Large-scale genome-mining analyses have revealed that microbes potentially harbor a huge reservoir of unchar-acterized natural product(NP)biosynthetic gene clusters(BGCs),and this has spurred a renaissance of novel dr...Large-scale genome-mining analyses have revealed that microbes potentially harbor a huge reservoir of unchar-acterized natural product(NP)biosynthetic gene clusters(BGCs),and this has spurred a renaissance of novel drug discovery.However,the majority of these BGCs are often poorly or not at all expressed in their native hosts under laboratory conditions,and thus are regarded as silent/orphan BGCs.Currently,connecting silent BGCs to their corresponding NPs quickly and on a large scale is particularly challenging because of the lack of universal strategies and enabling technologies.Generally,the heterologous host-based genome mining strategy is believed to be a suitable alternative to the native host-based approach for prioritization of the vast and ever-increasing number of uncharacterized BGCs.In the last ten years,a variety of methods have been reported for the direct cloning of BGCs of interest,which is the first and rate-limiting step in the heterologous expression strategy.Es-sentially,each method requires that the following three issues be resolved:1)how to prepare genomic DNA;2)how to digest the bilateral boundaries for release of the target BGC;and 3)how to assemble the BGC and the capture vector.Here,we summarize recent reports regarding how to directly capture a BGC of interest and briefly discuss the advantages and disadvantages of each method,with an emphasis on the notion that direct cloning is very beneficial for accelerating genome mining research and large-scale drug discovery.展开更多
microRNA（miRNA）是生物中一类起负调控作用的非编码的小分子RNA,主要在转录后水平上调节生物体的生长与发育。直接克隆法是鉴定植物中miRNA最常用也是最有效的方法之一。利用直接克隆法分离了陆地棉开花后0~10 d（days post anthesis,...microRNA（miRNA）是生物中一类起负调控作用的非编码的小分子RNA,主要在转录后水平上调节生物体的生长与发育。直接克隆法是鉴定植物中miRNA最常用也是最有效的方法之一。利用直接克隆法分离了陆地棉开花后0~10 d（days post anthesis,DPA）胚珠中总RNA,从中筛选17~24 nt小分子RNA构建cDNA文库,对初筛得到的阳性克隆进行测序。通过与miRNA数据库比对,最终获得4个保守的miRNA。进一步对他们的转录表达进行分析,发现所有miRNA在棉花胚珠纤维不同发育时期均能表达。其中miR167a、miR172c和miR394b在棉花幼苗的根茎叶中有不同程度的表达。预测得到55个miRNA的靶基因,多数靶基因编码转录因子、代谢酶类以及发育相关蛋白,进一步证明miRNA参与调控棉花纤维的形态建成、发育等各项生理活动。展开更多
基金supported by the National Key Research and Development Program of China(2021YFC2100600)the National Natural Science Foundation of China(31870034)the Science and Technology Commission of Shanghai Municipality(20ZR1469100).
文摘Large-scale genome-mining analyses have revealed that microbes potentially harbor a huge reservoir of unchar-acterized natural product(NP)biosynthetic gene clusters(BGCs),and this has spurred a renaissance of novel drug discovery.However,the majority of these BGCs are often poorly or not at all expressed in their native hosts under laboratory conditions,and thus are regarded as silent/orphan BGCs.Currently,connecting silent BGCs to their corresponding NPs quickly and on a large scale is particularly challenging because of the lack of universal strategies and enabling technologies.Generally,the heterologous host-based genome mining strategy is believed to be a suitable alternative to the native host-based approach for prioritization of the vast and ever-increasing number of uncharacterized BGCs.In the last ten years,a variety of methods have been reported for the direct cloning of BGCs of interest,which is the first and rate-limiting step in the heterologous expression strategy.Es-sentially,each method requires that the following three issues be resolved:1)how to prepare genomic DNA;2)how to digest the bilateral boundaries for release of the target BGC;and 3)how to assemble the BGC and the capture vector.Here,we summarize recent reports regarding how to directly capture a BGC of interest and briefly discuss the advantages and disadvantages of each method,with an emphasis on the notion that direct cloning is very beneficial for accelerating genome mining research and large-scale drug discovery.
文摘microRNA（miRNA）是生物中一类起负调控作用的非编码的小分子RNA,主要在转录后水平上调节生物体的生长与发育。直接克隆法是鉴定植物中miRNA最常用也是最有效的方法之一。利用直接克隆法分离了陆地棉开花后0~10 d（days post anthesis,DPA）胚珠中总RNA,从中筛选17~24 nt小分子RNA构建cDNA文库,对初筛得到的阳性克隆进行测序。通过与miRNA数据库比对,最终获得4个保守的miRNA。进一步对他们的转录表达进行分析,发现所有miRNA在棉花胚珠纤维不同发育时期均能表达。其中miR167a、miR172c和miR394b在棉花幼苗的根茎叶中有不同程度的表达。预测得到55个miRNA的靶基因,多数靶基因编码转录因子、代谢酶类以及发育相关蛋白,进一步证明miRNA参与调控棉花纤维的形态建成、发育等各项生理活动。
