The Study On Rice Transformation Of Recombined Bt Genes Derived From DNA Shuffling Library

Rice is one of the most important crops and the main food for two billions people of the world. Insect damage is the major cause of yield loss. The chemical pesticides have significantly contributed to decreasing yield losses caused by the pests in modern agriculture. However, the long-term employment of synthesized pesticides has inevitably led to the environmental pollution and human health problems. As a novel pest control technology, genetic modified (GM) insect-resistant crops began to grow on a large scale.Currently Bt gene is the most widely used, and the toxic protein can damage the insects in the intestinal epithelial cells, with a high degree of specificity of the resistance. However, long-term use of Bt insecticide produced tolerance of target insects to Bt toxic protein, natural Bt gene is not always able to achieve a good insecticidal effect. In order to solve this problem, we use DNA Shuffling to restructure the Bt gene technology, and getting a batch of clones. In vitro toxicity bioassay showed that the toxic of recombinant Bt genes improved greatly than the original Bt genes.On this basis, fourteen recombinant Bt genes were transformed into a japonica rice ZhongHua11 by Agrobacterium-mediated transformation methods in order to express efficient insecticidal protein. A number of transgenic lines with single insertion copy and good resistance were obtained. The main results are as follows:1. After the reorganization of Cry1Ab, Cry1Ac, Cry1C, Cry9C and Cry2A, we selected 14 sub-gene (named 722, 723, 724, 738, 906, 983, 385, 932, 936, 185, 897, 977, 901, 119, respectively) and constructed plant expression vectors. Then these vectors were transformed into ZhongHua11 callus, using Cry1Ab, Cry1Ac and Cry2A as a control. We obtained 80 resistant transgenes per construct.2. Through PCR analysis , about 75% of these transgenes are positive.3. The copy numbers of T₀ transgenic lines were detected by Southern blotting, ranging from 1 to 12.4. RNA expression levels of T₀ transgenic lines was detected by Northern blotting.5. Insect bioassay in the laboratory showed that part of transgenic lines exhibited high resistant against main rice insect pests.6. Analysising 14 Bt genes by ClustW software, we found that they showed above 90% homology with original genes and the chang happened in domain 1 or domain 2.