A Study On Transformation Of The New Bt Gene Into Indica Rice By Agrobacterium Tumefaciens-mediated

Rice is one of the most important grain crops in the world, for about40%of the world’s population taking it as their staple food. However, insect damage is one of the main reasons to decrease the rice yield. There are a large variety of rice pests in China. The rice production loss is more than5%of total yield because of insect damage. At present, the main method to control rice pest is to utilize chemical pesticides. However, it caused a lot of problems, such as environmental pollution, higher production cost, food safety, etc. Breeding new rice varieties that produce insecticidal proteins by themselves is undoubtedly the most economical and security strategy. On the basis of previous reports, it is difficult to solve the problem by traditional breeding because of rice germplasm resources are scarce. Given this, it is more important to use genetic engineering to breed resistant rice.In recent years, pest resistant rice with Bt genes have already been on a wide range of research. With large-scale cultivation of insect-resistant transgenic rice, Bt gene insect-resistant spectrum is relatively narrow and tolerance to pests and other problems have been attracted the attention of people. Therefore, separating and cloning new pest resistant genes from the natural world become more and more effective methods to solve these problems.Indica rice is a major subspecie of Asian cultivated rice, and more than80%of the rice cultivars in the world belongs to indica rice, so it is important significance to study tissue culture. Tissue culture of indica rice is generally poor and regeneration rate is very low, so that a large varieties with some production value is still difficult to successfully transform or frequency low. Therefore, the study of high quality indica rice callus formation and plant regeneration ability of genetic transformation is necessary.Agrobacterium tumefaciens-mediated transformation has technique advantages including low copies, and has been widely used in the study of insect-resistant transgenic rice. However, the relatively large genetic differences between different varieties of rice, and the transformation strong dependence on rice genotypes, increase complexity and randomness of the Agrobacterium tumefaciens-mediated genetic transformation in rice. Therefore, the optimization of Agrobacterium tumefaciens-mediated rice transformation system to improve transformation rates is necessary.This research did the following work:1. Sequences of new Bt genes Cry30Fal and Cry54Aal that had been added cleavage site sequences at both ends, were sent to gene company to synthesis. Then all of target gene fragments and middle cloning vector pUPROK were cut separately by two enzymes and connected to form middle carriers of the target gene fragments. Positive vectors were got by sequences identification, and were digested by endonuclease to separate target gene fragments. Then target gene fragments were inserted into pCDMAR-Hyg vector to form eventual expression binary vectors pCDMAR-Cry30Fal-Hyg and pCDMAR-Cry54Aal-Hyg. All of positive plasmids were identified by digestion and sequencing.2. By optimizing callus subculture times and different concentration of Ascorbic acid (Vc) in subculture media, callus growth state was adjusted and the number of embryo callus was increased. Results showed that when the callus of R125and R818were subcultured2to3times, the differentiation rate was the highest; And subculture media with40mg/L Vc could reduce the rate of R818callus browning with no effect on their differentiation capacity.3. By drying callus after washing Agrobacterium and before differentiation, the system of Agrobacterium tumefaciens-mediated transformation was improved. Results showed that after washing Agrobacterium and the caulls were dried2days on filter papers before regeneration, differentiation speed was accelerated and differentiation rate was increased.4. Agrobacterium tumefaciens-mediated transformation of exogenous genes Cry30Fal and Cry54Aal into caulls of rice restorers R125and R818, then identified gentic plants by PCR and Hyg B solution. As a result, R125was no transgenic plants; R818was only transformed into the gene of Cry30Fal. There were233transgenic plants, but only46plants of them were positive transgenic plants identified by PCR and Hyg B solution.