Expression And Biochemical Characterization Of Carboxylsterase Gene TCE2in Tetranychus Cinnabarinus (Boisduval)

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), widely distributed in China, is one of the most important pests, causing severe damage to a number of agriculture products. Currently, pesticide is mainly used to manage this mite. But with the widely spraying of pesticide without planning, the resistance of T. cinnabarinus has developed quickly for its short generation cycle, high fecundity and inbreeding.The esterase (EC3.1.1.1) is a hydrolase, can catalyze the hydrolysis of carboxylic lipid. In insect (mites), esterase can detoxify exogenous and endogenous toxins, or combined with the toxic substances and prevent them from reaching the targets. It plays an important role in pests (mites) resistance.In previous study, the correlation between T.cinnabarinus esterase gene TCE2and resistance has been proved. Based on the results, the aim of this research is to construct prokaryotic expression plasmid of TCE2gene, purify the recombinant protein, study its metabolism or combination function of acaricide (avermectin), and provide direct evidence for the resistance mechanism of T.cinnabarinus mediated by TCE2. The main results are as follows:1. Recombinant plasmid (pCold II-TCE2、pET-28a-TCE2, pET-28a-TCE2q and pCold II-TCE2q) were constructed and induced for expression. Among them:pCold II-TCE2, can not express the recombinant protein after induction; pET-28a-TCE2, pET-28a-TCE2q expressed the recombinant protein after induction, but the protein assumed to be as inclusion body; only pCold-TCE2q expressed soluble protein in E.coli BL21(pKJE).2. After purification, the activity of recombinant protein was assayed with its specific substrate alpha naphthyl acetate (α-NA). The optimum reaction condition (T, pH) was identified as30℃, pH7.4. Under The optimum reaction condition, the specific activity was483.3±71.8nmol/mg pro.min-1. Km is30.5±5.9nM, and Vmax is448.2±75.2nmol/mg pro.min-1.3. The metabolism or combination function analysis results showed that, with the increase of the amount of recombinant protein, the amount of avermectin showed a reduced trend in the reaction system, but different reaction time showed no significant effect on the amount of avermectin.