茶尺蠖和灰茶尺蠖内共生菌Wolbachia的分子检测及序列分析
Molecular detection and sequence analysis of Wolbachia strains inEctropis obliqua and Ectropis grisescens (Lepidoptera: Geometridae)
周孝贵1, 2, 3** 付建玉2, 3 刘守安2, 3毛腾飞2, 3 肖 强2, 3***
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DOI：10.7679/j.issn.2095-1353.2016.097
作者单位：1. 浙江大学昆虫科学研究所，杭州 310058；2. 中国农业科学院茶叶研究所，杭州 310008；3. 农业部茶树生物学与资源利用重点实验室，杭州 310008
中文关键词：茶尺蠖，灰茶尺蠖，Wolbachia，16S rRNA，ftsZ，wsp，序列分析
英文关键词： Ectropis obliqua, Ectropis grisescens, Wolbachia, 16S rRNA, ftsZ, wsp, sequence analysis
中文摘要：
【目的】 对茶尺蠖Ectropis obliqua及其近缘种灰茶尺蠖E. grisescens体内共生菌Wolbachia进行分子鉴定，确定两者体内Wolbachia的感染率及其进化地位，为进一步探讨其对茶尺蠖和灰茶尺蠖的潜在影响提供科学依据。【方法】 采用Wolbachia的16S rRNA、ftsZ和wsp基因特异性引物，通过PCR扩增法检测了我国3个茶尺蠖地理种群（浙江杭州、余杭和江苏无锡）和3个灰茶尺蠖地理种群（浙江新昌、湖北浠水和江西南昌）中Wolbachia的感染情况，并进行测序和序列分析。【结果】 茶尺蠖和灰茶尺蠖都感染了Wolbachia，灰茶尺蠖的Wolbachia感染率为100%，但茶尺蠖的Wolbachia感染率在22%～95%，且PCR产物电泳得到的条带微弱。wsp序列在茶尺蠖和灰茶尺蠖种间、种内无差异；但16S rRNA序列在茶尺蠖和灰茶尺蠖种间、种内差异为0.362%～0.727%之间；茶尺蠖样本未成功扩增出ftsZ序列，灰茶尺蠖样本获得2条ftsZ基因序列差异为1.647%。基于Wolbachia的16S rRNA和wsp基因构建的系统发育树表明，本研究中茶尺蠖和灰茶尺蠖种群所感染的Wolbachia全部属于B组的Pip亚组。【结论】 茶尺蠖和灰茶尺蠖均被B组Pip亚组的Wolbachia感染，但感染率相差很大，这为研究Wolbachia对茶尺蠖和灰茶尺蠖生物学及生态学的影响奠定了基础。
英文摘要：
[Objectives] The infection rates and taxonomic relationships between Wolbachia strains in the sister species Ectropis obliqua and E. grisescens (Lepidoptera: Geometridae) were identified by molecular methods in order to reveal the potential effects of this endosymbiont on these species. [Methods] Wolbachia infection in three populations of E. obliqua (Hangzhou and Yuhang, Zhejiang province; Wuxi, Jiangsu Province) and three populations of E. grisescens (Xinchang, Zhejiang Province; Xishui, Hubei Province; and Nanchang, Jiangxi Province) were detected using PCR with three specific primers for Wolbachia 16S rRNA and the Wolbachia ftsZ and wsp genes. All Wolbachia 16S rRNA, ftsZ and wsp genes from the three E. grisescens populations and some Wolbachia 16S rRNA and wsp genes from the three E. obliqua populations, were sequenced and analyzed. [Results] All E. obliqua and E. grisescens populations were infected with Wolbachia. Infection rates of the three E. grisescens populations were all 100%, but ranged from 22% to 95% among the three E. obliqua populations, with positive PCRs showing only weak bands in the electrophoretogram. There were no intra- and interspecific differences in the wsp sequences of E. obliqua and E. grisescens, however, intra- and interspecific differences in 16S rRNA sequences were between 0.362% and 0.727%, respectively. There was a 1.647% difference in two ftsZ haplotypes from E. grisescens, moreover, we were unable to successfully amplify ftsZ from all E. obliqua individuals. A phylogenetic tree based on the 16S rRNA and wsp sequences showed that all strains of Wolbachia detected belong to the Pip subgroup of the B group. [Conclusion] All three E. obliqua and E. grisescens populations tested are infected with Wolbachia strains belonging to the Pip subgroup of the B group, but the infection rate varies significantly between E. obliqua and E. grisescens. These results provide the basis for future study of the effects of Wolbachia on the biology and ecology of E. obliqua and E. grisescens.