The Constructon Of Bivalent Plant Expression Vector With Two Broad-spectrum Disease Resistance Genes Of Chi And HrpZpsta And Transformation In Soybean

Soybean (Glycine max) is an important oilseed crops in our country, rich in protein and oilseeds, nutrients completely, including minerals and vitamins. But, fungal diseases have severelylimited the improvement of soybean yield and quality in production. For example, as animportant disease of soybean duction areas, gray leaf spot can reduce yield by10%in general,even up to30%~50%when disastrous outbreak, while the protein and oil contents of grainreduced in varying degrees. In addition, both soybean downy mildew and soybean rust causedby fungal pathogens are the main factors limiting soybean production.In recent years, the development of plant genetic engineering technology provided a quickand effective way for crop resistance breeding. Chitin (also known as chitin) was majorcomponents of the fungal cell wall. Chitinase encoded by Chitinase gene from kidney bean cancatalyzes the degradation of chitin glucosidase and hydrolysis of β-1,4glycosidic bond,therefore it can inhibited the growth of fungus.So chitinases play an important role in thedefense system of plants. The hrpZpstagene from mutant of tobacco Pseudomonas syringaecodes harpin protein. With pollen tube way, transformed foreign gene hrpZpstainto the differentspecies of soybean, The protein can stimulate an allergic reaction in higher plants, which couldcasue the necrosis of infected region and surrounding cells, and protect plants against pathogensinfringement purposes. The study constructed two different resistance mechanisms andbroad-spectrum resistance genes bivalent plant expression vector pCAMBIA3301-chi-hrpZpstaincluding chi and hrpZpsta. Two broad-spectrum resistance gene were transferred into the soybeangenome by agrobacterium-mediated soybean cotyledon method, which expected to achieve twobroad-spectrum disease-resistant genes in the cell level and extracellular levels of synergyeffects in bacteria,as well as laying the foundation for new soybean that have strongerbroad-spectrum disease resistance and wider disease-resistant.The results are as follow:1. This research constructed two vectors, including a intermediate cloning vectorpBI121-35S-chi-nos and bivalent broad-spectrum antiviral gene expression vectorpCAMBIA3301-chi-hrpZpstathat was based on glufosinate herbicide resistance bar gene asselectable marker. The resistance genes including chi and hrpZpstawas transformed andintegrated into soybean species Jinong28by making use of agrobacterium-mediated method. 2. Positive plants had been gained through screen. PCR, southern blot hybridizationtechniques had been performed to detect the integration of the exogenous anti-disease genes.qRT-PCR was conducted to detect the expression level of the gene. According to the molecularexperiment, chi and hrpZpstawere integrated to genome of transgenic plants successfully. Thegenes expressed nomarly in the plant and could be inheritanted to next generation.3. Transgenic soybean plants was transformed two exogenous genes, which obtained7strainsPCR-positive plants of T0generation, including4strains chi gene of PCR-positive plants and3strains chi-hrpZpsta of bivalent PCR-positive plants.27strains were detected PCR-positiveplants by molecular techniques, which contains15strains chi gene of PCR-positive plants and12strains chi-hrpZpsta of bivalent PCR-positive plants. Therefore, exogenous bivalent geneshad been transformed to the genome of soybean receptor successfully, and it has been proved tobe stable after several subcultures.