梭菌(Clostridium)是一類與人類關(guān)系非常密切的細(xì)菌,。其中既有許多致病菌，如產(chǎn)生外毒素的破傷風(fēng)梭菌和肉毒梭菌等,；也有一些具有重要工業(yè)應(yīng)用價(jià)值或潛力的梭菌,，如丙酮丁醇梭菌和熱纖梭菌等。
基因失活或基因刪除是細(xì)菌功能基因組學(xué)研究的基本手段,。近年來,，基于乳酸乳球菌II型內(nèi)含子剪切機(jī)制開發(fā)的梭菌內(nèi)含子系統(tǒng)，顯著提高了梭菌基因插入失活的效率,。但若要在梭菌中通過雙交換同源重組來刪除基因,，仍然非常困難，鮮有成功案例,。
中科院微生物研究所的賈開志博士與其合作導(dǎo)師李寅研究員受到II型內(nèi)含子可高效插入梭菌基因組的啟發(fā),，設(shè)計(jì)了一個(gè)將逆轉(zhuǎn)錄轉(zhuǎn)座與同源重組結(jié)合起來的基因刪除策略。這一策略的第一步是在II型內(nèi)含子的下游引入與擬刪除基因上游同源的一段序列（簡稱為同源序列）,；第二步是通過逆轉(zhuǎn)錄轉(zhuǎn)座作用,，將攜帶有同源序列的II型內(nèi)含子插入梭菌基因組；第三步是篩選單交換同源重組的基因型,，獲得目標(biāo)基因,、操縱元或基因簇得以刪除的突變株。
采用這一新策略,，研究人員對位于丙酮丁醇梭菌染色體及大質(zhì)粒上的兩個(gè)操縱元進(jìn)行了有效刪除,。他們將這一策略稱為“II型內(nèi)含子錨定的基因刪除”——通過II型內(nèi)含子的高效逆轉(zhuǎn)錄轉(zhuǎn)座作用，增加了II型內(nèi)含子攜帶的同源序列與基因組DNA發(fā)生同源重組的可能性,。理論上,，只要能夠應(yīng)用II型內(nèi)含子插入失活系統(tǒng)的梭菌（甚至其他細(xì)菌），都可以采用這一策略來實(shí)現(xiàn)基因刪除,。
(生物谷Bioon.com)
生物谷推薦英文摘要：
PLoS ONE 6(1): e16693. doi:10.1371/journal.pone.0016693
Group II Intron-Anchored Gene Deletion in Clostridium
Kaizhi Jia, Yan Zhu, Yanping Zhang, Yin Li*
Clostridium plays an important role in commercial and medical use, for which targeted gene deletion is difficult. We proposed an intron-anchored gene deletion approach for Clostridium, which combines the advantage of the group II intron “ClosTron” system and homologous recombination. In this approach, an intron carrying a fragment homologous to upstream or downstream of the target site was first inserted into the genome by retrotransposition, followed by homologous recombination, resulting in gene deletion. A functional unknown operon CAC1493–1494 located in the chromosome, and an operon ctfAB located in the megaplasmid of C. acetobutylicum DSM1731 were successfully deleted by using this approach, without leaving antibiotic marker in the genome. We therefore propose this approach can be used for targeted gene deletion in Clostridium. This approach might also be applicable for gene deletion in other bacterial species if group II intron retrotransposition system is established.
