棉铃虫CYP6B7基因介导氰戊菊酯抗性的机制研究

In the previous studies, we found that cytochrome P450 CYP6B7 played an important role in the pyrethroid insecticide fenvalerate resistance of HDFR strain in the cotton bollworm, Helicoverpa armigera (Hübner) from China; overexpression of CYP6B7 gene combined with relevant substitution of amino acid residues (Val144→Met, Glu256→Lys and Cys319→Tyr) might be responsible for fenvalerate resistance in H. armigera. However, above three point mutants were spatially located in the variable regions of CYP6B7 protein, but not in the characteristically conserved domains of P450 enzyme. Whether those mutants of CYP6B7 in the HDFR strain of H. armigera are directly involved in the resistance toward fenvalerate is the matter of concern. Thus, this project will be undertaken to study on the following three aspects. Firstly, seven variants of CYP6B7 protein (CYP6B7v) carrying a single mutation, the double mutant and the triple mutant will be constructed by site-directed mutagenesis using the method of polymerase chain reaction. The wild-type of CYP6B7 (CYP6B7wt) will be expressed in baculovirus-infected fall armyworm, Spodoptera frugiperda Sf9 Cell lines as well as this same P450 mutagenized in order to introduce the mutations alone or in combination. Secondly, the catalytic activity of the enzymes CYP6B7wt and CYP6B7v for substrates, such as 7-ethoxycoumarin, 7-benzoyl-oxycoumarin and p-nitrophenol, and their degradations against pyrethroid insecticides including fenvalerate and beta-cypermethrin, will be determined. Thirdly, the mRNA expression levels of CYP6B7, the larval susceptibility of the HDFR strain to pyrethroid insecticides, and the catalytic activity of the microsomes P450 enzymes against substrates were determined after siRNA treatment. In this project, the relationship between the cytochrome P450 CYP6B7 gene in H. armigera and fenvalerate resistance will be further investigated. The important roles of three substitutions of amino acid residues of CYP6B7 protein and overexpression of CYP6B7 gene in the HDFR strain of H. armigera resistant to fenvalerate will be also clarified.

申请人前期研究发现，抗氰戊菊酯棉铃虫(HDFR) CYP6B7协同氨基酸突变（M144V、K256E、Y319C）的过量表达可能与氰戊菊酯的抗性水平上升相关。然而，以上氨基酸突变位于CYP6B7蛋白的可变区，而非P450的特征保守域，因此其是否直接与氰戊菊酯抗性相关，值得深入研究。为此，本项目研究将在原有基础上，以该基因为研究对象，采用定点突变(SDM)技术及RNA干扰策略，拟围绕以下三个方面的内容展开研究：①异源表达野生型及突变型CYP6B7蛋白。②比较研究上述蛋白对底物的催化活性及其对菊酯类药剂的降解能力。③检测CYP6B7基因沉默后其 mRNA表达水平、棉铃虫对菊酯类药剂的敏感性变化及微粒体总P450对底物的代谢能力。研究结果将验证棉铃虫CYP6B7基因与氰戊菊酯抗性的相关性，并阐明CYP6B7蛋白中3个氨基酸残基突变及CYP6B7基因过量表达在该药剂抗性中的重要作用。

申请人通过定点突变(SDM)技术、RNA干扰策略及实时荧光定量PCR技术，研究了抗氰戊菊酯棉铃虫(HDFR) CYP6B7协同氨基酸突变（M144V、K256E、Y319C）的过量表达与氰戊菊酯抗性的相关性，结果发现：①筛选得到介导RNAi沉默效应高达60.13%的siCYP6B7-2片段(位于CYP6B7基因ORF的1130-1152 bp)，并探明其触发CYP6B7基因沉默的最低作用剂量为1.0 µg/头；基因沉默后，HDFR品系棉铃虫对氰戊菊酯和高效氯氰菊酯的敏感程度明显下降，且LD50值较空白对照组（TE buffer）分别下降了21.03%-22.77%和25.11%-37.63%；此外，siCYP6B7-2注射处理组的微粒体总P450对底物（7-乙氧香豆素和对硝基苯酚）的催化活性明显下降。②Sf9细胞系异源表达的单个氨基酸突变的CYP6B7蛋白中CYP6B7vY对底物（7-乙氧香豆素和对硝基苯酚）的催化活性好于野生型CYP6B7蛋白，其他两个位点的氨基酸突变（CYP6B7vM、CYP6B7vK）对底物的催化活性影响不大；2个氨基酸突变（CYP6B7vMK、CYP6B7vKY、CYP6B7vMY）对底物的催化活性明显优于野生型；3个氨基酸突变的CYP6B7vMKY对底物的催化活性最高。③重组的突变型CYP6B7蛋白对氰戊菊酯、溴氰菊酯及高效氯氰菊酯的降解活性大于野生型CYP6B7蛋白，其中以CYP6B7vMKY的活性最高。以上研究结果进一步验证了棉铃虫CYP6B7基因与氰戊菊酯抗性的相关，同时阐明CYP6B7蛋白中突变的Y319残基对底物及菊酯类药剂的代谢能力存在影响。