Cloning A Novel 5-enolpyruvylshikimate-3-phosphate Synthase Gene Conferring Increased Glyphosate Tolerance From Halomonas Variabilis And Its Expression In Escherichia Coli And Its Glyphosate-tolerant Mechanism

A bacterial strain HTG7 was isolated from extremely glyphosate-polluted soil, and it could grow in Mops medium containing up to 900 mmol/L glyphosate. It was identified as Halomonas Variabilis by phenotypic and phylogenetic analysis based on 16 s rDNA.A plasmid carrying 1.35 kb DNA fragment was cloned from cosmid library of strain HTG7. It was able to complement with E. coli arok mutant, ER2799, and it also grew well in 80 mmol/L glyphosate Mops medium. The arok gene, which encoding 5-enolpyruvylshikimate -3-phosphate synthase (EPSPs), was little homologous to nucleotide acid of those which have been reported, and had less than 46% amino acid similarity to glyphosate-tolerant EPSPs from twenty two genus which were involved in US patents.The specialized features of the encoded protein, including Blocks and conserved domain and subcellular localization sites and membrane-spanning regions and secondary structure and protein proteolytic enzyme or reagent cleavage digest, were predicted and analyzed. All the predictions suggested that the gene had novel structure and well-defined function which conferring glyphosate-tolerance. The gene was enrolled in GenBank and its accessionnumber was AY573186.PET 28a(+) aro A fusion expression vector was constructed and transformed into E coli BL21(DE3) cells. Overexpression in soluble form was achieved with isopropyl -D-thiogalactoside(IPTG) induction at 30 . SDS-PAGE showed that molecular weight of expressed product was 51 kD. Enzyme activity measurement revealed that EPSP synthase had specific activity. EPSP synthase was purified using affinity chromatography. Western Blot analysis detected that purified target protein was conjugated specifically with antibody of T7 Tag AP LumiBlotTM Kit.In order to find some glyphosate-tolerant sites, the novel EPSPs gene from Halomonas Variabilis strain HTG7 was amplified by random error-prone PCR. Two EPSPs mutants which conferring little glyphosate-tolerance were screened using complement of E. coli ER2799. By compared No. 1 mutant with original gene, there were two differences of nucleotide acids (245 T mutated 245 G, and 1293 T mutated 1293G). It resulted in one difference of amino acid (82V (codon GTG) altered 82G (codon GGG) ).In comparison with No. 2 mutant and original one, there were five differences of nucleotide acids (179 A mutated179G, 802A mutated 802T, 872T mutated 872A, 669G mutated 669A, 1134T mutated 1134A, respectively). Those resulted In three differences of amino acids (60Q (codon CAA) altered60!? (codon CGA); 268T (codon ACC) altered 268S (codon TCC); 291V (codon GTT) altered 291D (codon GAT) .respectively ).Tertiary structure of the three EPSPs were predicted and aligned. The backbone was same. But the torsion angle between peptide plane and N atom linking with.C were quite different in the original and mutated amino acid position. All suggested that the backbone decided the EPSPs function, and torsion angle maybe induced delicate change of tertiary structure. The delicate alteration led to the loss of glyphosate-tolerance of EPSPs.