关键词: 二斑叶螨, 多重抗性, 解毒酶基因, RT-qPCR

Abstract: 【Objective】Tetranychus urticae is an important mite in agricultural production, which is easy to produce resistance to pesticide because its small size and rapid propagation. The objective of this study is to discuss the change of mRNA relative transcript level, find out multiple resistant mechanism of T. urticae and provide a theoretical basis for its integrated management (prevention and control). 【Method】SS and Mp-R strain of T. urticae were raised in laboratory. Mp-R strain was continuously selected with median lethal concentration (LC₅₀) of mixture of spirodiclofen, fenpropathrin and abamectin. After spraying insecticide for 4-5 times, biological assay was done referring to leaf-residue method which recommended by FAO, LC₅₀ and resistance ratio were calculated, and a selected cycle was recorded. After 3 cycles, acaricides concentrations were gradually increased, then toxicity equation, LC₅₀, resistance ratio, and chi-square value were calculated by PoloPlus software. The activities of CarEs, GSTs and MFOs were measured by biochemical analysis in egg, larva, nymph and adult of SS and Mp-R strains of T. urticae. Ten ditoxifying genes expression which were related to resistance were measured with RT-qPCR technology using ELFn as reference gene through comparing Ct value method.【Result】After 50 generations of selection, LC₅₀ of T. urticae to abamectin, spirodiclofen and fenpropathrin reached 1 103.55, 5 993.33 and 2 345.62 mg·L^-1, and the resistance ratios were 603.03, 167.65 and 51.77 times, respectively. Activities of MFOs in egg of Mp-Rstrain increased significantly compared with the SS strain, but CarEs and GSTs activities had no significant difference. CarEs and GSTs activities at other stages of Mp-R strain increased significantly compared with the SS strain, but MFOs activity did not increase. In Mp-R strain, CarEs and GSTs activities of female adult mite and MFOs activity in egg increased significantly compared with other stages, and compared with the SS strain, the expression of TuGSTd05, TuGSTd06 and TuGSTd09 genes were significantly up-regulated by 1.8 times at all developmental stages of Mp-Rstrain, and the expression of TuGSTd01 gene was significantly up-regulated by 1.63 times in nymph of Mp-R strain, but at other developmental stages had no significant difference. CYP392E10 gene expression significantly increased by 5.87 times in egg, significantly increased by 2.15 times in larva and 2.09 times in nymph, but in adult non-significant. CYP392A6 gene expression significantly increased by 1.89 times in egg, 1.64 times in larva and 1.59 times in nymph, but in adult non-significant. CYP392A16 gene expression significantly increased by 6.97 times in egg, 8.20 times in larva, 8.88 times in nymph, 7.34 times in male adult and 8.59 times in female adult. CYP392D8 was significantly up-regulated by 2.18 times in egg, 2.00 times in larva, 2.03 times in male adult and 2.41 times in female adult, but in nymph non-significant. TuCCE35 significantly increased by 1.58 times in nymph, 1.86 times in male adult and 2.65 times in female adult. TuCCE36 significantly increased by 1.73 times, 1.89 times in male adult and 2.14 times in female adult.【Conclusion】Ten detoxifying genes expression had various changes at different developmental stages of Mp-R strain in T. urticae compared with SS strain. CYP392A16 might participate the resistance formation of different developmental stages of T. urticae, but other genes’ expression had no obvious change, whether or not involved in the metabolism of the 3 acaricides about these genes need to be further verified.

Key words: Tetranychus urticae, multiple resistance, detoxification enzyme genes, RT-qPCR