烟粉虱不同发育阶段解毒代谢酶基因的特异性表达

Differential expression of the detoxification enzyme genes in different developmental stages of the whitefly,Bemisia tabaci( Hemiptera: Aleyrodidae)

【目的】基于烟粉虱Bemisia tabaci转录组数据,系统分析了烟粉虱解毒代谢酶基因在噻虫嗪抗性品系中的表达模式,探讨了这些基因在烟粉虱不同发育阶段差异表达的生物学意义。【方法】分别收集室内长期饲养的烟粉虱噻虫嗪抗性和敏感品系的卵、4龄若虫和刚羽化1 d的雌成虫,在烟粉虱转录组数据库中挑选8 394条解毒代谢相关基因设计探针,通过探针杂交,得到烟粉虱噻虫嗪抗性品系表达谱芯片,比较了这些基因在抗性烟粉虱3个不同发育阶段的表达情况。并随机挑选了9个基因,在抗感品系间3个不同发育阶段进行了荧光定量PCR验证。【结果】在抗性烟粉虱的卵和4龄若虫发育阶段,共有3 424个差异表达基因,其中489个是编码3类解毒代谢酶(羧酸酯酶、谷胱甘肽-S-转移酶和细胞色素P450多功能氧化酶)的基因;有14个基因在4龄若虫发育阶段过量表达,其中10个为P450基因,4个为GST家族基因。在抗性烟粉虱的4龄若虫和雌成虫发育阶段,总共有1 273个差异表达基因,193个为3类解毒代谢酶家族的基因,其中有9个P450家族基因在雌成虫期的表达量超过4龄若虫期的10倍。此外,表达谱芯片分析还筛选到了一些候选抗性基因。qRT-PCR验证显示在这些候选基因中,与敏感品系相比,9个基因在抗性烟粉虱的3个不同发育阶段表达上调,其中GST基因家族的p_06027和P450基因p_06013在抗性品系的卵和4龄若虫中过量表达;p_05885和p_07806和编码CYP6家族蛋白的p_00988在抗性品系的4龄若虫期的表达量上调;p_05916和p_00478在抗性品系卵和4龄若虫期表达量很低,而在成虫期过量表达;而p_00059和p_00428在抗性品系雌成虫发育阶段表达量显著上调,其中编码CYP4C1的p_00059的差异表达倍数在雌成虫期约为15.15倍。【结论】表达谱芯片分析结果提示,CYP6和CYP4C1基因的过量表达可能会是烟粉虱抗性产生的机制之一。解毒代谢酶基因在烟粉虱不同发育阶段的特异性表达,可能与其在抵御杀虫剂胁迫时体内能量的分布及有效利用率有关,也可能是害虫在环境选择压下的一种适应机制。

【Aim】Based on the transcriptome data of the whitefly,Bemisia tabaci,this study aims to systematically analyze the expression profiles of genes encoding detoxification enzymes in thiamethoxam resistant strain,and to focus on discussing the biological significance of developmental stage specific differential expression of these genes. 【Methods】Thiamethoxam resistant（ TH-R） and susceptible（ THS） strains of B. tabaci were maintained in a greenhouse,and individuals at three developmental stages（ egg,the 4th instar nymph and 1 day-old unmated female adult） were collected from the TH-R and THS strains. The array design was based on 8 394 ESTs from transcriptome of B. tabaci with putative association with insecticide resistance. By using a custom made microarray,the gene expression in eggs,nymphs and female adults of the TH-R strain of B. tabaci was compared. Nine ESTs were randomly selected for quantitative real-time PCR（ qRT-PCR） analysis to validate the microarray response between the TH-R and TH-S strains at three different developmental stages. 【Results】In the TH-R strain,a total of 3 424 ESTs were differentially expressed between the egg and 4th instar nymphal stages. Among them,489 ESTs encode detoxification enzymes（ carboxylesterase,glutathione S-transferases,cytochrome P450monooxygenases）. Fourteen ESTs（ 10 ESTs encoding P450 s, and 4 ESTs encoding GSTs） were overexpressed in the 4th instar nymphal stage. There were 1 273 ESTs which were differentially expressed between the 4th instar nymphal and female adult stages. Among them,193 genes encoding detoxification enzymes and nine ESTs encoding P450 s showed higher expression level（ 10-fold） in the female adult stage rather than the 4th instar nymphal stage. The qRT-PCR results revealed that nine ESTs were upregulated in the TH-R strain at three developmental stages compared to those in the TH-S strain. The EST p_06027 and the EST p_06013 encoding P450 s were overexpressed in egg and the 4th instar nymphal stage of the TH-R strain. The ESTs p_05885,p_07806 and the EST p_00988 encoding P450 s were highly overexpressed in the 4th instar nymphal stage of the TH-R strain. The ESTs p_05916 and p_00478had low expression levels in the egg and 4th instar nymphal stages but overexpressed in the female adult stage of the TH-R strain. The ESTs p_00059 and p_00428 were significantly up-regulated in the female adult stage but had low expression level in the other two immature stages of TH-R strain,and the EST p_00059 encoding CYP4C1 with the highest expression level（ - 15. 15-fold） was seen in the female adult stage. 【Conclusion 】 The results suggest that the overexpression of CYP4C1 and CYP6 B may underlie the resistance to thiamethoxam in B. tabaci. According to the microarray data analyzed,the detoxification enzyme genes are differentially expressed in different developmental stages of B. tabaci,which may be associated with the distribution and effective utilization of energy under the stress of insecticides and also an adaptive mechanism under the environmental selection pressures.