Identification On Insect-resistant Gene Of Cry1Ab13 Transformation Into Glycine Max L. Merr

Soybean is the main source of edible oil in China, and it is also an important plant protein resource, which has great potential for development. Soybean pest is an important factor that affects the yield and quality of soybean, to express the insect resistance genes in plants by using transgenic technology is one of the important ways to control pests synthetically.Bacillus thuringiensis(Bt) is a microorganism with high toxicity to a variety of Lepidoptera pests. It can form a specific insecticidal crystal protein(crystal proteins Insecticidal,referred to as ICP) when the spore formed. After the insects were fed on ICP, the activated ICP combined with the specific binding proteins in the insect’s gut, then all or part of ICP integrated into the cell membrane and forced the cells to produce some holes or ion channels, caused the cell rupture because of the damaged osmotic pressure. As the process occurs, the insects will no longer eat and finally die. Therefore, Bt is widely used in plant insect resistance breeding.Up to now, there are 42 kinds of cry1 Ab genes, and the amino acid sequence homology of ICP is between 75% and 95%. The cry1Ab13 gene which used in this text is one of the cry1 Ab genes, but it had not been used in soybean. In this text the pCambia3300-35s-cry1Ab13 plant expression vector with herbicide as screening marker was constructed by using the obtained cloned vector. Through pollen tube pathway method and Agrobacterium mediated transformation, the plant carrier was transferred into the receptor soybean "Ji Nong 28".The positive plants were ascertained by PCR detection, then positive for Southern blot, quantitative PCR, pest identification to verify the function of the gene and screen the fine lines of transgenic insect resistant which can be stably and efficiently expressed, provided the basis for breeding new insect resistant transgenic soybean varieties.The main results of this experiment are as follows:1. The plant expression vector pCambia3300-35s-cry1Ab13 with Bar gene as screening marker was constructed by using the sub-clone technique.2. Through pollen tube pathway method the vector was transferred into the soybean variety of "Ji Nong 28", 15 T1 positive plants were detected by PCR, the conversion rate reached 2.8%.And 4 T0 generations of transgenic plants were obtained by using Agrobacterium mediated method, the positive rate was 16%.3. 15T1 PCR positive plants were identified by Southern blotting. When cry1Ab13 gene was used as hybridization probe,the results showed that 5 strains appeared hybridization signals and integrated into the soybean genome in the form of the single copy.4. The results of the quantitative PCR dedicated that the cry1Ab13 gene in the transgenic plants were expressed in both stems and leaves. The expression of cry1Ab13 gene were different in each transgenic plants, and the expression in leaves was higher than that in stems.5. Inoculated soybean pod borer larvae by disk division, preliminary identified insect resistance of T1 generation positive plant seed, the results showed that the insect resistance of the transgenic plants was significantly increased, and it had obvious inhibiting effect on the growth of the non-transgenic soybean grain.