Transgenic Wheat Expressing Fusarium Graminearum Chitin Synthase 7 Gene RNAi Fragment And Fhb Resistance Evaluation

Fusarium Head Blight（FHB）is a destructive fungal disease in wheat,caused by Fusarium graminearum（F.g）worldwide.In 2016,there are several wheat planting regions have outbreak of FHB and the incidence of disease is more serious than the previous years,now FHB has become one of the most serious threat to wheat yield.Engineering technology can obviously broaden the selection range of target genes.Besides,the government is increasing expenditure to genetically modified organism popularize science,the citizen will be more clearly on genetically modified organism technology and genetically modified organism production.Sum up,the Engineering technology is a promising method to protect wheat against FHB.The topic of my study is transgenic wheat expressing F.graminearum chitin synthase 7 gene RNAi fragment and FHB resistance evaluation.The main results are as follow.1.Selection of wheat transformed with F.graminearum chitin synthase 7 gene RNAi fragment geneWheat variety（var.）Yangmai 158（Y158）,transformed with chs7as3as4 gene and pmi gene serving as selective marker gene,one positive transgenic plant in the T₀generation,named YL1line.There was no selective marker gene from T₂ to T₆generations.2.Evaluation of FHB resistance in transformed with F.graminearum chitin synthase 7 gene RNAi fragment lines（1）.YL1 line had significant resistance to FHB in the T₃ and T₄（grown in green house）,T₅ and T₆（grown in field）generation.The q RT-PCR confirmed that expression level of chitin synthase gene in F.graminearum was efficiently down-regulated in transgenetic lines compared to control lines.（2）.Several wheat were transformed with chs7as3as4 gene were obtained by the previous students.All the 10 lines were identified by PCR.Eight of them were homozygous and two of them were still in the stage of gene segregation.Planting all the homozygous lines into the field and injected with F.graminearum,the number Y line had significant resistant to FHB both in the T₅ and T₆generation.3.Identification of transgenic lines in gene segregation generation and FHB resistance evaluation.（1）.SYP,CZI,SZP,YB3,Z1,Z2,R2a and R2b lines were obtained by the previous students.Those lines were identified by PCR,based on the PCR results,we found SYP and CZI lines were still have gene segregation in the T₆ generation.SZP,YB3,Z1,Z2,R2a and R2b lines were homozygous.（2）.The homozygous lines were planted into the field and evaluation of FHB resistance.We found YB3 and R2b lines had significant resistant to FHB.（3）.Z5 and Z7 lines were obtained by the previous students.Both of them were identified by PCR and then based on the PCR results,selecting the positive plants as father to cross Huamai 13.Now the Z5 line is BC9F1,the Z7 line is BC8F1.4.Selection of wheat transformed with antimicrobial peptide or miRNA genes（1）.The target gene ACE,Zm GC and Ech42 were co-transformed into Y158 by gene-gun mediated transformation,pmi as selective marker gene.Only one positive transgenic plant was obtained and named YBa line.（2）.The target gene TamiR（4）and the selective marker gene pmi were introduced into Y158 by Agrobacterium-mediated transformation.One positive transgenic plant was obtained and named TR4 line.（3）.The target gene ubi-8miR7 and 35s-8miR7 were co-transformed into Y158 by gene-gun mediated transformation,pmi as selective marker gene.Nine transgenic plants were positive in T₀generation and named 8m R12a～8m R12i lines.（4）.The target gene 35s-chs3b5×2 and cmps-chs3b5×2 were co-transformed into Xiangmai76（X76）by gene-gun mediated transformation,pmi as selective marker gene.Three transgenic plants were positive in T₀generation and named 3Ba,3Bb and3Bc lines.