Study On The Construction Of Transformation Vector With PEAS Gene And Its Agrobacterium-mediated Rice Transformation

Phytoalexin, which is a kind of secondary metabolite with low molecular weight, is synthesized by plants when induced by biotic elicitors (eg.pathogens) or abiotic elicitors and its production is one of plant pathogen resistant mechanism developed during the long course of plant evolution. With genetics engineering technology, Phytoalexin gene could be transformed into target plants and the transgcnic plants could possibly synthesize heterogeneous phytoalexin, and thus improve its resistance to diseases, which is very likely to be efficient strategy for improving plant resistance to disease in plants genetics engineering.In this study, the PEASC (Pepper epi-aristolochene synthase cDNA) gene, which is cloned from pepper, was constructed to plasmid vector pGA1621.0n the other hand, high frequency Agrobacteriums-mediated genetic transform system of rice, involved in this experiment, was developed. With the expression vector and the high frequency genetics transform system, Agrobaclerium-mediated rice transfromation was conducted and transgenic rice plants were acquired. With the transgenic rice plants, it is possible to further evaluate practical value of PEASC in crop pathogen resistance genetic engineering. The main results of this study are as follows:(1) NB callus-inducing medium, which is good for rice varieties in this experiment, was screened from three candidate media (NB/MS/2N6). According to the experiment on different concentration matches with NAA to 6-BA, optimal NB differentiation medium was selected for rice differentiation, and the relationship between differentiation and callus generations was also studied, and a high frequency transformation system for the rice varieties involved in this experiment was established in this way.(2) Target gene PEASC, which was previously cloned into pBSK plasmid, was acquired through digestion by KpnI/SacI enzymes, electrophoresis and recover, and then, the acquired PEASC gene was inserted into the corresponding position of the expression vector pGA1621 and the plant expression vector which contained target PEASC gene was acquired. The recombinant plasmid was transformed into Agrobacterium tumefaciens by freezing-thaw method and the engineered clones were further confirmed by PCR.(3) With the high frequency Agrobacteriums-mediated rice genetic transformation system and the engineered Agrobacterium tumefaciens clones, the rice genetic transformation was conducted and 32 resistant plants were acquired.(4) Six positive plams were PCR-positive with special primers from randomly selected 8 resistant plants. Among the 6 plants, 2 plants was found to be positive by southern blotting analysis, one plant, with single copy of PEASC in its genome, was derived from DN950, die other plant, with two copies of PEASC in its genome, was derived from TJ8.