| 5-enolpyruvlshimimate-3-phosphate synthase (EPSP synthase) is a necessary enzyme of the shikimate pathway in prokaryote,yeasts,fungi,apicomplexan parasites,plants and algae. The product chorismate of this pathway is precursor to the synthesis of the aromatic amino acids phenylalanine,tyrosine,and tryptophan as well as other essential aromatic secondary metabolites such as ubiquinone,menaquinone and vitamin K. Phosphoenolpyruvate and shikimate 3-phophate are converted to 5-enolpyruvlshimimate-3-phosphate and phosphate. EPSP synthase is a attractive prime target for many antimicrobial agents and herbicides. Glyphosate is a foliar applied ,broad spectrum ,post-emergence herbicide capable of controlling annual and perennial grasses and dicotyledonous weeds. Due to the similarity structure between glyphosate and phosphoenolpyruvate,glyphosate can inhibit EPSP synthase by rivalrously uniting EPSP synthase with phosphoenolpyruvate. Plants can obtain high doses glyphosate tolerance if the enzyme of this gene is overproduced and accumulated or herbicide-insensitive enzymes produced due to some amino acid mutation in active sites.A pair of primers was designed based on the conserved regions of other higher plants' EPSP synthase through homology alignments. Two DNA fragments were first cloned from O.violaceus by performing PRC which used O. violaceus genome as the template. One has 798 nucleotidesand the other 1157 nucleotides,but they can encode the same amino acid sequence and have same extrons according to the GT-AG rule of characteristic sequence of enkaryoutic intron. Their accession numbers in GenBank respectively are AF440390,AF440391. Primers serving as RT-PCR primers were synthesized based on the two cloned fragments. Two partially overlapping cDNA fragments which were cloned from O. violaceus using the RT-PCR,3'RACE (rapid amplification of cDNA ends) and 5'RACE technique was assembled a 1758bp full cDNA sequence of EPSP synthase (accession number in GenBank:AF440389). The sequence encodes an open reading frame of 518 amino acids. There is a transit peptide of 74 amino acids in the N terminal of the putative amino acids sequence coded by the cDNA. The start codon is at 84-86bp,the stop codon at 1658-1661bp. The homologous comparison of the deduced amino acid sequence with other plant EPSP synthase proteins shows the identity to those of B.napus,A.thaliana,Tomato,N.labacum respectively are 92%,91%,80%,65%. |
