近期,，中科院武漢病毒研究所周寧一研究員課題組在基因組內(nèi)16S rRNA基因的差異性而導(dǎo)致的原核生物多樣性高估研究方面取得重要進(jìn)展,。相關(guān)結(jié)果發(fā)表在10月的微生物學(xué)刊物Applied and Environmental Microbiology上，并被本期雜志選為亮點(diǎn)文章（Spotlight: Articles of Significant Interest Selected from This Issue by the Editors）,。
自從Carl Woese應(yīng)用16S rRNA基因來(lái)確定原核生物的親緣關(guān)系，這個(gè)方法已成為原核生物系統(tǒng)發(fā)育和分子生態(tài)學(xué)研究中公認(rèn)的經(jīng)典標(biāo)準(zhǔn),。然而，該基因在原核生物內(nèi)往往同時(shí)存在多個(gè)拷貝,，而且拷貝之間的基因序列并不完全一致。因此,，在原核生物生態(tài)學(xué)研究中,，基于16S rRNA基因的菌群多樣性分析會(huì)引起一定程度的高估,。該組從目前2013個(gè)測(cè)序的原核生物基因組中,，對(duì)16S rRNA基因的拷貝數(shù)及基因組內(nèi)部異質(zhì)性做了詳細(xì)的分析研究，發(fā)現(xiàn)細(xì)菌基因組中包含1-15個(gè)拷貝,，古菌基因組中包含1-4個(gè)拷貝,。在952個(gè)基因組（隸屬于585個(gè)種）內(nèi)發(fā)現(xiàn)16S rRNA基因存在異質(zhì)性,。并發(fā)現(xiàn)16S rRNA基因不同區(qū)域存在不同程度的異質(zhì)性，因而利用不同區(qū)域的序列進(jìn)行基于焦磷酸測(cè)序的分子生態(tài)學(xué)研究會(huì)造成不同程度的多樣性高估,。
本研究指出，對(duì)于經(jīng)常用來(lái)進(jìn)行焦磷酸測(cè)序的區(qū)域中，V4-V5區(qū)域顯示了最低的高估程度（約為3.0%）,，而V6區(qū)域的高估程度最高（約為13%）。因此在原核生物分子生態(tài)學(xué)研究中,，16S rRNA基因的V4-V5區(qū)域更適合作為焦磷酸測(cè)序的目的片段,。這是迄今為止最全面的關(guān)于16S rRNA基因的基因組內(nèi)差異的研究,，本研究不但提供了在針對(duì)不同的16S rRNA基因區(qū)段時(shí)不同豐度的高估程度,，而且提出了可利用V4-V5區(qū)來(lái)降低這一高估（生物谷Bioon.com）,。
生物谷推薦的英文摘要
Applied and Environmental Microbiology doi: 10.1128/AEM.01282-13
Intragenomic Heterogeneity of 16S rRNA Genes Causes Overestimation of Prokaryotic Diversity
Dong-Lei Suna,， Xuan Jianga,， Qinglong L. Wub and Ning-Yi Zhoua
Ever since Carl Woese introduced the use of 16S rRNA genes for determining the phylogenetic relationships of prokaryotes,， this method has been regarded as the “gold standard” in both microbial phylogeny and ecology studies. However， intragenomic heterogeneity within 16S rRNA genes has been reported in many investigations and is believed to bias the estimation of prokaryotic diversity. In the current study,， 2,，013 completely sequenced genomes of bacteria and archaea were analyzed and intragenomic heterogeneity was found in 952 genomes (585 species),， with 87.5% of the divergence detected being below the 1% level. In particular， some extremophiles (thermophiles and halophiles) were found to harbor highly divergent 16S rRNA genes. Overestimation caused by 16S rRNA gene intragenomic heterogeneity was evaluated at different levels using the full-length and partial 16S rRNA genes usually chosen as targets for pyrosequencing. The result indicates that， at the unique level,， full-length 16S rRNA genes can produce an overestimation of as much as 123.7%， while at the 3% level,， an overestimation of 12.9% for the V6 region may be introduced. Further analysis showed that intragenomic heterogeneity tends to concentrate in specific positions,， with the V1 and V6 regions suffering the most intragenomic heterogeneity and the V4 and V5 regions suffering the least intragenomic heterogeneity in bacteria. This is the most up-to-date overview of the diversity of 16S rRNA genes within prokaryotic genomes. It not only provides general guidance on how much overestimation can be introduced when applying 16S rRNA gene-based methods， due to its intragenomic heterogeneity,， but also recommends that,， for bacteria， this overestimation be minimized using primers targeting the V4 and V5 regions.
