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棉花学报 ›› 2022, Vol. 34 ›› Issue (1): 23-32.doi: 10.11963/cs20210027

• 研究与进展 • 上一篇    下一篇

基于改进的荧光原位杂交技术定位B型烟粉虱内共生菌PortieraWolbachia

卢珍华1(),闫晓会1,马亚杰1,单永潘1,宋贤鹏1,刘青梅2,王丹1,胡红岩1,*(),马艳1,*()   

  1. 1.中国农业科学院棉花研究所/棉花生物学国家重点实验室,河南 安阳 455000
    2.安阳县农业农村局,河南 安阳 455000
  • 收稿日期:2021-04-24 出版日期:2022-01-15 发布日期:2022-05-31
  • 通讯作者: 胡红岩,马艳 E-mail:luh_2053@163.com;huhongyan1986@163.com;aymayan@126.com
  • 作者简介:卢珍华(1993―),男,硕士研究生, luh_2053@163.com
  • 基金资助:
    国家自然科学基金(31801751);中央级公益性科研院所基本科研业务费专项(1610162021029);中国农业科学院科技创新工程(CAAS-ASTIP-CCRI)

Localization of endosymbionts Portiera and Wolbachia in Bemisia tabaci based on improved fluorescence in situ hybridization

Lu Zhenhua1(),Yan Xiaohui1,Ma Yajie1,Shan Yongpan1,Song Xianpeng1,Liu Qingmei2,Wang Dan1,Hu Hongyan1,*(),Ma Yan1,*()   

  1. 1. Institute of Cotton Research, Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology, Anyang, Henan 455000, China
    2. Anyang County Bureau of Agriculture and Rural Affairs, Anyang, Henan 455000, China
  • Received:2021-04-24 Online:2022-01-15 Published:2022-05-31
  • Contact: Hu Hongyan,Ma Yan E-mail:luh_2053@163.com;huhongyan1986@163.com;aymayan@126.com

摘要:

【目的】共生菌Wolbachia在烟粉虱种群分化和物种形成中起重要作用。旨在采用荧光原位杂交技术,明确初生共生菌Portiera和次生共生菌Wolbachia在烟粉虱体内的分布。【方法】利用2种共生菌的特异性荧光探针对烟粉虱整虫进行荧光原位杂交,研究其在烟粉虱不同发育阶段的分布;在磷酸缓冲液中对烟粉虱成虫腹部进行组织解剖,利用优化的荧光原位杂交流程对获得的组织样本进行荧光染色、观察。【结果】对烟粉虱整虫进行荧光染色,在烟粉虱各个发育阶段虫体中均检测到PortieraWolbachia的荧光信号;在卵和若虫中,这2种共生菌集中分布于含菌细胞中,在成虫中,这2种共生菌所在的具体位置不清晰;成虫腹部组织染色结果显示,PortieraWolbachia除集中分布于含菌细胞中外,在卵巢中也有分布。【结论】利用本研究优化建立的昆虫组织染色方法,可以清晰观察烟粉虱成虫卵巢及含菌细胞中PortieraWolbachia的分布,可为其他昆虫及组织内共生菌的荧光染色观察提供参考。

关键词: 烟粉虱; 内共生菌; Portiera; Wolbachia; 定位; 荧光原位杂交; 方法改进

Abstract:

[Objective] Whitefly, Bemisia tabaci is one of the most invasive insect pest, and its endosymbiont Wolbachia plays an important role in population differentiation and speciation of this pest insect. The aim of this study is to identify the localization and distribution of the primary endosymbiont Portiera and the secondary endosymbiont Wolbachia, by using the fluorescence in situ hybridization (FISH). [Method] Whitefly samples were subjected to whole-mount FISH using two specific fluorescent probes to detect Portiera and Wolbachia in the B. tabaci at different developmental stages. The abdominal tissue samples of adult B. tabaci dissected in phosphate buffer saline (PBS) were stained using an optimized FISH protocol to recognize the localization of the endosymbionts. [Result] When whitefly samples were subjected to whole-mount FISH, hybridization signals were observed at different developmental stages of the B. tabaci. Portiera and Wolbachia were localized inside of the bacteriocytes in eggs and nymphae. However, the localization of these two bacteria in adults of B. tabaci was obscured. The staining results of the dissected samples showed that Portiera and Wolbachia were located inside of the bacteriocyte and ovary of B. tabaci. [Conclusion] An optimized fluorescence staining method of insect tissue was established in this study. The method can be used for the observation of the localization of these two endosymbionts in the bacteriocyte and ovary of B. tabaci, and can provide references for the observation of fluorescence staining of other insects and tissue.

Key words: Bemisia tabaci; endosymbiont; Portiera; Wolbachia; localization; fluorescence in situ hybridization; method improvement