Screening And Identification Of Wheat Plants Teansformed With Small RNA And Antimicrobial Peotides Genes

Wheat is a main food crop world-wide.Fusarium Head Blight（FHB）is a great threat to wheat production and food safety in China and many other countries in the world.Because of lacking of natural resistance germplasm against wheat FHB,the progress of traditional breeding wheat varieties resistant to FHB is slow.With the development of genetic engineering and molecular biology,genetic engineering breeding provides a new method for generating wheat varieties with high efficiency,persistence and safety.In this study,miRNA and RNAi genes with FHB resistance were transformed into wheat varieties Yangmai158（Y158）and Xiangmai76（X76）by Agrobacterium-mediated transformation and particle bombardment.These small RNA molecules target growth-related genes of Fusarium graminearum（Fg）.The main purpose is to obtain multiple types of transgenic lines in order to provide new materials for breeding wheat varieties with improved FHB resistance.Main results are as follows:1.Transformation and identification of wheat with RNAi fragments and FHB resistance assay.（1）By Agrobacterium-mediated transformation,RNAi fragments of Fg chitin synthases gene Chs7 and marker gene Pmi were transformed into Y158.For the gene lem2-Chs7As3-Chs7As4,four transgenic lines were obtained and named from S1 to S4.PCR identification showed that transgenic genes can be stably inherited from T₁ to T₄.We also identified transgenic materials without marker gene.For the gene ubi-cmps-Chs7As3-Chs7 As4,seven transgenic lines were obtained and named from U1 to U7.Single-floral inoculation indicated that FHB resistance of T₁transgenic line was significant increased compared with that of non-transgenic control Y158.（2）By particle bombardment,RNAi fragments of Fg glucan synthase gene-Gls and Myosin gene-Myo2 were transformed into the X76 together with marker Pmi gene.These lines are in T₂ and T₁ generations.Transgens can be stably inherited.（3）Selection and identification three transgenic lines that was transformed with RNAi-Pkc-Chs3b genes.PCR analyses showed that all genes were still segregating.Single-floral inoculation indicated that FHB resistance of T₇transgenic line L was significantly increased compared with that of non-transgenic control X76.（4）Selection and identification three transgenic lines that was transformed with RNAi-Pkc-Chs7 genes.PCR analyses showed that all genes were inherited to T₅ generation.The transgenic lines with promoter ubi were hybridized with and transgenic lines with promoter lem2,and the PCR identification indicated that F₁ hybrids obtained parental genes.2.Transformation and identification of wheat with antimicrobial peptides genes and FHB resistance assay.（1）Eleven transgenic lines were transformed with Hvrip and Bar genes.PCR analyses showed that all genes were inherited to T₄ generation.Single-floral inoculation of T₄ transgenic lines（Y158 background）indicated that FHB resistance was significantly increased compared with that of non-transgenic control Y158.T₄ transgenic lines（Z9023 background）did not have significant resistance compared with non-transgenic control Z9023.（2）Five transgenic lines that were transformed with Hvglu and Bar genes.PCR analyses showed that these genes were inherited from T₂ to T₄generations.Three transgenic lines have no marker gene.Transgenes were linked to marker gene in two transgenic lines.3.Transformation and identification of wheat with miRNA genes that are resistant to FHB.Different promoters were combined with miRNA fragments that were derived from FHB resistance genes,which were used to transform X76 and Y158 together with marker gene Pmi.After screening and identification,eight positive T₀ plants with six different combinations of transgenes were obtained.Details are as follows:Transgenic line 7A2 contained Lem1-8mR7,Lem2-8mR7,cmps-8mR7 and ubi-Pmi,and these materials are in T₁ generation;Transgenic line mR1 contained ubi-8mR7,35SN-8mR7 and ubi-Pmi.These materials are in T₂ generation;Transgenic line 8C contained ubi-8mR7,cmps-8mR7 and ubi-Pmi.These materials are in T₁ generation;Transgenic line 11D1,11D2 and 11D3 contained Lem2-8mR7,cmps-8mRFD10787,ubi-8mR12-47 and ubi-Pmi.These materials are in T₀ generation;Transgenic line 11R1 contained Lem1-8mRF06326,35SN-8mRF08568,ubi-8mR12-47 and ubi-Pmi.These materials are in T₀generation;Transgenic line11E1 contained Lem2-8mR7,ubi-8mRFD10787,cmps-8mR12-47 and ubi-Pmi.These materials are in T₀ generation.