苏云金芽胞杆菌转录调控因子CodY的体外靶基因的筛选

Genome-wide identification of CodY target genes in Bacillus thuringiensis by in vitro binding analysis

摘要:

[目的] 利用Avi-tag和Pull down技术建立体外靶基因筛选的新方法,在苏云金芽胞杆菌YBT-1520基因组范围内进行体外高通量筛选转录调控因子CodY的靶基因,为深入研究CodY在苏云金芽胞杆菌中的功能及调控网络打下基础。[方法] 用Avi-tag技术对CodY蛋白进行体外生物素标记,酶切苏云金芽胞杆菌YBT-1520总DNA并在两端加上便于测序的adapter序列,用链霉亲和素树脂筛选与CodY蛋白相互作用的靶基因。[结果] 在苏云金芽胞杆菌中得到了46条CodY可以直接作用的靶序列,分析发现CodY在苏云金芽胞杆菌中主要调控氨基酸代谢、糖代谢、脂肪酸代谢、转运、调控、DNA修复、双组份调控等的转录。[结论] 本研究采用的Avi-tag技术具有特异性强、操作简单、成本低等优势,为转录因子体外靶序列的高通量筛选提供新的方法;苏云金芽胞杆菌中CodY靶基因的筛选为其他微生物中CodY的功能研究打下基础。

Abstract:

[Objective] This study is aimed to establish an in vitro pull down method combining Avi-tag technology to screen and indentify the CodY targets in the whole genomes of Bacillus thuringiensis strain YBT-1520. The method offers a potential biocatalyst forscreening target sequences of transcription factors in vitro. [Methods] CodY proteins were labeled with biotin using Avi-tag technology. B. thuringiensis strain YBT-1520 genomes were enzymatically digested to 100-400 bp DNA fragments that had adapters with known sequences on both ends. Then, the target genes of CodY protein were screened by streptavidin biotin resin. [Results] The 46 targets of CodY were mainly involved in amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, transport, regulation, DNA replication repair, and two component regulatory systems. [Conclusion] Compared with chromatin immune co-precipitation technology in vivo, this pull down method is simple, cheap, fast and specific to biotin streptavidin system.