Study On The Preparation Of Transketolase Protein From Setaria Viridis And The Interaction Of Chemicals

Weeds harm the normal growth of crops and compete with them for water,nutrients,light,and space,causing huge economic losses to agriculture.Nowadays,the problem of weed resistance to herbicides is becoming increasingly serious,and the development of weed resistance management and new herbicide targets is urgent.Transketolase widely exists in various animals,plants and microorganisms.It plays an important role in the pentose phosphorylation cycle and the reduced pentose phosphate cycle of photosynthesis.The Calvin cycle involved in plant photosynthesis,as the limiting factor of plant maximum photosynthetic rate,plays an important role in regulating plant growth and development,and is irreplaceable in this pathway,so it is a potential herbicide target.Setaria grass is a serious weed in the gramineae family,widely distributed in corn fields and other farmland across China.Its resistance to herbicides such as nicosulfuron has been widely present.Studying new herbicide targets with Setaria grass as the experimental object has profound significance and prospects.1.In this paper,the total RNA of Setaria was extracted from the leaves of Setaria,and then the cDNA of Setaria was synthesized through reverse transcription kit.Specific primers were designed to amplify the Transketolase(TKL)gene of Setaria.Through the construction of prokaryotic expression vector and screening of induction conditions,it was found that the best induction condition of Transketolase protein of Setaria viridis was that the protein expression was the highest when it was induced at 16℃ for 16 h;In the purified protein,the optimal elution concentration was determined by gradient elution of imidazole with a buffer of 300 mM imidazole.2.Study on the interaction between Transketolase and chemicals in Setaria viridis:Based on the pH based Transketolase activity measurement method,a high-throughput screening method for potential Transketolase inhibitors was constructed.The prokaryotic expressed Transketolase protein was incubated with different chemicals,and then the enzyme activity was measured.The specific activity of Transketolase was quantitatively analyzed through the change of absorbance,so as to screen out the agents that specifically inhibit Transketolase,And the screening results were verified through fluorescence quantitative amplification experiments.3.Research on the interaction between Transketolase mutant protein of Setaria viridis and medicament:based on the previous research,five key amino acids related to the enzyme activity were selected,and five point mutation(all mutated to alanine)Transketolase expression vectors were constructed.The protein was expressed,purified,and the enzyme activity was measured.By comparing with the enzyme activity measurement results after incubation of hydroxyphenylpyruvate(HPP),a Transketolase inhibitor reported in the literature,Sort the intensity of the impact of five key amino acids on enzyme activity:H139A>H376A>H179A>H587A>S497A.4.The Transketolase transgenic vector of Setaria viridis,the Transketolase transgenic vector of Arabidopsis thaliana,and the Transketolase gene knockout vector of Arabidopsis thaliana were constructed,and the Transketolase gene knockout vector of Arabidopsis thaliana was applied.Through the method of Agrobacterium tumefaciens infecting the inflorescence of Arabidopsis thaliana,more than 20 T1 generation positive seedlings were obtained through hygromycin plate screening,laying the foundation for the subsequent acquisition of homozygous gene knockout strains.