华贵栉孔扇贝MSTN基因启动子的功能分析

Functional analysis of MSTN promoter in scallop(Chalmys nobilis)

为了解肌肉生长抑制素myostatin (MSTN)在华贵栉孔扇贝(Chalmys nobilis)闭壳肌肌肉生长和发育过程中所起的负调控作用,对华贵栉孔扇贝的MSTN启动子序列进行了生物信息学分析。结果显示,MSTN启动子序列长1 358 bp,有4个转录起始位点。核心启动子区为–100～–51 bp。有1个TATA-box (–92～–86 bp)和2个Ebox等顺式作用元件;潜在的转录因子结合位点有MEF2、MEF3、FoxO、MTBF和MyoD等;启动子区域无CpG岛。成功构建了6个MSTN启动子不同长度片段的荧光素酶表达载体,瞬时转染到293T细胞并进行双荧光素酶报告基因活性检测,表明6个启动子片段均有转录活性,PGL-534的活性最高,其次为PGL-274、PGL-22和PGL-102,最低的为PGL-995。–216～–364区域可能存在负调控基因表达的转录因子结合位点,–364～–825区域可能存在正调控基因表达的转录因子结合位点。

In order to investigate the negative regulater role of myostatin (MSTN) in growth and development of adductor of Chalmys nobilis , we analyzed the promoter sequence of MSTN of C. nobilis. The results show that the promoter sequence was 1 358 bp in length, including four transcription start sites (TSS). The core promoter region was located from-100 bp to-51bp. Two kinds of cis-regulatory element, a TATA-box (located from-92 bp to-86 bp) and two E-boxes, were detected in promoter. Potential transcription factor binding sites including MEF2, MEF3, FoxO, MTBF, MyoD and so on were found in promoter. No CpG island was found. Six luciferase expression vectors with different lengths of MSTN promoter were successfully constructed and transiently transfected into 293T cells for an analysis of the activity of dual luciferase reporter gene. It is shown that all the six promoter sequences had transcriptional activity, with PGL-534 the highest, followed by PGL-274, PGL-22 and PGL-102, and PGL-995 the lowest. There might be some binding sites of potential negative transcription factors from-216 bp to-364 bp, and potential positive transcription factors from-364 bp to-825 bp.