Transfer Of Chitinase Gene And Ribosome Inactivating Protein Gene Into Rice

Rice is one of the most important crops, but fungi diseases affect its yield and attribute seriously. It was testified that culturing the fungi-resistant varieties is the most economical and basic way. However, the conventional breeding can't meet the need of cultivating the fungi-resistant varieties because of its long period, short fungi-resistant genes and disappearing resistance with the changes of pathogen. Gene engineering is an available method to breed the fungi-resistant varieties. But there are some problems in rice engineering to resist fungi. One is that a single gene only can restrain a few varieties fungi, and the other is that it is difficult to obtain a large quantity of transgenic plants because of lower rate of transformation.In this study, four cultivars of rice were choosed. Bivalent plant expression vector with chitinase(chi) and ribosome inactivating protein (rip) gene was constructed, and then transferred into rice mediated by Agrobacterium Tumefaciens and particle bombardment. Some factors in plant regeneration and gene transformation were optimized, and plant regeneration system and gene transformation system with high efficiency were established. It could be the base of molecular breeding to resist diseases for rice and other grasses.On the other hand, it could provide gene resources for sexual hybridization breeding.The main results were summarized as follows.(1) Univalent plant expression vectors and bivalent plant expression vector were constructed.Five vectors were constructed in the experiment: One was intermediate clone vector (pUchB), one was plant expression vector cassette (p33EG), one was bivalent plant expression vector (p33ECR), and the other two was univalent plant expression vectors (p33EC and p33ECB).(2) Regeneration system of rice was Optimized.Desiccation can increase differentiation of calli of rice obviously. Differentiation efficiency of rice could be increased 4.6 times at most than control if fifty percent fresh weight were lost. Survival rate of regenerated plants were increased obviously.(3) Transformation system was optimized and transgenic plants were obtained.Bivalent plant expression vector was constructed and transformed into calli of rice.Calli of rice were not sensitive to PPT during their propagating period, but they were sensitive at differentiating period. So the concentration of PPT for selection is 0.7mg/L in the period of shoot differentiation. In the condition, 34 of 327 regenerated plants were PCR positiveafter transforming by Agrobacterium Tumefaciens, and positive rate is 10.40%. However, 8 of 561 regenerated plants were PCR positive after transforming by particle bombardment if they were screened during calli were propagated, and positive rate is only 1.43%..