›› 2011, Vol. 54 ›› Issue (6): 664-674.doi:

• 研究论文 • 上一篇    下一篇

基于16S rDNA序列的柑桔木虱体内共生菌多样性研究

 殷幼平, 刘婷婷, 田圣超, 胡修峰, 吴东, 王中康   

  • 收稿日期:2010-02-01 出版日期:2011-06-20 发布日期:2011-06-20
  • 通讯作者: 王中康 E-mail:zkwang646@sina.com
  • 作者简介:殷幼平,女,1955年生,重庆长寿人,博士,教授,主要从事昆虫学及昆虫与微生物互作相关研究, E-mail: ypy128@sina.com
  • 基金资助:

    国家自然科学基金项目(309771875); 校企合作科研课题 (200706-201312)

Microbial diversity in Diaphorina citri (Homoptera: Psyllidae) estimated by 16S rDNA analysis using DGGE and RFLP

YIN You-Ping, LIU Ting-Ting, TIAN Sheng-Chao, HU Xiu-Feng, WU Dong, WANG Zhong-Kang   

  • Received:2010-02-01 Online:2011-06-20 Published:2011-06-20
  • Contact: WANG Zhong-Kang E-mail:zkwang646@sina.com

摘要: 昆虫消化道内是一个复杂的微生态系统, 有大量的微生物存在。这些微生物对寄主发育、 营养吸收和防御方面都起着重要的作用。本文利用基于16S rRNA基因的PCR-RFLP指纹图谱法和变性梯度凝胶电泳(denaturing gradient gel electrophoresis, DGGE)的方法对柑桔黄龙病虫-媒柑桔木虱Diaphorina citri体内细菌菌群多样性进行了研究。经PCR-RFLP分析显示31条序列与变形菌门的假单胞菌科、肠杆菌科、黄单胞菌科、伯克氏菌科、立克次氏菌科和根瘤菌科细菌具有较高同源性。柑桔木虱内共生细菌的优势菌群依次为合胞体共生菌(syncytium endosymbiont)(同源性99%, 5条序列, 分离频率31%)、 Candidatus Carsonella ruddii和Mycetocyte 内共生菌(同源性98%, 5条序列, 分离频率31%)以及亚洲韧皮杆菌(Candidatus Liberibacter asiaticus)和内共生菌Wolbachia。对柑桔木虱内生细菌16S rDNA V3区序列的PCR-DGGE分析, 条带相似性的UPMAGA聚类分析表明, 采自九里香Murraya paniculata的柑桔木虱内生细菌大多聚为一支, 而来自柑桔的聚在另一支, 说明寄主差异对柑桔木虱内生细菌菌群构成的影响大于地理位置的影响。将PCR-DGGE条带测序, 序列经GenBank序列比对发现柑桔木虱内生细菌主要属于变形菌门假单胞菌科、 立克次氏菌科、 肠杆菌科、 黄单胞菌科以及厚壁菌门链球菌科和芽孢杆菌科。合胞体共生菌(条带3-4)因其稳定存在于木虱体内且不随柑桔木虱寄主和地理位置的改变而变化, 可能是柑桔木虱体内的优势共生菌; 内共生细菌Wolbachia也在柑桔木虱内稳定存在, 表明我国柑桔木虱感染Wolbachia是普遍现象。PCR-RFLP和PCR-DGGE两种方法相结合较好地反映了柑桔木虱内生细菌菌群的多样性, 而且均显示出柑桔木虱内的合胞体共生菌是主要的优势菌群。

关键词: 柑桔木虱, 共生菌, 16S rDNA, 细菌菌群多样性, 变性梯度凝胶电泳(DGGE), RFLP分析

Abstract: The internal gut of insects is a complex micro-ecosystem, in which inhabits a large and varied microbial community. This microbial community plays an important role for their hosts’ growth, development, digestion and absorption of nutrition, colonization resistance against invasion of exotic microbes. In this paper, the diversity of bacterial flora of citrus psyllids (Diaphorina citri), which is a vector of the Huanglongbing pathogen Candidatus Liberibacter asiaticus, was investigated based on analysis of 16S rDNA sequences by restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). The PCR-RFLP result showed that 31 sequences share high homology with Proteobacteria, including Pseudomonadaceae, Enterobacteriaceae, Xanthomonadaceae, Burkholderiaceae, Rickettsiaceae and Rhizobiaceae. The dominant bacterial flora in D. citri included syncytium endosymbiont (5 sequences, homology 99%, isolating frequency 31%), Mycetocyte endosymbiont and Candidatus Carsonella ruddii (5 sequences, homology 98% and isolating frequency 31%), Candidatus Liberibacter asiaticus and Wolbachia. The analysis of 16S rDNA V3 region sequencing by PCR-DGGE revealed that the inner bacteria of D. citri collected from Murraya paniculata were clustered in one branch and those from citrus were clustered in another branch, suggesting that the host plant has more obvious impact on the bacteria flora of D. citri than the geographic location. The sequencing of the isolated bands of DGGE and GenBank alignment showed that the bacteria of D. citri mainly belong to Proteobacteria and Firmicutes, including Pseudomonadaceae, Rickettsiaceae, Enterobacteriaceae, Xanthomonadaceae, Streptococcaceae and Bacillaceae. Syncytium endosymbiont (band 3-4) may be predominating population because it can be stably isolated from D. citri no matter what the host plant is and where the geographic location. The high isolation rate of Wolbachia inside of citrus psyllids revealed that D. citri being infected with Wolbachia is a common phenomenon in China. Both PCR-RFLP and DGGE analysis indicated that the syncytium endosymbiont of D. citri is a dominant bacterial flora and the two methods in combination can reveal the microbial diversity in D. citri conveniently.

Key words:  Diaphorina citri, endosymbiont bacteria, 16S rDNA, microbial diversity, PCR-DGGE, RFLP