Construction Of Plant Expression Vector Containing HrpZ_Psgl2 Gene And Genetic Transformation Into Rice

Rice is one of the most important food crops in the world, in order to promote the resisting adversity property, the ability to resist disease and insect resistance of rice, transgenic technology have widely used. Sources of breeding for disease resistance were widely distributed. Hrp gene comes from microorganism and it is generally found in Gram-negative bacteria, which can cause HR in resistant host plants or non-host plants. Harpins encoded by hrp have similar to the other harpins:aboundant glycine, stable to heat, and sensitive to proteinase. In this study, hrpZPsg12 derived from strain Psg12 of Pseudomonas syringae pv. glycinea. The obtained systemic resistance to damping off and powdery mildew of cucumber were induced by the harpin coded by hrpZPsg12. Hypersensitive response of nonhost was induced by the harpin. Plants acquire systemic resistance and growth potential could be elicited by the harpin. It will be an effective way to cultivate resistance rice by transplanting the gene into rice.The main results are as follows:1. The pGM-hrpZPsg12 and vector pCAMBIA1300 digested with SacI/XbaI, then the hrpZPsg12 gene and pCAMBIA1300 ligated with T4DNA ligase. The expression vector pCAMBIA1300-bar-hrpZPsg12 was successfully constructed.2. Based on pCAMBIA1300-bar-hrpZpsg12, the plant expresion vector was constructed by substituting pmi for bar gene. The security selection marker expression vector was successfully constructed and named pCAMBIA1300-pmi-hrpZPsg12. The two recombined vector was transferred into DH5α. After verifying correct orientation of genes, this transgenic construction was introduced into Agrobacterium tumefaciens strain EHA105 by using frozen-melt method.3. The tissue culture system was optimized. Sterile seeds inoculate on N6D medium with 2 mg/L 2,4-D, cultured under continuous light at 32℃for 10 d.Bacteria cell concentration was adjusted to 0.6 at OD600, immerse callus in this Agrobacterium for 30 min, co-cultivate the callus at 25℃in the dark for 3 d.,and selective culture for 14 d.The explants were transferred to MS culture-medium containing 0.1 mg/L NAA,2 mg/L KT and 5 mg/L PPT(or 10 g/L mannose)(major inorganic constituents:185 mg/L MgSO4·7H2O,1.72 mg/L ZnSO4·7H2O,3.38 mg/L MnSO4·H2O and 0.005 mg/L CuSO4·5H2O),culture under at 28℃for light 16 h and dark 8 h.The resistant callus come into being after 10 d,and resistant plantlets were screened about 40 d. Drying treatment of calli for 16 h and culture dark for one week before differentiation,which can increase plant regeneration efficiency and reduce browing. Then, transfer resistant plantlets to root media for inducing roots about 7 d. 4. Transformation was confirmed by PCR, totally 3 regenerated plants were obtained.