| Wheat scab is an important disease in wheat production in china,which has seriously happened in the middle and lower reaches of the Yangtze River,the northeast china. However,wheat breeding for scab resistance has developed slowly,and the application of transgenic technology will play an important role in wheat breeding for scab resistance.In this research,disease resistance genes NPR1、MsrA1 and Pep3 were used for monocotyledon expression vectors construction and vectors contraining NPR1 and antimicrobial peptide-antibody fusion protein ACE-D2 were transferred to wheat cultivars mediated by Agrobacterium,with bar and pmi as the selection marker genes.After selection,transgenic wheat plants were obtained.The main results are followings:1.Wheat expression vectors pTRAkc-BAR-NPR1,pTRAkc-BAR-MsrA1 and pTRAkc-BAR-Pep3 were constructed successfully and plasmids of pTRAkc-BAR-NPR1, pTRAkc-BAR-ACED2 and pTRAkc-BAR-PMI were transferred to Agrobacterium GV3101.2.ACE-D2 gene was transformed to three wheat cultivars successfully and 54 PCR positive plants of Yangmai 158,17 of Zheng 9023 and 10 of Huamai 13 were obtained. The transformation efficiency were 2.33%,1.36%and 0.44%.Some transgenic plants of T1 generation were identified by PCR and Southern blotting.The results confirmed that the foreign gene was intergrated into wheat genome and inherited to filial generation.3.The effects of wheat genetypes,preculture time of calli and high osmotic treatment to transformation efficiency were studied during the process of transformation. Comparing differences of the three cultivars in callus induction efficiency,regeneration efficiency and transformation efficiency,the results indicated that three cultivars had no big difference in callus induction efficiency,but the status of calli from Yangmai 158 and Zheng 9023 immature embryos were better than Huamai 13,also the regeneration efficiency and transformation efficiency were higher,and the Yangmai 158 was the best in all of three.Transformation efficiency of Yangmai 158 was compared after calli precultured 3 d,10 d,20 d and 30 d,and transformation efficiency was higher when calli precultured 20 d were transformed.High osmotic treatment on Yangmai 158 calli enhanced transformation efficiency for 0.44%.4.PMI/mannose selection system and BAR/PPT selection system were compared with Zheng 9023 10 d and 18 d precultured calli as transformation receptors.The results showed:mannose as the selective agent was less harmful for calli and the selection efficiency of mannose was apparently higher than PPT selection.Therefore, PMI/mannose selection system has great application prospects in wheat genetic transformation. |
