Cloning And Characterization Of Glutathione S-Transferases From Echinochloa Crusgalli

Weeds compete with crop plants for nourishment, moisture, light and space, thus reduces crop yield and quality. The use of herbicide can greatly improve the working efficiency and relieve labor intensity. However, it might cause damage to crops as well. At present, breeding herbicide-resistant crop varieties is the key to solve this problem.Glutathione transferases (GSTs) exist widely in plants and play an important role in plant abiotic stresses response and metabolic detoxification. This thesis studied two different GST genes cloned from E. crusgalli, EcGSTZl and EcGSTF1. The gene cloning, prokaryotic expression, fluorescent quantitative analysis, GSTs activity and bioinformatics analysis of the two genes were conducted, and the main results were as follows:1. Cloning of EcGSTs:Two different GST genes were cloned, designated as EcGSTZl and EcGSTFl. The EcGSTZ1is747bp in length with639bp in the encoding region. EcGSTZl encoding a protein of212ammo-acids and the calculated protein molecular weight is23.67KDa, GenBank accession code for EcGSTZ1is JX122857. The EcGSTF1is661bp in length with645bp in the encoding region. EcGSTF1encoding a protein of214amino-acids and the calculated protein molecular weight is23.79KDa. GenBank accession code for EcGSTF1is JX122858. The PSI-BLAST comparative analysis and phylogenetic analysis showed that EcGSTF1belongs to a glutathione transferase Phi family and EcGSTZl belongs to Zeta family.2. Prokaryotic expression analysis:Two fusion expression vectors of pET28b-EcGSTFl and pET28b-EcGSTZl were successfully constructed. Two fusion proteins expressed in E.coli cells induced by IPTG, and the expression level was increased along with the induction time. The molecular weight of these two expressed protein was consistent with theoretic values.3. qPCR analysis:The expression of GSTs gene before and after herbicide treatment in different traits of barnyard grass was analyzed by real-time quantitative PCR. β-Actin was used as the reference gene. The results showed that EcGSTFl and EcGSTZl were both expressed and the expression levels of these two genes were increased after the herbicide treatment. The expression level of the EcGSTFl was higher than the EcGSTZ1, and their expression level in resistant barnyard grass was higher than that in sensitive barnyard grass, which suggested that the EcGST genes expression were closely related to the metabolic detoxification of plant.4. Enzyme assays of GSTs:The study of GSTs (glutathione S-transferases) activity showed that the GSTs activity of the resistant barnyard grass, were higher than that of the sensitive ones before the herbicide treatment. The activity of the two E. crusgalli showed the downtrend in a short time after treatment, and the resistant barnyard grass response faster than the sensitive ones. With the time prolonging, the GSTs activity of the resistant barnyard grass keeps increasing, while the activity of the sensitive barnyard grass increased firstly and then decreased. Finally the GSTs activity of the resistant barnyard grass was higher than that of the sensitive barnyard grass. Analysis shows that relationship between the GSTs activity and the resistance of E. crusgalli to herbicides, the difference in GSTs activity is an important cause of herbicide metabolism ability of in difference resistant barnyard grass.To sum up, the qPCR analysis showed that the expression level of EcGSTs gene in resistant barnyard grass was higher than that in sensitive barnyard grass, while the GSTs activity was higher than that in sensitive barnyard grass, the EcGST genes expression were closely related to the metabolic detoxification of plant. Two of EcGST genes were cloned and the prokaryotic expression vectors were successfully constructed, thus providing important foundation for further analysis of GSTs gene in plant body detoxification mechanisms of exogenous toxic substances.