Molecular Detection And Insect Resistance Evaluation Of Polymeric Insect - Resistant Gene And Cry2A * And Cry1C * Early Japonica Rice

Rice is one of the most important food crops in the world, more than half of the world’s population on rice as the staple food. Insect pests pose a serious threat to rice production which causing huge economic losses every year. Bacillus thuringiensis(Bt) is a gram-positive bacterium, it can produce the crystal protein for lepidoptera and other insects with specific toxicity. In order to prevent and control of lepidoptera insects, many Bt genes has been successfully transferred into rice, there has achieved many transgenic rice lines with insect-resistant properties at the present time.In this paper, bivalent Bt insect-resistant rice Kongyu 131 and Songjing 9 as the research objects, using the method of PCR, real time PCR, ELISA and Southern blot, studied at the level of DNA, mRNA and protein, from which to choose the genetic stability, low copy number and high Bt protein expression, significant effect of indoor insect resistance of the transgenic rice lines. The main results are as follows:1. Using the EHA105(pbar-cry2A*) and EHA105(pbar-cry1C*) of agrobacterium tumefaciens to infect the Songjing 9, obtain co-transformed plants which contain cry2A*/cry1C*/bar; The Kongyu 131(cry2A*/bar)and Kongyu 131(cry1C*/bar) as the parent, through sexual hybridization to obtain transgenic plants. Using Basta resistance screening, strip testing, PCR, Southern blot and other methods, after T0 ~ T7 generation of screening, obtained 60 bivalent Bt insect-resistant rice lines(cry2A*/cry1C*/bar) successfully, including 10 lines of Kongyu 131 and 50 lines of Songjing 9.2. Real time PCR and ELISA test results show that even the same organization of different lines, the transcription level of Bt gene is differences; in the meanwhile, The same line of different organizations, there are significant differences of Bt gene transcription level; In addition, at the transcription level, cry2A* gene transcription level obviously higher than cry1C*. Enzyme-linked immunosorbent assay(ELISA) result showes that the expression of Bt protein is positively correlated with the transcription level of Bt gene. The same line of Bt protein content in different tissues from high to low in turn for leaf > ear > stem-sheath > brown rice.3. Indoor insect resistance result shows that higher protein content of Bt lines which induced more high fatality rate of stem borer larvae. Southern blot result shows that after continuous passages, the genetics to be stable of foreign genes, and the copy number of the exogenous gene is relatively low, most of the lines between 1 and 2, at the same time, single copy transgenic lines showed more stable, high insecticidal activity, The cultivation of new materials has the vital significance for breeding.4. This experiment using the inverse PCR method to amplify cry2A* flanking sequence of 2-1 lines of transgenic rice lines which shows a better insect resistance and cotains a single copy, through the NCBI database to determine which is located in no. 2 chromosome.