Cloning,Expression And Functional Analysis Of Glutathione S-Transferase Genes (GSTs) In Daktulosphaira Vitifoliae (Hemiptera:Phylloxeridae)

The grape phylloxera Daktulosphaira vitifoliae?Fitch??Hemiptera:Phylloxeridae?is a worldwide monophagous pest that only harms the genus Vitis.It is also one of the important phytosanitary pests in China that can cause devastating hazard.Glutathione S-transferases?GSTs?are an important class of multifunctional enzymes in aerobic organisms.They are mainly involved in the detoxification of endogenous and exogenous compounds.They are also involved in intracellular transport,hormone biosynthesis and antioxidant applications radical protection.In-depth study of the function of GSTs gene in grape phylloxera is of far-reaching significance to the pest management methods that interfere with its detoxification process.This article takes Daktulosphaira vitifoliae?Fitch?as the research object.Seven cytoplasmic Dvi GSTs were obtained by gene cloning,and on this basis,prokaryotic expression,protein purification,enzyme kinetic parameter determination of recombinant protein,antioxidant capacity detection,and pesticide in vitro inhibition of recombinant protein were determined.The main findings are as follows:1. Gene cloning:Seven cytoplasmic Dvi GSTs were obtained by cloning,namely Dvi GSTd1,Dvi GSTd2,Dvi GSTt1,Dvi GSTo1,Dvi GSTs1,Dvi GSTs2 and Dvi GSTs3,belonging to four different families,namely Delta,Theta,Omega and Sigma,sequenced Verify that the sequence of each gene is consistent with the sequence in NCBI.The seven GSTs gene open reading frames range in length from 606 to 717 bp,encode 201 to 238amino acids,have a molecular mass of 58.73 to 70.22 KD,and have a theoretical isoelectric point range of 5.15 to 5.23.2. Prokaryotic expression and protein purification:p ET28a?+?-Dvi GSTs recombinant expression plasmid was obtained by constructing a prokaryotic expression system,and then transformed into Rosetta gami 2?DE3?competent cells to successfully construct expression bacteria.After induced expression by IPTG and purification by Ni column,seven soluble target proteins with a size of 25-40 k D were finally obtained.3. Analysis of enzymatic characteristics of Dvi GSTs:the Michaelis-Menten constant K_m of Dvi GSTs1 is the minimum,and the Michaelis-Menten constant K_m of Dvi GSTt1 is the maximum;Through the comparison between different families,it was found that both Dvi GSTd1 and Dvi GSTd2 in delt family showed strong activity,and the lowest V_max value was Dvi GSTo1.4. Antioxidant analysis of Dvi GSTs:Examining the antioxidant activity of seven Dvi GSTs,only Dvi GSTs2 and Dvi GSTs3 in the Sigma family were found to have antioxidant properties,and the antioxidant activity of Dvi GSTs3 was higher than Dvi GSTs2.5. In vitro inhibition analysis of Dvi GSTs activity:To determine the activity inhibition rate of GSTs structure specific inhibitors and insecticides acetamiprid and fipronil on recombinant protein Dvi GSTs.It was found that among the 7 Dvi GSTs,only the activities of Dvi GSTd1,Dvi GSTd2,and Dvi GSTs1 were shown to be inhibited by the agent.And the insecticides acetamiprid and fipronil showed certain inhibitory activity on Dvi GSTd1,Dvi GSTd2,Dvi GSTs1,but the effect was far less than the structurally specific inhibitors of GSTs.This study helps to understand the process of GSTs protecting cells from oxidative damage and to analyze the detoxification and metabolic process of GSTs to insecticides.It provides a theoretical basis for studying the resistance of grape phylloxera,and it also provides a new idea for the development of new compounds that interfere with the detoxification process and the chemical control strategies for grape phylloxera.