Construction Of Bivalent Plant Expressed Vector Of The NPR1and HrpZpsg12Genes And Its Transformation In Soybean

Soybean (Glycine max), as one of the most important crop for food and oil in the world, isthe main source of vegetable protein and edible oil. It has important economic values and greatmarket demands. But the yield of soybean is decreased obviously by the disease during theirgrowth and development.The soybean phytophthora root rot caused by phythophthora sojae isone of the most destructive diseases. It can lead to the yield of susceptible cultivars decreased by50%~100%. The Cercospora sojina Hara caused by fungus can make the soybean yielddecreased by12%～15%in general years and by30%in severe occurrence years.The loss canbe as high as50%in some especially serious years. The direct economic losses caused by allsorts of soybean disease can reach hundreds of millions yuan at the most serious time.There are mainly two methods of the prevention of soybean disease at present.They are theprevention and control of chemical pesticides and breeding of disease-resistant varieties. Thecontrol effection of chemical pesticide is obvious, which improving the efficiency of agriculturalproduction to some extent, but the long-term excessive use of chemical pesticides may pollutethe atmosphere, water and soil, and upset the ecological balance. In order to solve the diseaseproblem, the most fundamental and most effective way is to cultivate the broad-spectrumdisease-resistant varieties. Traditional breeding methods have many limitations, such as longbreeding cycle, limited genetic resources, limited resistance genes and so on, which limiting theapplication of crop resistance. With the development of the genetic engineering research, theimprovement of resistance varieties has been applied in many crops by transgenic technology.The most effective way to improve the resistance of soybean is the combination of transgenictechnology and traditional breeding methods.The current study found that NPR1and HrpZpsg12genes can effectively resists soybeandiseases. NPR1gene (nonexpressor of pathogenesis-related genes1) is an important regulator toimprove the plant disease resistance. It can regulate many kinds of plant disease resistancemechanisms and control the broad-spectrum resistance. HrpZpsg12genes encoding Harpinprotein derived from soybean bacterial spot pathogen. It can stimulate plant to resist diseases,including viruses, bacteria, fungi and so on.The bivalent resistance genes of plant expression vectorpCAMBIA3301-NPR1-HrpZpsg12was built by genetic engineering technology.，The methodsof agrobacterium-mediated and the pollen tube pathway are utilized to inject the plants expression vector which contain NPR1genes and HrpZpsg12genes into soybeans. After thescreening tests, the transformed plants will be detected by PCR detection, Southern-blot,Real-time quantitative PCR, expecting to have a new gene-modified soybean which hascomprehensive ability of disease-resistance.The main results were as follows:(1) The plant expression vector pCAMBIA3301-NPR1-HrpZpsg12was successfully obtainedusing the basic expression vector pCAMBIA3301-NPR1and pCAMBIA3301-HrpZpsg12provided by our laboratory, which replacing gus gene for HrpZpsg12gene.(2) Agrobacterium-mediated method was used to obtain4T0generation of gene-modifiedJILIN30soybean which consist of NPR1and HrpZpsg12genes; the method of pollen tubepathway was used to get814seeds of T0generation of JINONG28. All above T0generationseeds were cultivated to get the T1generation.(3) Soybean strains were transformed by Agrobacterium-mediated method, got4strains and69grains of T0transformed soybean,37strains and406grains of T1transformed,31strains and414grains of T2transformed. By pollen tube pathway, got21strains and428grains of T1transformed,12strains and237grains of T2transformed.(4) PCR, Southern blot and Real-time quantitative PCR results showed that the transgenic plantswere obtained successfully. However, to detect whether the NPR1and HrpZpsg12genes couldinherit and have comprehensive ability of disease resistance, further disease resistance andagronomic traits detection of subsequent generation are needed in order to obtain soybeanstrains of efficiently broad-spectrum disease resistance.