Cloning,Expression Purification And Activity Determination Of Several Proteins As Potential Herbicide Target

as one of the three major pesticide.Herbicides play an important role in the agriculture production.With increase of the weed resistance and the strengthening of environmental protection regulations,the development of herbicides is facing enormous challenge.The new two commercial herbicide mode of actions,that is,auxin transmission pathway target and 4-hydroxyphenyl pyruvate deoxygenase(HPPD),are also found in 20 years ago.The long-term repeated usage of several kinds of action modes has led to the rapid development for resisitance of the weeds.Therefore,the study of herbicide based on new target is urgent need of current pesticide development,which is an effective way to solve the current weed resistance and meet the ecological and ecological requirements.In recent years,three kinds of plant proteins have been identified as potential herbicide targets,which are transport inhibitor response protein 1(TIR1),4-coumaric acid:coenzyme A ligase(4CL1)and 4-diphosphocytidyl-2C-methyl-D-erythritol synthase(IspD).These three proteins have also become hot topics in the development of new herbicides.These three proteins play an important role in plant growth and metabolism.They have been identified as potential herbicide targets.TIR1 is recognized as auxin receptor protein,which can affect auxin signaling;4CL1 is a key enzyme in the turning point of Phenylpropane metabolism and lignin-specific synthesis pathway;and IspD protein is also a key enzyme in the non-mevalonic acid pathway(MEP).Therefore,it is important to study these three potential herbicide targets for the development of new herbicides.In this paper,we studied the cloning,expression,purification and activity determination of three potential target proteins based on genomics and molecular cloning techniques.We futher studied their activity and enzymatic properties and inhibitors,which lays the foundation for the subsequent study of molecular probe interaction with target proteins and inhibitors screening.The main work as follows:1.Herbicides,herbicide targets and the methods of target protein were outlined.In addition,the research progress of TIR1,4CL1 and IspD were reviewed in detail.2.The auxin receptor gene TIR1 gene was cloned from Arabidopsis thaliana cDNA by polymerase chain reaction(PCR).A several of recombinant expression vectors were constructed.Eventually,the modified double-label expression vector pGEX-4T-1-TIR1(codon optimization)was selected.3.The 4CL1 gene was cloned from the cDNA of Arabidopsis thaliana by PCR with the same method.The pCold I-4CL1 expression vector was constructed and protein was expressed.The protein was purified by and affinity chromatography(Ni-NTA).The activity of the recombinant enzyme was detected by spectrophotometry,which lays a foundation for the subsequent screening of inhibitors with the protease as the target.4.The IspD gene was obtained from the cDNA of Arabidopsis thaliana with the same method.Two recombinant expression plasmids pCold I-IspD and pCold I-IspD(76-302)were constructed and transformed into host bacteria to express the proteins.However pCold I-IspD is an inclusion body that needs to be obtained by denaturation and renaturation.The expression of pCold I-IspD(76-302)protein was expressed as soluble protein.We obtained the protease with high purity and concentration by affinity chromatography.The activity of the protein was detected by high performance liquid chromatography and spectrophotometry respectively.We established a high-throughput method for inhibitor screening by the detection of phosphoric acid with malachite green and ammonium molybdate,Which lays the foundation of screening inhibitors and studying the interaction between the photoreactive molecular probe with the target protein.