The Clone Of EPSPs Gene And The Identity Of Base Pairs Related To Resistant To Glyphosate

The enzyme, known as EPSPS (5-enolppyruvylshikimate-3-phosphate synthetase), is found in all plants, fungi, and bacteria, but is absent in animals. EPSPS is an important enzyme for the process of synthesizing the aromatic amino acids such as phenylalanine, tryptophan, and tyrosine. Production of these amino acids is required for the essential plant functions of protein synthesis, cell wall formation, defense against pathogens and insects, and the production of hormones. Inhibition of the EPSPS enzyme by glyphosate results in an inability of the plant to perform these functions, and quickly leads to death.Glyphosate (N-phosphonomethylglycine) which is a herbicide lacking specificity between weeds and crops, has been used as a selective agent for microorganisms and higherplant cells. These biological systems use two different mechanisms to overcome EPSPS inhibition by glyphosate. One is to produce glyphosate-tolerant, mutant EPSPS enzyme which has a different affinity for one or both substrates as well as for glyphosate. Selected lines from salmonellas typhumurium, Aerbacteraero genes and Escherichia coli are examples. The second mechanism is to produce elevated levels of EPSPS enzyme activity as has been reported for A. acrogenes and plant cells. The moleculasr bases of the EPSPS activity increase can either by an increase in gene expression or by gene amplification carrot and tobacoo.The total DNA was extracted from glyphosate polluted soil directly and digested with resistant Sau3AI, and then liagted with pACYC184. The ligated products were transformed into E.coli ER2799, which lacks of the EPSPs gene. The clone E. coli strain named ERAM1, which included the reconstructed plasmid and has the ability of growing in the 150mM of glyphosate concentration culture medium. The results of extra segment in the ERAM1 sequenced and analyzed in DANMAN shows that it is 3.6kb and there is a integrity ORF which can be translated into EPSPs. The base and amino acid sequence of the EPSPs gained compared with that in the US patents , the result indicated that these gene has no identity. This result shows that the EPSPs gene in the ERAM1 strain gained is a new one.Error prone PCR (EPPCR) technology can introduce random mutations in gene fragment. The EPSPs gene was processed and mutated by EPPCR. The mutated mixture was digested and ligated with the vector. Then the ligated products were transformed into E.coli ER2799, which lacks of the EPSPs gene. The ability of glyphosate tolerance declined obviously contrasted with that of the wild strain. The cloned E. coli strain was named AZ100. The sequence comparison shows that there are two bases have been mutated in the EPSPs gene of AZ100 contrasted with that of the wild. In the mutated gene,the 792nd base pair is G instead of T, and the 1203rd base pair is G instead of C. In the responding changed sequence of amino acid, the 264th Gly was mutated into Val and the 401st Ala was changed into Gly. The both changed amino acids locates in the sites where EPSPs and its two substrates combined together by the prediction of the second construe of the protion in internet. These mutation can abolish EPSPs activity under the circumstance with glyphosate. Compare these two sites with that of patents of America and found that neither of them is in these patents.The L35 strain has the ability of growing in the 350mM of glyphosate concentration culture medium was selected from unpolluted glyphosate soil by PCR used with strains directly instead of DNA. The primes used in this PCR was designed based on the EPSPs gene from G2 strain which has the ability of growing in the 450mM of glyphosate concentration culture medium. The sequence of EPSPs gene from the L35 strain has the high identity compared with that from the G2 strain. The amino acid sequence of the EPSPs gene comparison shows that there are two different amino acid in the EPSPs gene of L35 contrasted with that of G2. In EPSPs gene of L35, the 83td the 83th Ser instead of Pro in that of G2 and the 94th Asp instead of Gly.There was not...