核桃举肢蛾和桃蛀螟幼虫肠道细菌多样性的PCR-DGGE和T-RFLP分析

doi:10.11707/j.1001-7488.20160609

摘要/Abstract

摘要： [目的] 了解危害核桃果实青皮的核桃举肢蛾和桃蛀螟幼虫肠道细菌群落结构与多样性。[方法] 采用基于16S rRNA基因的变性梯度凝胶电泳(PCR-DGGE)和末端限制性片段长度多样性(T-RFLP)技术分析2种害虫幼虫肠道细菌群落特征。[结果] 1)PCR-DGGE分析显示核桃举肢蛾幼虫肠道细菌主要有沃尔巴克氏体属、肠球菌属、肠杆菌属、根瘤菌属和假单胞菌属,其中沃尔巴克氏体属是优势菌群,占测得序列的40.0%;桃蛀螟幼虫肠道细菌主要有沃尔巴克氏体属、肠球菌属、根瘤菌属和假单胞菌属,其中肠球菌属是优势菌群,占测得序列的62.9%。2)DGGE条带的系统发育分析显示,核桃举肢蛾幼虫肠道中,72.5%的菌株属变形菌门,12.5%的菌株属于厚壁菌门,15.0%的菌株属于拟杆菌菌门。桃蛀螟幼虫肠道中,37.0%的菌株属于变形菌门,63.0%的菌株属于厚壁菌门,未检测到拟杆菌门细菌。3)T-RFLP分析显示76 bp和122 bp T-RFs分别是桃蛀螟和核桃举肢蛾幼虫肠道的优势菌群,T-RFLP的主成分和聚类分析显示核桃举肢蛾和桃蛀螟幼虫肠道细菌群落结构明显地分为2类。4)PCR-DGGE和T-RFLP 2种方法均显示核桃举肢蛾幼虫与桃蛀螟幼虫肠道细菌多样性差异不显著但种类组成所占比例有较大差异。[结论] 初步揭示了危害核桃果实的2种不同食性昆虫幼虫肠道细菌组成特点,可为进一步探讨其肠道微生物与其食性之间的关系提供理论基础。

关键词: 核桃举肢蛾, 桃蛀螟, 肠道微生物, PCR-DGGE, T-RFLP

Abstract: [Obiective] This study was aimed to exam the structure and diversity of intestinal bacteria community from Atrijuglans hetaohei Yang and Dichocrocis punctiferalis Guenee larvae consuming walnut's green husk. [Method] The 16S rRNA PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and the terminal-restriction fragment length polymorphism (T-RFLP) were employed to investigate the characteristics of intestinal bacteria community from the two main pests. [Results] 1)PCR-DGGE showed that the intestinal tract of A. hetaohei larvae had Wolbachia sp., Enterococcus sp., Enterobacter sp., Rhizobium sp. and Pseudomonas sp., with phylotypes of Wolbachia being the predominant group, accounted for 40% of the measured sequence; the intestinal tract of D. punctiferalis larvae had Wolbachia sp., Enterococcus sp., Rhizobium sp. and Pseudomonas sp., with phylotypes of Enterococcus being the predominant group, accounted for 62.9% of the measured sequence. 2)Phylogenetic analysis of the DGGE band revealed that in all the isolated strains, 72.5% of the strains belonged to Proteobacteria, 12.5% to Firmicutes, and 15% to Bacteroidetes in the intestinal tract of A. hetaohei larvae; 37% of the strains belonged to Proteobacteria and 63% belonged to Firmicutes in the intestinal tract of D. punctiferalis larvae.3)T-RFLP data showed 76 bp and 122 bp T-RFs were the predominant groups in the intestinal tract of A. hetaohei and D. punctiferalis larvae, respectively. Principal component analysis (PCA) and cluster analysis indicated that there were two structure categories for the intestinal bacteria communities in A. hetaohei and D. punctiferalis. 4)Both DGGE and T-RFLP analyses showed the diversity differences of intestinal bacteria from A. hetaohei larvae and D. punctiferalis larvae were not significant, and there was a difference in the proportion of bacteria composition. [Conclusion] This study revealed the characteristics of the intestinal microbial composition in two different insects, which provided a theoretical basis for further study on the relationship between the diets and intestinal microbes of the two different diet larvae consuming walnut's green husk.

Key words: Atrijuglans hetaohei, Dichocrocis punciferalis, intestinal bacteria, PCR-DGGE, T-RFLP

中图分类号:

S718.83

王娇莉, 南小宁, 任争争, 明洁, 唐光辉. 核桃举肢蛾和桃蛀螟幼虫肠道细菌多样性的PCR-DGGE和T-RFLP分析[J]. 林业科学, 2016, 52(6): 76-85.

Wang Jiaoli, Nan Xiaoning, Ren Zhengzheng, Ming Jie, Tang Guanghui. Diversity of Intestinal Bacteria Communities from Atrijuglans hetaohei (Lepidoptera: Heliodinidae) and Dichocrocis punctiferalis (Lepidoptera: Pyralidae) Larvae Estimated by PCR-DGGE and T-RFLP Analysis[J]. Scientia Silvae Sinicae, 2016, 52(6): 76-85.

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