Graham Kirsty M, Sparagano Olivier A E, Finn Robert D
Department of Applied Sciences, Faculty of Health and Life Sciences, Northumbria University Newcastle, Ellison Building, Ellison Place, Newcastle Upon Tyne, NE1 8ST, UK.
Coventry University, Alan Berry Building, Coventry University, Priory Street, Coventry, CV1 5FB, UK.
Ticks Tick Borne Dis. 2016 Jun;7(4):614-23. doi: 10.1016/j.ttbdis.2016.01.012. Epub 2016 Jan 21.
The monooxygenase complex is composed of three key proteins, a cytochrome P450 (CYP), the cytochrome P450 oxidoreductase (CPR) and cytochrome b5 and plays a key role in the metabolism and detoxification of xenobiotic substances, including pesticides. In addition, overexpression of these components has been linked to pesticide resistance in several important vectors of disease. Despite this, the monooxygenase complex has not been isolated from the Southern cattle tick Rhipicephalus (Boophilus) microplus, a major disease vector in livestock. Using bioinformatics 115 transcriptomic sequences were analyzed to identify putative pesticide metabolizing CYPs. RACE-PCR was used to amplify the full length sequence of one CYP; CYP3006G8 which displays a high degree of homology to members of the CYP6 and 9 subfamilies, known to metabolize pyrethroids. mRNA expression levels of CYP3006G8 were investigated in 11 strains of R. microplus with differing resistance profiles by qPCR, the results of which indicated a correlation with pyrethroid metabolic resistance. In addition to this gene, the sequences for CPR and cytochrome b5 were also identified and subsequently isolated from R. microplus using PCR. CYP3006G8 is only the third CYP gene isolated from R. microplus and the first to putatively metabolize pesticides. The initial results of expression analysis suggest that CYP3006G8 metabolizes pyrethroids but further biochemical characterization is required to confirm this. Differences in the kinetic parameters of human and mosquito CPR in terms of NADPH binding have been demonstrated and could potentially be used to design species specific pesticides. Similar differences in the tick CPR would confirm that this is a characteristic of heamatophagous arthropods.
单加氧酶复合体由三种关键蛋白质组成，即细胞色素P450（CYP）、细胞色素P450氧化还原酶（CPR）和细胞色素b5，在包括农药在内的外源性物质的代谢和解毒过程中发挥关键作用。此外，这些组分的过表达与几种重要疾病传播媒介对农药的抗性有关。尽管如此，单加氧酶复合体尚未从南方牛蜱微小扇头蜱（Rhipicephalus (Boophilus) microplus）中分离出来，微小扇头蜱是家畜中的一种主要疾病传播媒介。利用生物信息学分析了115个转录组序列，以鉴定假定的农药代谢CYP。采用RACE-PCR扩增了一种CYP的全长序列；CYP3006G8与已知可代谢拟除虫菊酯的CYP6和9亚家族成员具有高度同源性。通过qPCR研究了11个具有不同抗性谱的微小扇头蜱菌株中CYP3006G8的mRNA表达水平，结果表明其与拟除虫菊酯代谢抗性相关。除了该基因外，还鉴定了CPR和细胞色素b5的序列，并随后通过PCR从微小扇头蜱中分离出来。CYP3006G8是仅从微小扇头蜱中分离出的第三个CYP基因，也是第一个假定可代谢农药的基因。表达分析的初步结果表明CYP3006G8可代谢拟除虫菊酯，但需要进一步的生化表征来证实这一点。已证明人类和蚊子的CPR在NADPH结合方面的动力学参数存在差异，这可能潜在地用于设计物种特异性农药。蜱的CPR中存在类似差异将证实这是吸血节肢动物的一个特征。
