| Rice is one of the most important food crops of human being, which feed more than half of the people in the world. China is the largest rice producing country and its rice yield is one third of the world’s total output of rice. Sheath blight disease caused by the soil-borne fungal disease Rhizoctonia solani Kiihn is one of the most destructive diseases for rice. Under the most favorable environment conditions, rice sheath blight can cause yield losses up to50%. Gastrodia antifungal protein (GAFP), isolated from traditional medicinal plants Gastrodiae elataBl., is a broad-spectrum antifungal protein with the strong inhibit effect to many plant fungal disease. It has a greatly potential value in plant antifungal genetic engineering.In this study, by the method of Agrobacterium mediated transformation, eight vectors from4GAFP genes, were introduced into three varieties, they were R6547(Indica), Xudao3(Japonica) and the highly susceptible cultivar "Shuanggan LMNT", respectively. Finally, we obtain transgenic positive plants by adopting the technologies of tissue culture and molecular detection. For obtaining transgenic varieties or intermediate materials with resistance to rice sheath blight, the pure lines with GAFP genes were evaluated by inoculated artificially Rhizoctonia solani in field.The main results as follows:1、The verification of plant expression vectors in Agrobacterium tumefaciens.The plasmid DNA was extracted firstly from different Agrobacterium clones integrated successfully different plant expression vectors. Then they were cloned in the competent E.coli line. Some verification technologies, including the specific primer PCR, enzyme digesting and sequencing, were used to verify if the foreign GAFP genes had been integrated into the expression vector or not. The results showed that the plant expression vectors with GAFP genes had been successfully transformed into Agrobacterium tumefaciens strain AGL-1.2、Molecular detection of transgenic rice plants.The transgenic positive plants were identified by using the specific primer PCR and Southern blot in DNA level. The expression levels of GAFP genes in transgenic plants were detected by the RT-PCR. Also, we used Western Blot to detect the integration of Gastrodia antifungal protein. Finally, we confirmed the GAFP genes had been integrated and expressed in transgenic plants.3、Evaluating the resistance levels and main agronomic phenotype of transgenic lines The resistance levels of transgenic lines obtained from eight expression vectors were evaluated in field tests. The eight vectors are G1, SG1, G2, SG2, G3, SG3, G4, SG4, respectively. We used three evaluating indicators to evaluate the resistance level of transgenic plants to rice sheath blight. The results demonstrated that the resistance levels of transgenic plants with GAFP genes were significantly higher than that of control. The resistance levels between vectors with or without signal peptides also reached to significant difference level. The transgenic lines transformed SGI and SG2vector showed the highest level of resistance to sheath blight. The average disease scores of transformed SG1and SG2lines were lower1.59and1.55than that of the wild control in2013, respectively. There was no significant difference between SGI and SG2transgenic lines.In addition, we investigated partial main agronomic traits of transgenic plants transformed SGI, SG2and SG3vectors, which had better resistance to rice sheath blight. The results showed that there were no differences in the agronomic traits among of GAFP transgenic lines and wild Xudao3. So, we considered that trasforming GAFP genes were very meaningful to breed new transgenic varieties or lines against to rice sheath blight. |
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