Cloning, Expression And Analysis Of Glyphosate Resistance Gene From The Deep-Sea Bacterium

Glyphosate-resistant species screening and resistant gene mining have been the focus of herbicide resistance research. But studies on glyphosate-resistant Gene selected from marine microorganism are few. To get a valuable glyphosate-resistant Gene from marine microorganisms, two bacteria with high-activity glyphosate-resistance were screened from deep-sea marine microorganisms. Two glyphosate-resistant genes were obtained from the genomic library of these two bacteria strains. These two genes were expressed in E.coli, and their catalyze mechanisms were analyzed. The main results of this experiment were as follows:1. Two high activity glyphosate-resistance strains of bacteria were obtained from deep-sea marine microorganisms. The marine bacteria are labeled as AJ415377 and X74723 respectively, which can be cultivated in 100 mM glyphosate medium. This means the bacteria were resistant to 100 mM glyphosate; these two strains were identified by comparing their 16S rDNA, which were obtained byPCR using the genome DNA of the strains as template. Sequence alignment shows that AJ415377 have the highest homology with Brachybacterium paraconglomeratum and the identity is 97.973%, which indicates that strain AJ415377 belongs to Brachybacterium paraconglomeratum Spp; X74723 has the highest homology with Vibrio proteolyticus and the identity is 99.588%, which demonstrates that the strain AJ415377 belongs to Vibrio proteolyticus Spp.2. In this study, two positive clones were screened from Brachybacterium paraconglomeratum and Vibrio proteolyticus, which have glyphosate-resistance. The cloned gene fragment from Brachybacterium paraconglomeratum is named Bp-nupCc, and the gene fragment from Vibrio proteolyticus is named Vp-Cglh. Sequence analysis shows that the fragment Bp-nupCc contains an incomplete ORF(495 bp) encoding 165 amino acids and a full functional domain. The content of C+G is 51.6% and the amino acids showed 95% identity with nucleoside permease NupC, which belongs to the GATE superfamily. Some researches demonstrated that proteins of GATE superfamily function as recognition and identification information between the nuclear pores and nuclear membranes. In addition, NupCc shows 25% identity with sarcosine oxidase chain C. The homologous region distributed in the first and second domain. The mechanism needs further study. Sequence analysis shows that the fragment Vp-Cglh which is from Vibrio proteolyticus contains two complete ORF, ORFO gene fragment has 495 bp, encoding 165 amino acids, its content of C+G is 55.6% and it is named Cglh0.There is no further research on CglH0 because of its inclusion body when induced in BL21. ORF1 gene fragment has 996 bp and its content of C+G is 55.9%. The homology is under 30% compared with known proteins on amino acid level, so it improves that Cglh is a new glyphosate-resistant gene.3. Primers were designed separately to amplify genes (NupCc, Cglh and CglhO) by PCR. Then genes NupCc, CglhO and Cglh were inserted into E.coli expression vector pGEX-6P-1, respectively. The recombinants were transformed into E.coli BL21 (DE3). Both of the GST-NupCC and GST-CglH0 proteins was found as inclusion bodies. The GST-CglH are purified by GST purification kit and the purified protein GST-CglH was obtained. The concentration of protein was determined as 0.28 mg/ml by protein determinate Kit.4. The filter color rendering experiment shows that the enzyme reaction product with ninhydrin hydration is positive. It indicated that the imino in glyphosate moleculer treated by CglH has been changed. This inferred CglH can directly act on glpyhosate. From the show layer chromatography experiment, it was found that the amount of glyphosate was reduced significantly after treated by the enzyme, accompanied with new material formation. To understand the composition of the new material, the products of the enzyme reaction were analysed by LC-MS, and one more component was found when the product was protonized (The substance has 150 Molecular mass). According to the analysis of glyphosate degradation pathway, the substance which has 150 Molecular mass was found that it has the same molecular weight as the substance that formed by glyphosate taking off a molecular of water. The way of CglH acting on glyphosate may be described as dehydration. However, the confirming of its mechanism needs further study.This study has not only provided candidate gene for the glyphosate Resistant gene screening, but laid the foundation for further study.