高通量分析技术在资源微生物及功能基因发掘中的应用

Application of high-throughput techniques in the discovery of microbial resources and functional genes

摘要:

随着新的微生物资源不断被发现以及微生物基因组测序数据的积累和完善，目前研究重点和难点是如何从大量数据中快速发现和鉴定与微生物重要表型相关的功能基因，这就需要高通量的分析研究手段，主要涉及建库和筛选两个主要技术单元。其中，建库是指构建能够覆盖微生物全基因组的突变或干扰文库，所涉及的技术包括宏基因组、转座子插入突变、RNA干扰(RNA interference，RNAi)、反转录子文库重组工程(retron library recombineering，RLR)、CRISPR抑制(CRISPRi)和CRISPR激活(CRISPRa)等。筛选则是通过某种胁迫压力来促使文库菌群的差异化生长，并结合高通量测序全面发掘与特定表型相关的功能基因，从而为后续研究提供有效信息。本文对功能基因组学研究中现有的高通量分析技术进行了梳理、总结和展望，以期为这类技术方法的拓展、优化以及应用提供参考。

Abstract:

In light of the continuous discovery of microbial resources and the accumulating microbial genome data, it is an urgent task to screen and identify functional genes associated with important microbial phenotypes from the enormous data, for which high-throughput techniques are indispensible. The techniques are mainly involved in library construction and screening. Library construction refers to the establishment of mutant or interference libraries covering the whole genome of target microorganisms with techniques such as metagenomics, transposon insertion, RNA interference (RNAi), retron library recombineering (RLR), CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa). As for the screening, a certain stress is often used to allow the differential growth of microbial individuals of a mutant library and then the causal relationships between specific genes and phenotypic outcomes at genomic level are identified by high-throughput sequencing. In this review, we briefly summarize the current high-throughput techniques used in functional genomics research, hoping to provide a reference to the development and optimization of these techniques in the future.