Study On The Transgenic Wheat With Fusarium Head Blight Resistance-related Genes By Particle Bombardment

Wheat is one of the widely cultivated crop plants in the world, whose yield and area rank forefront of the crops. Fusarium head blight(FHB) is a devastating fungal disease of wheat worldwide, which is primarily caused by Fusarium graminearum.The pathogens that infect wheat result in the yield reducations and the accumulation of mycotoxin in mature grains, which will threat to the health of humans and animals. Conventional breeding is an important approach to improve agronomic traits and FHB resistence of wheat. Since the germplasm resources are scarce, it is difficult to breed wheat varieties with FHB resistence by conventional approaches. With the development of plant genetic engineering technology and the improvement of the plant tissue culture technology, it is a brand-new way to create the FHB resistance and enrich the FHB resistance germplasm resource with transgenic method.In this study, immature embryos of Zhengmai 9023, Yangmai 158 and Xiangmai 76 were used as the transformation receptors. We obtained the transgenic plants with FHB resistant genes via particle bombardment. Bar or PMI were used as the selectable marker gene and transgenic plants were seleted with Bialaphos or mannose selection system, the results of PCR proved that positive transgenic plants are obtained.The main results are as follows:1. Lem2-UEch42-D2 and Ubi-AFP2 genes were co-transformed into Yangmai 158. The minimal expression cassettes of Lem2-UEch42-D2 and Ubi-AFP2 with Bar were transformed into Yangmai 158 by particle bombardment. After Bialaphos selection and PCR identification, we obtained three transgenic wheat plants in T0 generation, the PCR identification of T1 generation showed that only one T0 line containing Lem2-UEch42-D2 and Ubi-AFP2 genes can be stably inherited. PCR identification of T2 generation showed that the alien genes can be stably inherited, and RT-PCR identification of T2 generation showed that the alien genes can express at the RNA level. The result of single-floret inoculation showed that the FHB resistance of transgenic wheat was enhanced in greenhouse condition.2. Cmps-Fen A and Cmps-Fen B genes were co-transformed into Zhengmai 9023. The minimal expression cassettes of Cmps-Fen A and Cmps-Fen B with Bar were transformed into Zhengmai 9023 by particle bombardment. After Bialaphos selection and PCR identification, we obtained one transgenic wheat plant in T0 generation containing Cmps-Fen A and Cmps-Fen B. PCR identification of T1 and T2 generations showed that Cmps-Fen B was separated and Cmps-Fen A can be stably inherited. PCR identification of T3 generation showed that the segregation ratio of target gene gradually narrow and some transgenic wheat only with target gene.3. Lem2-UEch42-D2, Ubi-Blf-A6 and Amt-Blf genes were co-transformed into Xiangmai 76. Four minimal expression cassettes including Lem2-UEch42-D2, Ubi-Blf-A6, Amt-Blf and PMI were transformed into Xiangmai 76 by particle bombardment, and the seedings were selected by mannose-containing medium. We obtained five transgenic wheat plants by PCR identification in T0 generation, and three of them carry three target genes including Lem2-UEch42-D2, Ubi-Blf-A6 and Amt-Blf, and two of them carry two target genes including Lem2-UEch42-D2, Ubi-Blf-A6. The result of PCR identification of T1 and T2 generation proved that target genes can be stably inherited in 4 transgenic wheat lines that produced seeds normally, and the segregation ratio of target genes became gradually narrow. RT-PCR identification indicated that Lem2-UEch42-D2 and Ubi-Blf-A6 were expressed in the RNA level.4. Ubi-Pkc As3-Chs3As4 and Amt-Blf genes were co-transformed into Xiangmai 76. We transformed three minimal expression cassettes including Ubi-Pkc As3-Chs3As4, Amt-Blf and PMI into Xiangmai 76 via particle bombardment. After selection by mannose selection system and identifion by PCR, 3 transgenic wheat plants were obtained, two of them carry target genes including Ubi-Pkc As3-Chs3As4 and Amt-Blf, and one of them only carris one target gene Ubi-Pkc As3-Chs3As4. PCR identification of T1 and T2 showed that the trget genes were segregated or rearranged in transgenic wheat, two transgenic lines carries Ubi-Pkc As3-Chs3As4 and one transgenic line carry Ubi-Pkc As3-Chs3As4 and Amt-Blf genes can stable inherit, the FHB resistance of transgenic wheat were detected by single-floret inoculation method, the results showed that target gens expression can enhance FHB resistance in greenhouse condition.5. Dubiubi-Chs3As3-Chs3As4 gene was co-transformed Xiangmai into 76. Two minimal expression cassettes including Dubiubi-Chs3As3-Chs3As4 and selectale marker gene PMI were transformed into Xiangmai 76, we obtained 4 transgenic wheat plants, however only the target gene from three of them can be stably passed on to its offsprings.