Construction Of The Resistant Fusarium Head Blight Vectors And Expression Analysis In Arabidopsis Thaliana

Fusarium head blight(FHB)is a devasating disease caused by Fusarium graminearum.These diseases cause huge yield losses across millions of hectares in global wheat production regions.Moreover,the pathogens produce various mycotoxins which are harmful to human and animal.However,the methods for control this disease are not efficient enough.Therefore,alternative strategies should be found to protect crops from Fusarium pathogens.RNA interference(RNAi)is a conserved mechanism that silences target genes by specific antisense RNA.Plant expression of antisense or hairpin RNAi constructs directed against the target transcripts of Fusarium has a great potentiality to control FHB.In this study a series of Agrobacterium-mediated RNAi vectors were constructed that can be used for plant transformation;Arabidopsis thaliana was used to express RNAi vectors.The disease resistance of transgenic Arabidopsis thaliana was analyzed by leaf inoculation in vitro.The genes or gene segments are identified as new resistant resources against FHB.In addition,this study constructed some new Agrobacterium-mediated transformation vectors used for wheat transformation;propergating and identifing a batch of advanced transgenic wheat lines by PCR.The results are as follows:i:Expression and analysis of chitin synthase RNAi segments derived from F.graminearum in Arabidopsis thaliana.The gene-Chitin synthase gene CHS of F.graminearum was selected as the target of RNAi.A double ring or more stem ring segments was constructed,and then connected to the laboratory existing Arabidopsis thaliana erpression vector named pTRAc.Through Agrobacterium strain GV3101 into Arabidopsis thaliana,transgenic plants were screened by Kana resistance.By cutting the leaf of T3 generation transgenic Arabidopsis plants,inoculating B51066 spores in vitro and analysis the wounded leaf areas,the results indicated that resistance of the transgenic plants is significantly higher than that of wild type.By the method of real-time quantitative PCR detection between transgenic RNAi-CHS7 and wild-type,the expression levels of the gene targeted by RNAi in Fusarium graminearum were significant lower in transgenic RNAi-CHS7 than that in wild-type.These resuls suggested that the small RNA of transgenic RNAi-CHS7 Arabidopsis entered into the colonizing F.graminearum,silencing the target genes and leading to the lower expression and resistance.ii:Construction of wheat expression vectors.Wheat expression vectors were constructed,which includes double promoters of double broder EHA105 Agrobacterium-mediated transformation.Promoters used included CAMV35S and Ubi;also double promoters of singel broder EHA105 expression vector of wheat were constructed.Promoters used included CAMV35S,Ubi and Cmps.The screening marker was 6-mannose phosphate isomerase gene PMI for all the vectors.The results showed that plasmids entered into the Agrobacterium EHA 105 as identified by bacterial PCR.iii:Propagation and identification of transgenic wheat lines.T2 and T3 transgenic wheat lines containing BLF-A6,RNAi-CHS7 and RNAi-chs3b-A2S2-A3S3 were propagated.DNA was extracted from these wheat plants and PCR was used for identification of the wheat plants.Transgenic wheat seeds of T4 and T5 generations have been generated.