Cloning And Functional Verification Of EPSPS

The enzyme 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is essential for the biosynthesis of aromatic compounds in prokaryote, fungi, plants and algae. It is the unique target of the herbicide glyphosate. Glyphosate is a broad-spectrum herbicide. 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.In this study, a apair of primers are designed based on the conserved regions of other higher plants' EPSP synthase through homology alignments. One DNA fragment is first cloned from Abutilon theophrasti Medic by performing PCR which used Abutilon theophrasti Medic genome as the template. This DNA fragment has 1446 nucleotides. RNA of Abutilon theophrasti Medic is been isolated by Improvemently Trizol Reagent. cDNA fragments of EPSP synthase which are cloned from Abutilon theophrasti Medic using the RT-PCR, 3′RACE and 5′RACE (rapid amplification of cDNA ends) technique is assembled a 1886bp full cDNA sequence of EPSP synthase, 5′UTR 64bp, 3′UTR 280bp, poly (A) tail in 3′end, the content of G+C% is 44.80%. The sequence encodes an open reading frame of 523 amino acids.The homologous comparison of the deduced amino acid sequence with other plant EPSP synthase proteins shows the identity to those of Fagus sylvatica, Oryza sativa Athaliana, phasedus vulgaris L respectively are 81%,81%,76%,74%.Using Point Mutation technology mutate epsps gene.Construction yeast and plant expression vector with original gene and mutated gene, transferred into tobacco plant by electroporation and Agrobacterium mediated transformation respectively, validating genic function. Under the glyphosate pressure, the transgenic yeast strains with good tolerance are screened. The mark named ATME-4-M and ATME-9 prives the attained genes have biological function. Transgenic tobacco young seedings are obtained in taking root culture medium .