Study on Cry30Fα1Gene in Transgenic Rice
|School||Sichuan Agricultural University|
|Course||Biochemistry and Molecular Biology|
|Keywords||rice Bt insecticidal crystal protein cry30Fa1 insect resistance protein detection|
Rice is an important crop in the world. There is a population of8hundred million taking rice as the staple food in China. At the same time, it’s the most serious pest in the crop year, harm of rice planthopper causes huge losses, the general damage is10-20%, sometimes even greater. Integrated control based on chemical control, play some role in the control of rice plant hopper harm. However, the long&wide use of chemical pesticides, generate a series of problems, such as pesticide residue, environmental pollution, insect resistance enhancement, which has become the focus of attention. With the development of biotechnology, breeding new varieties resistant ability by the means of genetic engineering has become the trend of development and the new way. At present, the exogenous gene modified crops resistance is Bt toxic protien gene mainly isolated from Bacillus thuringiensis. The main purpose of this study is to use genetic engineering methods to cultivate rice varieties of resistance to brown planthopper with independent intellectual property rights.In this study, taking excellent rice restorer line Shuhui818(R818) as receptor material, using Agrobacterium-mediated transformation method, it transfers cry30Fal gene cloned by our lab into it to obtain transgenic plants. Through the selective marker gene hygromycin resistance gene expression detection, detection of PCR, To positive transgenic plants were screened. Through the detection of gene in Ti generation plants the target gene and selective marker PCR, it screened positive plants without selective marker gene. Expression of Western was detected by Ti Hybridization Generation transgenic plant protein. Through the field observation, it collected positive transgenic plant with marker-free and good agronomic traits. Through the T2generation plants indoor feeding, it tested the preliminary determination of the resistant insect.The main results obtained in this study are as follows:1. It constructed the transformed binary vector pCDMAR-cry30.Fa7-Hyg with efficient expression, obtained233strains of transgenic plants by transforming into R818embryogenic callus, and obtained46positive strains containing cry30Fal gene by hygromycin and PCR detection.2. Through the detection of seedling leaves of transgenic plants to39lines of T1generation were1680strains of PCR gene,1049strains of the1680individuals contained the target genes of cry30Fal,631strains of them does not contain. In1049individuals with cry30Fal gene for hygromycin resistance gene detection in PCR,353individuals of them were detected with hygromycin resistance gene,696individuals were detected without hygromycin resistance gene. Test results show that, the transgenic plants in Ti generation, containing cry30Fal gene without the hygromycin resistance gene, were detected in41.43%of total plants. Genetic separation and detection of T1transgenic plants, through the x2test, plant genetic segregation of Ti generation was detected in64.10%with3:1separation.3. The determination results of protein content showed:target proteins, in the numbers1,2,3,4,10,11,12,13,17,25,29,30,31,32,33,36,37,38,39plants in T1generation have no expression or are low expressed. But expressed in T1transgenic plants are numbered as5,6,7,8,9,14,15,16,18,19,20,21,22,23,24,26,27,28,34and35, and the depth of target protein size, color reaction with the aim protein expression quantity.4. Indoor artificial feeding insect test results showed:Comparing high sensitive TN1died entirely and R818died mainly, the transgenic plants died partly. Detected by PCR, the first batch of experiments with objective gene plant survival rate was44.31%; no objective gene plant survival rate was16.52%. Insect resistance of the transgenic plants containing the target gene is2.68times than those without target gene. The second batches containing the target gene plant survival rate was48.06%; no objective gene plant survival rate was14.56%. Insect resistance of the transgenic plants containing the target gene is3.30times than those without target gene. The third batches of plants containing the target gene have the survival rate of46.91%; no objective gene plant survival rate was14.88%. Insect resistance of the transgenic plants containing the target gene is3.15times than those without target gene. The three summations show that, plants containing the target gene have the survival rate of46.41%; plants with no objective gene have the survival rate of15.34%. Insect resistance of the transgenic plants containing the target gene is3.03times than those without target gene. Therefore, in a certain range, it can be considered that the positive transgenic plants have the resistance to brown planthopper.