Isolation Of Glyphosate-resistant Fungus Strain And Cloning And Expression Of Epsps Gene

The glyphosate was a efficient, low toxicity, broad spectrum, conductive, no selective kind of herbicide. It was registered as one of the most consumption herbicides in the world in1974. The glyphosate could control most of weeds, but it had poisoning effect on the crops when it controled weeds, because of its non-selective so as to limit its wide application. If glyphosate-resistance of crops was improved, this was beneficial to application of glyphosate on agricultural production, and could reduce agricultural production cost. So the excavation and utilization of herbicide-resistant genes have was key of research on bioengineering field.The most important way is to isolate herbicide-resistanct genes from the micro-organisms. Isolation of glyphosate-resistant strains from the soil polluted by glyphosate was a efficient kind of method. On the research, a glyphosate-resistant fungus strain was isolated from sludge of glyphosate factory outfall by flask-foster enrichment technology. Based on its morphological, physiological and biochemical properties as well as the18S rRNA sequence analysis result, the strain was tentatively identified as Candida palmioleophila. A cDNA library was constructed by SMARTer technology of Clontech company. EPSPS gene from strain Candida palmioleophila TY-JM and Aspergillus flavus TZ1985with resistance to glyphosate was isolation and expression in the E. coli, which has not been reported all over the world since before. It is expected to provide new genes resources for cultivation of genetically modified herbicide-tolerance crops.The major results of this research are summarized as follows:1. A glyphosate-resistant fungus strain TY-JM, which can grow in concentrations of glyphosate900mmol/L, was isolated from sludge of glyphosate factory outfall by flask-foster enrichment technology. Based on its morphological, physiological and biochemical properties as well as the18S rRNA sequence analysis result, the strain was tentatively identified as Candida palmioleophila. Morphological, physiological and biochemical properties were the same as the research results of the prehominid.18S rRNA sequence similarity of Candida palmioleophila was as high as99%.18S rRNA sequence was uploaded to GenBank, and the accession number is JN656713.2. A cDNA library driven by the total RNA extracted from the strain TY-JM was constructed by SMARTer technology of Clontech company. The library quality was evaluated, and the results showed that the titer of primary cDNA library and amplified cDNA library were2.58×106cfu/ml and3.42×109cfu/ml, respectively, with the library volume of about2.58×106cfu and the recombination rate of97.6%. The inserted fragments were distributed from500bp to3kb, and the most cDNA fragments were distributed around1kb. These results indicate that the strain TY-JM cDNA library is constructed successfully.3. EPSPS gene was isolated from Candida palmioleophila and Aspergillus flavus by homology cloning approach. Length of sequence of this gene is1311bp and1358bp, encoding436and450amino of polypeptide, theoretical molecular weight is46kDa and48kDa, and isoelectric point is5.77and6.04, respectively. NCBI Protein BLAST program analysis TY-EPSPS and TZ-EPSPS gene deduced amino acid sequence shown, TY-EPSPS is consistent with the Candida tropicalis, Candida dubliniensis and Candida albicans EPSPS gene sequences, which was up to80%, EPSPS gene sequence reached more than66%consistency with Sclerotinia sclerotiorum and Aspergillus clavatus. TZ-EPSPS is consistent with the Aspergillus clavatus, Aspergillus fumigates, Aspergillus niger, Aspergillus kawachii and Aspergillus terreus EPSPS gene sequences, which was up to90%, EPSPS gene sequence reached more than68%consistency with Candida dubliniensis, Candida albicans and Candida tropicalis. Compared TY-EPSPS and TZ-EPSPS with other species by software MEGA4.0shown the evolutionary relationships of EPSPS. The relationship of TY-EPSPS and TZ-EPSPS and fungus is closer, but it is far away from plant and bacterium. The relationship of TY-EPSPS and Saccharomyces is closer, The relationship of TZ-EPSPS and Aspergillus is closer.4. Connect TY-EPSPS and TZ-EPSPS with pET-30a vector into E. coli BL21(DE3), the E. coli resistance to glyphosate increased from the original50mmol/L to75mmol/L, which was higher than the negative control plasmid. It showed that the recombinant plasmid transfered into E. coli, and successfully replicated its expression in vivo to improve E. coli resistance to glyphosate.