Screening, Identification And Its Gene Cloning Of Glyphosate Resistant Strains

5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target enzyme of glyphosate. Glyphosate-resistance transgenic crops are mainly developed through the transformation of exogenous EPSPS gene. The cloning, expression and its function verification of EPSPS gene is the research emphasis in modern molecular breeding. Most patents of glyphosate-resistance gene and its transformation techniques are belonged to biotech company in developed countries. Therefore, it is important to mine new glyphosate-resistance gene and own the independent intellectual property rights.One strain of glyphosate-resistance bacteria and three strains of fungi were isolated from the soil and water in southern china, with different concentration gradient of glyphosate selective culture. Resistant bacteria Klebsiella pneumoniae, tentatively named kpS001and three resistant fungi, Paeciloomyces lilacinus, Rhizomucor variabilis and Aspergillus flavus, which were named F35, F37, F62respectively, were identified through the observation of phenotypic characteristic and the system analysis of16S rDNA sequence and ITS sequence.The aroA gene from Klebsiella pneumoniae kpS001was cloned and sequenced, and named aroAs001. The gene had1284bp. It contained the functional conservation sequences-L-G-N-A-G-T-A-in the area of91-98and-A-L-L-M-T-A-P-L-A in175-183of EPSP synthase. Compared with NCBI NTUH-K2044full-length aroA encoding amino acid sequence of Klebsiella pneumoniae subsp., the76th site of amino acid of kpS001EPSP synthase changed from threonine to isoleucine. It is deduced that the mutation of this site is one of the reson for enhanced resistance of kpS001to glyphosate.Recombinant plasmid pProA-aroAs001was constructed with pProA vector, and was transferred into EPSPS gene defective host strains DH5a (DH5α/△aroA). A48kDa target protein was detected in recombinant strain. The recombinant strains with aroAs001grew obviously better than the control one on the M9medium containing different concentrations of glyphosate even up to350mmol/L. These results indicated that aroAsooi was the main reson for kpS001to have high resistance to glyphosate.