Construction Of Plant Expression Vector Carrying Ssbhmt Gene And Transformation To Arabidopsis Thaliana

With the development of genetic engineering, we can transfer some protein coding genes, which have important physiological functions but do not exist in plant into plants for adjusting develepment of plant and improving quality of plant etc. Betaine-homocysteine methyl transferase (BHMT), which is a methyl-metabolizing enzyme in animals and micro-organisms, can catalyze methyl of betaine shifting to homocysteine, forming dimethyl glycine and methionine. There is no report of its expression about plant until now. According to the character of SsBHMT that can promote synthesis of protein by regulating methionine metabolism. Met was substrate of adenosine methionine(SAM) which is dominate methyl donor in plant. Methyl have been taken part in metabolizing and adjusting of plant.BHMT was transferred into plant to seek for action to plant.RNA was extracted from fresh pig liver. SsBHMT gene was amplified by RT-PCR. After confirming that its sequence was correct by sequencing, prokaryotic expression vector pET30a-SsBHMT was constructed and transferred into E. coli ER2566and BL21(DE3). The objective protein was induced and purified. By SDS-PAGE electrophoresis analysis, it was found that the gene was not induced in E. coli BL21(DE3), but mainly expressed in the form of insoluble inclusion body in ER2566 and expressed a little in supernatant . Then the conditions of inducement including OD value of bacilli, induction temperature, and IPTG concentration were optimized .The expression of objective protein enhanced than before,but still expressed mainly in the form of insoluble inclusion body. Thus inclusion body dissolved with denaturing agent and was purtified. By SDS-PAGE electrophoresis analysis,there was a protein band between 45KDa and 66KDa, which size is the same as the expected,.Meanwhile the eukaryotic expression vector p3301-SsBHMT containing CaMV 35S promoter was constructed.This vector was transferred into Arabidopsis thaliana by flower dip method of Agrobacterium-mediated. 25 strains were screened by spraying repeatedly 0.5% (v / v) herbicide. Leaves DNA of 14 strains which were selected from 25 transgenic strains was extracted., SsBHMT and BAR gene of 14 strains were tested by PCR. The result indicated that SsBHMT had been transferred into Arabidopsis thaliana.SsBHMT gene was expressed,purified and transferred its plant expression vector into Arabidopsis thaliana, which provides a basis for further researching the role of animal's SsBHMT gene on growth development and resistance of plant.