference vector p FGC5941 and fungal expressionvector p BHt2-e GFP,by enzyme digest and blunted them,ligated to construct a new fungal RNA interference vector p BHt2-CHSA-Intron.The vector may be used to test the interference efficient in fungus,for the obvious interference gene we can ligate the fragment to the plant RNA interference vector p FGC5941 directly,so we can obtain transformed plants.So that RNAi between fungal and plant transformation are more convenient,this provides great convenience for research of plant disease.4 The study used the method of co-cultivation with Agrobacteriumm to transformation,and explored the amount of Agrobacterium for transformation,the time of co-culture with Agrobacterium,the concentration of As in co-cultured medium,and compared the transformation efficiency when the hypha dealing with lywallzyme to obtain an efficient transformation system.The study found that when co-culture with Agrobacterium for 3days,the Agrobacterium amount is 300?l(OD=0.35),the concentration of As in co-cultivation medium is 400?mo L/ L,the transformation efficiency of Gaeumannomyces mycelium is higher.After deal with Lywallzyme(500 U/m L),Gaeumannomyces cell wall produces some changes,made the transformation efficiency improved,the transformation efficiency is about 10%,make transformation efficiency a twice higher than without treatment by Lywallzyme.And the Lywallzyme treated for 6h can make a higher transformation efficiency than other time.After genetic transformation,transformants cultured after 5 generations still have resistibility of hygromycin B and can grow stable,indicating a stable T-DNA insertion.