Enzymatic Characteristics And Functional Analysis Of Glutathione S-transferase From Haemaphysalis Doenitzi And Hyalomma Rufipes

Glutathione S-transferase(GSTs)is a kind of metabolic detoxification enzyme distributed in organisms.GSTs mainly detoxifies exogenous and endogenous compounds to produce water-soluble and excretable metabolites,thus reducing the damage caused by toxic substances to the body.As an ectoparasite that can spread a variety of pathogens,ticks cause great harm to human health and animal husbandry.The irrational use of chemical insecticides for tick prevention and control for a long time resulted in the generation of tick resistance to insecticides,which made the prevention and control work more difficult.In this study,the glutathione S-transferase genes of Haemaphysalis doenitzi and Hyalomma rufipes were cloned,prokaryotic expression,enzymatic characteristics and functional analysis,which is helpful to understand the molecular mechanism of tick resistance to insecticides and provide a theoretical basis for tick control.In this study,glutathione S-transferase genes of H.doenitzi and H.rufipes were cloned by PCR amplification,which were named HdGSTm1 and HrGSTm1respectively.The open reading frame of HdGSTm1 was 693 bp,encoding 230 amino acids,and its isoelectric point and protein molecular weight were 6.83 and 26.22 k Da,respectively.The open reading frame of HrGSTm1 was 672 bp,encoding 223 amino acids,and its isoelectric point and protein molecular weight were 8.22 and 25.62 k Da,respectively.Phylogenetic tree analysis showed that both HdGSTm1 and HrGSTm1were GSTs genes of Mu family,both of which had no signal peptides and were cytoplasmic GSTs.Soluble recombinant proteins HdGSTm1 and HrGSTm1 were expressed and purified from E.coli BL21(DE3)by prokaryotic expression technique,and their sizes were about 45 k Da and 44 k Da,respectively.By calculating the activity of CDNB and GSH as substrates,the conjugated reaction between GSH and CDNB was catalyzed by the two recombinant proteins.The Vmax and Km of HdGSTm1 were 1.397±0.07?mol/min/mg and 1.254±0.12?M,respectively.The Vmax and Km of HrGSTm1were 3.367±0.81?mol/min/mg and 2.208±0.76?M,respectively.HdGSTm1 and HrGSTm1 showed different activity at different temperature and p H.HdGSTm1showed high activity below 40?,and the highest activity at 30?.HrGSTm1showed high activity below 70?,and the highest activity at 60?.The enzyme activities of HdGSTm1 and HrGSTm1 increased first and then decreased with the increase of buffer p H,and reached the highest at p H 7.0 and 8.0,respectively.The antioxidant results showed that both HdGSTm1 and HrGSTm1 had concentration dependent antioxidant ability.The relative expressions levels of HdGSTm1 and HrGSTm1 in different periods and tissues were determined by RT-qPCR.The results showed that the relative expression level of HdGSTm1 in egg,larval and adult ticks was significantly higher than that in nymph ticks(P<0.05);The relative expression of HrGSTm1 in egg stage was significantly higher than that in larval and adult stage(P<0.05).Both HdGSTm1and HrGSTm1 were highly expressed in the malpighian tubules,which was significantly higher than that in other tissues(P<0.05).To explore the effect of glutathione S-transferase on the biological characteristics of ticks by RNAi technology.The results showed that the interference of HdGSTm1 and HrGSTm1genes affected on the average mortality,average spawning rate,average engorgement weight and average egg weight of ticks.Compared with the control group,there were significant differences in the feeding time of H.doenitzi and the average engorgement weight of H.rufipes(P<0.05).Three sublethal concentrations of cypermethrin,LC₀₅,LC₁₀and LC_50,were selected by bioassay of tick impregnation method,which were 0.075 mg/ml,0.135mg/ml and 1.076 mg/ml respectively.The effects of three sublethal concentrations of Cypermethrin LC₀₅,LC₁₀and LC₅₀on the transcription level of HrGSTm1 in midgut,ovary,salivary gland and malpighian tube of H.rufipes were determined by RT-qPCR.Compared with the control group,the expression levels of HrGSTm1 in midgut,ovary and salivary gland were up-regulated in varying degrees with the increase of the three sublethal concentrations of cypermethrin;However,the expression level in malpighian tube was up-regulated first and then down-regulated.The expression response pattern of HrGSTm1 was different in different tissues after different sublethal concentrations of cypermethrin,so it was speculated that the detoxification metabolism of HrGSTm1 might be different in different tissues.