Cloning NBS Resistance Gene Analogs In Soybean And Preliminary Study Of Genetic Transformation

Soybean is an important high-protein feed and oil crops, which is also a kind oftraditional food in china. Nowadays，it is very popular in our life attricuting to thekind of soybean producs is extremely various and becomes an indispensable food.However，a variety of plant diseases and insect pests exert an influence on its yieldand quality. Moreover，there is a huge challenge to native non-GMO(non-GeneticallyModified Organism) soybean because of a large number of foreign transgenic soybeaninvasion. The nucleotide-binding site (NBS) being conservative structural domainlocates in some plant disease resistance protein, providing the probability that theNBS disease resistance genes are cloned from soybean. Meanwhile, it is quitesignificant that NBS resistance genes are isolated from some strong of diseaseresistance, high protein soybean and transformed into the other genetypes of breed,which contributing to resistant breeding of soybean, the linkage mapping of resistantgenes, make clear the function of NBS structural domain and the mechanism ofdisease resistance. Our research results are as follows:First, the homologous fragment of NBS resistant gene was cloned. According to theconservative structure more than100fragments from the cDNA of Hunan and Fujianblack soybean by three degenerate primers. Sequence analysis shows two fragmentsnamed FNBS1and FNBS3can be read and own NBS conservative structural domain.The size of FNBS1is534bp and encodes181amino acid, including p-loop, kinase-2,HD. While the size of FNB3is513bp and encodes169amino acid, including p-loop,HD. They have a NB-ARC structural domain (Associated with disease resistance inplants). In addition, the homology of FNBS1with RPM1is97%, and the homologyof FNBS3with TMV protein is99%.Second, the system of agrobacterium-transformed soybean is optimized. In theexperiment, we research some factors (genetypes, different organs, antioxidants) exertan influence on transformation and optimize the transformation system of soybean.Compared to the cluster bud ability of3genetypes, the results showed that the cluster bud rate of Hunan green soybean reached to60%and the average cluster bud capacityis more than3. Compared to different organism of Fujian green soybean, the resultsdiscovered that the cluster bud rate of cotyledonary node reached to36%. Theexperiment which the effect of antioxidant on transformation rate, indicates additionappropriate amount of antioxidant could boosted the instant expression rate of GUS,ranging from15%to43%，and effectively inhibited wound browning and the death ofthe organization.Third, the homology fragment of NBS resistant genes of soybean was transformedinto tobacco and soybean. Constructed the plant expression vector of pFGC5941-NBSand transformed into soybean and tobacco by agrobacterium,2soybean transgenicpositive strains and22tobacco transgenic positive strains were gained, themtransformation rate was1.08%and45.6%. The experiment of the herbicide daubtobacco found that the ability of herbicide resistance was obviously higher transgenicpositive strains than black plant.