摘要
目的克隆马蓝Baphicacanthus cusia吲哚类生物碱合成途径的色氨酸合成酶(tryptophan synthase,TSB)基因,命名为BcTSB(GenBank登录号AYM45644.1),对其生物信息学和表达进行分析。方法基于前期马蓝转录组数据库,获得BcTSB基因开放阅读框(ORF),运用生物信息学手段对基因功能做出初步预测,构建pET32a-BcTSB原核表达载体,并转化大肠杆菌BL21(DE3),IPTG诱导表达后进行SDS-PAGE检测。通过实时荧光定量PCR(qRT-PCR)技术检测马蓝不同组织(根、茎、叶)中TSB表达水平。结果克隆的BcTSB基因ORF长度为1452bp,编码483个氨基酸,生物信息学预测为亲水性蛋白,定位于叶绿体,该蛋白相对分子质量为51 665.89,pET-32a载体包含18 000的标签。SDS-PAGE分析在70 000处出现目的蛋白条带,与理论预期值大小一致。qRT-PCR分析显示BcTSB基因在马蓝不同组织中均有表达,且在茎中的表达量最高。结论通过对BcTSB基因的克隆及表达分析,为进一步研究该基因功能及调控奠定了实验基础。
Objective To clone the tryptophan synthase gene named as BcTSB(GenBank accession number AYM45644.1) involved in the synthesis pathway of indole alkaloids from Baphicacanthus cusia, meanwhile, the bioinformatics analysis and expression analysis were also performed. Method The open reading frame(ORF) of BcTSB gene was obtained by the database of prophase Baphicacanthus cusia transcriptome. The function of the BcTSB gene was preliminarily predicted by a series of bioinformatics tools. The entire protein-coding cDNA of BcTSB was cloned into the prokaryotic expression vector pET32a, then the recombinant plasmid was transformed into E. coli BL21(DE3) cells, with IPTG induction. SDS-PAGE was used to investigate the situation of expression. The expression of the gene in root, stem and leaf was determined by using real-time PCR(qRT-PCR). Results The open reading frame(ORF) of cloned BcTSB gene was 1 452 bp, and encoding 483 amino acids, it was predicted by bioinformatics analysis as hydrophilic protein, being located in the chloroplasts. Bioinformatics analysis of the amino acid sequence showed that the molecular weight of encoded protein was 52 kDa, because prokaryotic expression vector pET32a contained 18 kDa label, SDS-PAGE results showed that a protein band at 70 000 was in consistent with molecular weight of the predicted protein. The QRT-PCR revealed that BcTSB gene was expressed in different tissues of B. cusia, the expression level of BcTSB in stems was much higher than that in roots and leaves. Conclusion In this study, BcTSB gene of B. cusia was cloned and its expression was analyzed successfully, which laid an experimental foundation for further study on the function and regulation of the gene.
作者
马小毛
宁书菊
叶齐
胡永乐
蔡国倩
魏道智
MA Xiao-mao;NING Shu-ju;YE Qi;HU Yong-le;CAI Guo-qian;WEI Dao-zhi(College of Life Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China;College of Crop Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China)
出处
《中草药》
CAS
CSCD
北大核心
2020年第24期6328-6336,共9页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金面上项目(81573517)
福建省自然科学基金面上资助项目(2019J01827)。
作者简介
马小毛(1995-),女,硕士研究生,研究方向为分子生态学。E-mail:2592300779@qq.com;通信作者:魏道智(1960-),男,博士,教授,博士生导师,研究方向为药用植物资源学。E-mail:weidz888@sohu.com。
