浒苔(Ulva prolifera)共附生细菌群落结构分析的引物优化

Primers modification for analysis of Ulva prolifera-associated bacterial community

海藻共附生细菌密切参与宿主的生长发育、营养吸收等重要过程,浒苔(Ulva prolifera)是构成黄海绿潮的唯一优势种,对其共附生细菌开展群落结构分析,有望为成灾机制研究提供重要线索。由于植物叶绿体基因组同时具备原核特征和高拷贝数,会严重干扰共附生细菌基于16S rDNA的序列扩增。高等植物中开发了通用引物799F,利用其3′末端与植物同源区的非配对双碱基,可实现对细菌来源序列的特异性扩增。本文针对多个浒苔群体样本,首次评价了引物799F在藻类中的适用性,发现799F的3′末端与浒苔叶绿体同源区仅存在非配对单碱基,其扩增仍会受到浒苔叶绿体严重干扰。在引物799F的基础上,设计了新引物800F,与通用反向引物1492R配对扩增细菌16S rDNA中具高分辨率的V5-V9可变区,配合使用具热启动功能、同时不具校对功能的DNA聚合酶,可基本完全避免浒苔叶绿体来源序列的扩增,并阐明了方法的原理。优化的引物扩增方案可用于浒苔共附生细菌群落结构分析。

Macroalgal-associated bacteria play key roles in terms of algal growth and nutrition acquisition.Ulva prolifera is the only dominated species of green tides in the Yellow Sea.The study of the community structure of associated bacteria would contribute to reveal bacteria’s key function in the blooming of their host.Due to a pro-karyotic origin and high copy numbers,the genomic DNA of plant chloroplast usually interferes with the amplifica-tion of 16S rDNA fragments from associated bacteria.To overcome this issue in higher plants,a universal primer 799F,which has a two-bp mismatched 3’end with chloroplast DNA was developed to specifically amplify fragments only from bacteria.In this study,for the first time,the primer 799F was evaluated in seaweed considering different strains of U.prolifera.However,the failure of bacteria-bias amplification was revealed and attributed to only one-bp mismatched 3′end of primer 799F with determined U.prolifera chloroplast DNA.According to the com-parison and modification,a new primer 800F was designed based on 799F.Coupled with the universal reverse primer 1492R,the V5 V9 region from the 16S rDNA of bacteria could be amplified without almost any interference from Ulva chloroplast when hot-start and non-proofreading DNA polymerase were used.This study clarifies the principle of modified profile with new primer,which can be applied to the analysis of community structure of U.prolifera-associated bacteria.