Preparation of polyclonal antibody against PFK protein and application in identification of glyphosate resistance in Eleusine indica
Phosphofructokinase(PFK)is one of the indicator proteins for glyphosate resistance in goosegrass Eleusine indica as the content of PFK is positive correlation with the target 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS).In this study,the full-length of PFK gene was cloned from E.indica,the prokaryotic expression vector pGEX-4T-1-PFK was constructed and polyclonal antibodies were obtained by immunizing rabbits.Then,the specificity and sensitivity of the antibody were detected.Results showed that the full length of PFK gene is 1 656 bp,encoding 551 amino acids.A45 kD recombinant protein with the concentration of 3.5 mg/mL was obtained after IPTG induction and purification.The antibody could only specifically recognize the PFK protein of E.indica,and the antigen can still be detected when antibody was diluted by 512 000 times.The antibody could quickly identify the PFK protein and accurately identify the resistant goosegrass with different levels of PFK expression.These results suggested the PFK polyclonal antibody could identify glyphosate-resistant E.indica caused by EPSPS overexpression.This provides a practical tool for rapid identification of resistance to glyphosate for E.indica.
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