| Rice sheath blight (Rhizoctonia solani) and rice blast (Magnaporthe grisea) are two serious rice diseases in China. Rice cultivars with broad spectrum, high level and durable resistance to these two pathogens are not easy to obtained by traditional breeding methods due to the deficiency of disease-resistant germplasm to R solani and the complex genetic diversity of M grisea. Using chitinase and glucanase genes to alter rice resistance to fungal pathogens could be an effective approach to solve this problem. In the previous work of our laboratory, an endochitinase gene, ThEn-42(ech42), from a biocontrol agent, Trichoderma harzianum strain P1, was introduced into rice genome. The transgenic rice TO plants showed enhanced resistance to R. solani. The genetic stability analysis of this gene and further screening of resistant lines in transgenic offspring were carried out in this research. In order to acquire higher resistance to the pathogens, a stronger promoter, Actl, was used to replace the CaMV 35S promoter and three cell wall degrading enzyme genes(ech42, nag70 and glue78) in the different combination were transformed into rice.The results obtained are as following:1. T1 transgenic rice plants with ThEn-42 were detected with PCR and analyzed with hygromycin B resistance, and the results were the same. About 70 percent of the TI lines showed the segregation ratio of 3:1 (positive : negative), indicating that these lines had single integration locus. About 20 percent of the T1 lines displayed the segregation ratio of 15:1, indicating that these lines had two integration loci. Several lines showing resistance to hygromycin B were also analyzed by Southern blotting. The results further confirmed that ThEn-42 could be stably inherited toT1 generation. The PCR analysis and hygromycin B resistant analysis of some T2 plants also demonstrated that ThEn-42 gene had stably inherited to T2 generation.2. Comparing with the controls, most of the transgenic rice offspring lines showed enhanced resistance to .R. solani K(?)hn and M. grisea in greenhouse or nursery at different level. The selected Hne-Tp64 was even completely resistant to M. grisea but didn't show enhanced resistance to R. solani. The results revealed that the endochitinase gene ThEn-42 could be exactly expressed in transgenic rice and play an important role in increasing the disease resistance.3. In order to attain rice with a higher resistance against the fungal pathogens, three different cell wall degrading enzyme(CWDE) genes, ech42, nag70 and gluc78, from T. harzianum P1 were placed respectively downstream of Act1 promoter. Based on this work, 7 different plant expression vectors were constructed by cloning each gene alone or either two genes in combination or three genes together into binary vector pCAMBIA1305.2. The constructed vectors also carried HPT gene and Gus gene which was already placed in two sides of the multiple cloning sites of binary vector pCAMBIA1305.2, that provided a suit of systematic materials for further study on the influence of T-DNA size, gene combination and gene direction on transformation frequency and gene expression.4. Seven combinations of CWDE genes were transformed into Oryza saliva L ssp. Japonica cv. Ishikari-shiroge respectively mediated by Agrobacterium tumefaciens. More than 1,800 independent regenerated plantlets of all seven populations were obtained. PCR and Southern blot analysis of part of transgenic plants revealed that about 96 percent of them integrated with at least one of three exogenous genes, more than 80 percent had intact exogenous fragment, and most of the transgenic plants had one insertion site. Differentiation and regeneration frequencies in transformation process appeared a decreasing tendency along with the increase of T-DNA size of the different vectors. The differentiation and regeneration frequencies of vectors carrying gluc78 gene were significantly low, indicating that gluc78 gene had negative effects on calli growth.5. Survival of tr...
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