Cloning Of New Insect Resistant Gene Cry1AB13-2 And Its Genetic Transformation And Functional Identification In Soybean

Soybean production in China is seriously affected by insect pests every year.Insect pests can not only reduce the yield of soybean production in large area,but also have great influence on the quality of soybean.The traditional chemical methods for pest control not only cause serious pollution to the environment,but also affect the food safety of soybean.Therefore,the molecular breeding methods of genetically modified organisms are beginning to be favored by the breeders.Among them,Bt?Bacillus thuringiensis,Bt?insect resistant gene was first used in crop breeding,and good insect resistance was achieved,and it was quickly commercialized.Through transgenic technology,the Bt insect resistant gene was introduced into the crop to show its insect resistance.The method of breeding the insect resistant varieties by molecular technology to achieve the insect resistance is less harmful to the environment than the traditional chemical insecticidal method.In this experiment,the Cry1AB13-2 gene was cloned by error-prone PCR and constructed into an expression vector,which was successfully introduced into the receptor soybean variety“JN 28”,and its insect resistance function was identified.The test verified Cry1AB13-2 new insect resistant gene to soybean pod borer has significant insecticidal activity.In order to provide data reference for the selection of transgenic insect resistant varieties,the fine lines of resistance to Cry1AB13-2 gene were selected through the identification of insect resistance.The main results obtained in this experiment are as follows:1.By error prone PCR Cry1AB13 plasmid DNA induced mutation,finally cloned a novel gene of Cry1AB13-2.2.ThecloningvectorpMD-18T-Cry1AB13-2andtheexpressionvector pCAMBIA3300-35S-Cry1AB13-2 were constructed.Agrobacterium-mediated method and pollen tube pathway method were used to successfully introduce the plant recombinant overexpression vector into the receptor soybean variety“JN 28”.Five T₀-positive plants and 18T₁-positive plants were obtained by routine PCR.3.Using the Bar gene as a probe,the transgenic plant of Agrobacterium mediated transformation and pollen tube pathway shared by Southern blotting identification,of which 12strains showed hybridization signal,and in the form of single copy integration to the"JN 28"soybean genome.4.Fluorescence quantitative PCR detection showed that the target gene Cry1AB13-2 was expressed in 3 parts of root,stem and leaf,and the average relative expression of 3 target genes in each part was 1.64,3.34 and 6.87 respectively.It can be seen that the gene in root,stem and leaf of 3 parts of relative expression showed increasing trend,while the highest expression level in leaves.5.Indoor insect resistance identification showed that Cry1AB13-2 transgenic plants in the pod of Leguminivora glycinivorella survival was significantly lower than the number of live insects"JN 28"receptor material quantity and receptor material compared to transgenic plants for Spodoptera litura?Fabricius?susceptible index higher.6.Outdoor insect resistance identification results showed that the transgenic plants herbivory rate was 3.23%,belonging to the level of insect resistant?R-resistant,2-4%?;non transgenic plants herbivory rate was 6.72%,belonging to the susceptible level?S-susceptible,6-10%?.