肌肉特异性合成启动子文库构建及其高活性启动子元件组成与活性的关联性分析

Construction of a muscle-specific synthetic promoter library and correlation analysis of the element composition and activity of highly active promoters

摘要:

本研究旨在构建肌肉特异性合成启动子库，筛选出活性较高的肌肉特异性启动子，分析高活性启动子序列中启动子元件与其活性的关系，为人工合成启动子提供一定理论依据。选取19个源于肌肉特异性元件、保守元件、病毒调控序列的元件，随机连接构建肌肉特异性合成启动子库。构建荧光素酶质粒pCMV-Luc和pSPs-Luc，将以上质粒转染至成肌细胞系C2C12，通过荧光素酶活性检测评估合成启动子活性；利用另外2种非肌肉源细胞系HeLa和3T3验证高活性启动子的肌肉特异性，分析活性高、肌肉特异性好且测序比对正确的启动子序列，探索元件组成与启动子活性之间的关系。本研究成功构建肌肉特异性启动子库，筛选出有效合成启动子质粒321个，其中SP-301启动子活性为CMV活性的5.63倍；活性较高的15个启动子具有肌肉特异性；分析高活性且测序正确的启动子，发现元件组成与启动子活性之间存在一定关系。肌肉特异性元件在启动子中占比偏高，但与启动子活性强弱相关性较小，是组织特异性的决定元件；病毒元件在强活性启动子中含量均不低于20%，可能是影响启动子活性的关键元件；保守元件含量与启动子活性之间具有显著的相关性，其含量与启动子活性成正比。本研究为合成组织特异性高效启动子提供了一定理论基础，为原位基因传递体系的构建和应用提供了新思路。

关键词:合成启动子;筛选;相关性分析;启动子元件

Abstract:

The purpose of this study is to construct a muscle-specific synthetic promoter library, screen out muscle-specific promoters with high activity, analyze the relationship between element composition and activity of highly active promoters, and provide a theoretical basis for artificial synthesis of promoters. In this study, 19 promoter fragments derived from muscle-specific elements, conserved elements, and viral regulatory sequences were selected and randomLy connected to construct a muscle-specific synthetic promoter library. The luciferase plasmids pCMV-Luc and pSPs-Luc were constructed and transfected into the myoblast cell line C2C12. The activities of the synthesized promoters were evaluated by the luciferase activity assay. Two non-muscle-derived cell lines HeLa and 3T3 were used to verify the muscle specificity of the highly active promoters. The sequences of promoters with high activity, good muscle specificity, and correct sequences were analyzed to explore the relationship between the element composition and activity of promoters. We successfully constructed a muscle-specific promoter library and screened out 321 effective synthetic promoter plasmids. Among them, the activity of SP-301 promoter was 5.63 times that of CMV. The 15 promoters with high activity were muscle-specific. In the promoters with high activity and correct sequences, there was a relationship between their element composition and activity. Muscle-specific elements accounted for a high proportion in the promoters, while they had weak correlations with the promoter activity, being tissue-specific determinants. Viral elements accounted for no less than 20% in highly active promoters, which may be the key elements for the promoter activity. The content of conserved elements was proportional to the promoter activity. This study lays a theoretical foundation for the synthesis of tissue-specific efficient promoters and provides a new idea for the construction and application of in-situ gene delivery systems.

Key words:synthetic promoter;screening;correlation analysis;promoter elements