Study On Transgenic Wheat With Antibody-lactoferrin Fusion Gene And RNAi Segments By Particle Bombardment

Wheat is a world-widely grown cereal grain crops, playing a vitally important role in the human food supply. However, because of the Fusarium head blight(FHB), wheat yield is often severely affected. Wheat scab caused by Fusarium graminearum is a devastating fungal disease of wheat in the South of China, which brought great losses of wheat production especially in the mid- lower reaches of the Yangtze River valley. Infection of wheat spikes causes not only the yield decrease, but also accumulation of mycotoxins from the fungal metabolic products grains; these mycotoxins are harmful to human and animal health. To solve the problems caused by Fusarium head blight, the traditional methods of wheat breed ing can be achieved to a certain level, but the limited resistance germplasm resources for wheat scab makes it difficult to obtain a good variety with high FHB resistance. In this study, scab resistance-related genes were transferred into wheat by particle bombardment, transgenic wheat plants resistant to scab have been seleteced, which will be used as a basis for breeding scab resistance wheat and investigation of resistance mechanisms.FHB-resistant genes includeing BLF-A6, RNAi-chs3, RNAi-pkc-chs3, pls As6-chs3 b As1, and pkc As3-chs3 b As1 have been transferred into wheat varieties Xiangmai76 by particle bombardment. Some positive transgenic plants have been molecularly identified. The primary results are as follows:1. Antibody- lactoferritin fusion gene BLF-A6 and RNAi-chs3 were transformed into Xiangmai 76. Using multi- gene co-transformation technology, minimum expression cassettes carrying BLF-A6, RNAi-chs3 and selective maker gene Bar were co-transferred to Xiangmai 76 by particle bombardment. Through bialaphos screening, a certain number of positive plants were obtained. Four T0 generations positive plants were identified by PCR, three of which in the offsprings can be stably inherited and containing both transgenes. One transgenic plant only had one target gene RNAi-chs3 in the T1 generation. These transgenes are stably inherited in the subsequent offsprings, and now in the T3 generation.2. Antibody- lactoferritin fusion gene BLF-A6 and RNAi-pkc-chs3 were used to transform Xiangmai76.Minium expression cassettes containing BLF-A6, RNAi-pkc-chs3 and PMI were co-transformd into Xiangmai 76 by particle bombardment. After mannose selection and PCR identification, two T0 generations transgenic plants which only have RNAi-pkc-chs3. This transformation material is now in the T2 generation.3. Antibody-lactoferritin fusion gene BLF-A6, pls As6-chs3 b As1 and pkc As3-chs3 b As1 were used to transform Xiangmai 76. Also minium expression cassettes containing BLF-A6, pls As6-chs3 b As1, pkc As3-chs3 b As1 and PMI were transferred into Xiangmai 76 by bombardment. After mannose selection and PCR identification, five T0 generations transgenic plants were selected, two of which 2 have three taansgenes and the remaining plants contained two genes pls As6-chs3 b As1 and pkc As3-chs3 b As1. Leaf inoculation experiments indicated these transgenic plants had significantly improved scab resistance. This batch of transformation materials are now also in the T2 generation.4. FHB resistance-related gene pls As6-chs3 b As1, chs3b-A5-i-S5-chs3b-A1-i-S1 and chs3b-A2-i-S2-chs3b-A3-i-S3 were used to transform Xiangmai 76. Similarly mediated by bombardment, both PMI and Bar gene were used as selection markers. N ine positive transgenic plants in T0 generation were generated, two of which 2 were from the mannose selection, and the remaining seven were from the bialaphos selection. Three transgenic lines did not contain transgenes based on the PCR identification of T1 generation. At present these materials are now in T1 generation.