细菌在翻译过程中，mRNA受到损伤(如缺失终止密码子)时会使翻译提前终止，导致核糖体熄火，细菌自身会启动核糖体拯救途径。由tmRNA-SmpB介导的反式翻译系统是结核分枝杆菌中的核糖体拯救途径，对结核分枝杆菌的生长繁殖有重大影响。为探究分枝杆菌中反式翻译途径的启动及其功能特点，本研究选取耻垢分枝杆菌为实验菌株，分别以mCherry和egfp作为报告基因，通过在报告基因3′端添加大肠埃希菌终止子序列，构建能在菌体中反映反式翻译表达的报告体系，并初步探究该体系中报告基因的动态表达特点。结果显示，相比正常表达mCherry的对照菌株，实验菌株中表达的错误mCherry蛋白很快被水解，菌体颜色均明显浅于前者，增强绿色荧光蛋白(enhanced green fluorescent protein，EGFP)定量检测数据也显示错误EGFP水平显著低于正常表达的EGFP水平，表明两种反式翻译报告体系均构建成功。报告基因的动态表达数据显示，蛋白出现翻译异常时，耻垢分枝杆菌可在蛋白翻译过程中快速启动反式翻译途径，并于 40～45 h将不成熟错误蛋白完全水解。本研究构建的反式翻译报告体系可为后续开展分枝杆菌反式翻译途径的功能研究及抗结核药物筛选提供帮助。

During the translation process, the translation would be terminated prematurely when abnormal mRNA (such as the deletion of the stop codon) is translated, resulting in the ribosome stalling and causing the bacteria to initiate ribosome rescue pathway. The trans-translation system mediated by tmRNA-SmpB is the main ribosome rescue pathway in Mycobacterium tuberculosis (M. tuberculosis), which has a significant impact on its growth and physiology. In order to explore the initiation and other functional characteristics of the trans-translation pathway in mycobacteria, Mycobacterium smegmatis (M. smegmatis) was selected as the experimental strain and a reporting system that reflects trans-translational expression in cells by adding Escherichia coli (E. coli) terminator at the 3′ end of the reporter genes was constructed. mCherry and egfp were chosen as reporter genes respectively. The results showed that the immature mCherry protein expressed in the experimental strain was quickly hydrolyzed compared with the normal mCherry expressed in the control strain, and the color of the former strain was significantly lighter than that of the latter strain. In addition, the quantitative detection data of enhanced green fluorescent protein (EGFP) also showed that the error EGFP level was significantly lower. These results suggested that both trans-translational reporting systems were successfully constructed. Dynamic expression data of the reporter genes showed that M. smegmatis could initiate the trans-translation pathway and completely hydrolyze the immature error proteins at 40 h to 45 h. This study will help us carry out functional researches on the trans-translation pathway of mycobacteria and screen for new anti-tuberculosis drugs.