DNA的G-四链体(G-quadruplex，G4)是由富含串联重复的鸟嘌呤(guanine，G)的核酸序列折叠形成的四链体螺旋结构，目前认为其与基因表达调控和基因组稳定性有关。已有研究表明，结核分枝杆菌(Mycobacterium tuberculosis)的espK(Rv3879c)是构成ESX-1分泌系统的一个重要元件，其蛋白序列具有串联重复的GTPITP氨基酸序列多态性。本研究经核酸序列比对分析，确定该氨基酸序列多态性区域对应的模板链上存在G4序列，且该G4序列仅存在于结核分枝杆菌复合群。通过比对结核分枝杆菌临床分离株espK基因的核酸序列，发现espK基因的高频率G1573C突变位于G4序列。为研究该G4结构及基因表达调控功能，首先利用圆二色谱检测其核酸片段在钾离子存在条件下的光谱学特征，证实其可在体外形成具有顺式平行结构特征的G4，同义点突变G4会使其结构稳定性下降。采用重叠聚合酶链反应(overlapping polymerase chain reaction，overlapping PCR)构建含有G4突变的espK表达质粒，获得重组表达菌株。通过实时定量PCR测定espK重组表达菌株中基因转录水平变化，发现同义点突变G4后，其基因转录水平比野生型espK重组菌株提升 1.5 倍(P＜0.05)。此外，临床分离株中espK出现的高频率G1573C突变会破坏G4结构，但蛋白免疫印迹检测结果显示espK G1573C突变导致EspK蛋白表达水平上升。以上结果提示，espK的G4结构具有表达调控功能，该G4区域的序列多态性可能通过影响EspK表达水平来调节ESX-1分泌系统的活性。

Four-stranded helical structures called G-quadruplexes (G4s) formed by G-rich DNA sequences are non-canonical structures. G4s are now known to affect gene expression and genome stability. Studies have suggested that Mycobacterium tuberculosis (M. tuberculosis) espK is an important component of ESX-1 (ESAT-6 secretion system-1), and its protein sequence has tandem repetitive GTPITP sequence polymorphism. In this study, we identified a G4 sequence of espK (Rv3879c) on the template strand corresponding to GTPITP sequence polymorphism region, and this G4 sequence only existed in M. tuberculosis complex (MTBC). By comparing espK gene nucleic acid sequence in the clinical M. tuberculosis isolates, we confirmed a high-frequency G4 single nucleotide polymorphism (SNP), G1573C mutation in espK gene. The purpose of the study is to analyze the structure and expression regulation of espK G4. Firstly, espK G4 nucleic acid fragment in vitro could form parallel G4 structure in the presence of K^＋ by circular dichroism spectrum detection and the structural stability of G4 decreased after synonymous point mutation of G4. We constructed espK expression plasmid containing synonymous point mutant G4 by overlapping polymerase chain reaction (PCR), and further constructed recombinant strains with espK gene of synonymous point mutant G4 and wild-type espK. By quantitative real-time PCR detection, transcription level of recombinant strains with espK gene of synonymous point mutant G4 was significantly upregulated (P<0.05). At the same time, the high-frequency G1573C mutant in espK could destabilize G4 structure and resulted in increased protein expression level in the recombinant strain by Western blotting. It is thus concluded that G4 of M. tuberculosis espK could regulate expression, and the sequence polymorphism in espK G4 region may regulate the activity of ESX-1 secretion system through affecting the expression level of EspK.