Studies On The Transformation Of Wheat With Defense Genes Resistant To FHB By Microprojectile Bombardment

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of wheat in the warm and humid regions around the world. It not only causes quality and yield loss, but also produces a variety of mycotoxins which threat to human and animal health. Due to conventional breeding techniques are time-consuming and natural germplasm resource is deficient, the progress of improve FHB resistance is slow. Genetic engineering has thus been proposed to improve FHB resistance by transform exogenous defense genes into wheat.In this study, using the bar and pmi selection systems, minimal cassettes of defense genes were introduced into two different wheat elite cultivars Yangmai15 and Zhengmai9023 by biolistic co-bombardment. Several positive transgenic plants were obtained through identification by PCR、Southern blot analysis and RT-PCR. The main results were as follows:1. Immature embryos of two different wheat cultivars Yangmai15 and Zhengmai 9023 were bombarded, and comparative analyses of these two cultivars indicated that Zhengmai9023 has a higher efficiency of regeneration and transformation. These results suggested that immature embryo of Zhengmai9023 is more suitable for wheat transformation by microprojectile bombardment.2. With bar selection maker gene, antibody fusion genes Ech42-D2 and AFP2-D2 were used to co-transform wheat cultivar Yangmai15. One positive transgenic plant containing Ech42-D2 gene was obtained in To generation. PCR identification of this transgenic line from T1 to T3 generation showed that the transgene was inherited stably. RT-PCR analysis indicated that the transgene can express at the RNA level.3. With bar selection maker gene, a trichoderma chitinase gene UEch42、an antimicrobial peptide gene D4E1 and the antibody fusion gene AFP2-D2 were co-transfer into wheat cultivars Yangmai15 and Zhengmai9023.18 positive transgenic plants were obtained in To generation. Thereinto,5 transgenic plantlets were obtained from Yangmai15 and 13 transgenic plantlets from Zhengmai9023. PCR identification of these transgenic lines in T1 and T2 generation showed that most of the lines lose their transgenes, except that one transgenic line containing D4E1 and AFP2-D2 genes from Yangmai15 and another transgenic line containing UEch42 gene from Zhengmai9023 were inherited stably. Southern blot analysis and RT-PCR identification of these two stably transgenic wheat showed that transgenes had integrated into the wheat genome and expressed at the RNA level.4. With pmi selection maker gene, two chitinase genes Ech42, Hvchi and one antimicrobial peptide gene AFP2 were introduced into wheat cultivars Yangmai15 and Zhengmai9023. Only one transgenic plant containing Hvchi and AFP2 genes was obtained in To generation. However, the transgenes were lost by PCR identification in T2 generation.5. To further investigate the response of transgenic plants to FHB, the stable transgenic lines described above were subjected to single-floret inoculation with the macroconidia of F. graminearum. The results showed that some of the transgenic plants displayed a significantly enhanced resistance to FHB.