周浩, 路星星, 敖雯雯, 廖海民, 张明生, 强玮*. 钩藤STR基因及其启动子的克隆与分析J. 药学学报, 2022,57(5): 1526-1536. doi: 10.16438/j.0513-4870.2021-1700
引用本文: 周浩, 路星星, 敖雯雯, 廖海民, 张明生, 强玮*. 钩藤STR基因及其启动子的克隆与分析J. 药学学报, 2022,57(5): 1526-1536. doi: 10.16438/j.0513-4870.2021-1700
ZHOU Hao, LU Xing-xing, AO Wen-wen, LIAO Hai-min, ZHANG Ming-sheng, QIANG Wei*. Cloning and analysis of STR gene and its promoter from UncariaJ. Acta Pharmaceutica Sinica, 2022,57(5): 1526-1536. doi: 10.16438/j.0513-4870.2021-1700
Citation: ZHOU Hao, LU Xing-xing, AO Wen-wen, LIAO Hai-min, ZHANG Ming-sheng, QIANG Wei*. Cloning and analysis of STR gene and its promoter from UncariaJ. Acta Pharmaceutica Sinica, 2022,57(5): 1526-1536. doi: 10.16438/j.0513-4870.2021-1700

钩藤STR基因及其启动子的克隆与分析

Cloning and analysis of STR gene and its promoter from Uncaria

  • 摘要: 根据钩藤转录组中STR基因核心序列设计特异性引物,并进行RACE扩增,克隆到UrSTR基因(GeneBank:OL310251)的cDNA全长1 541 bp,编码345个氨基酸;采用染色体步移克隆到UrSTR基因启动子(GeneBank:OL310252)序列1 179 bp。系统进化发育树分析显示,钩藤UrSTR蛋白与同为茜草科的美丽帽柱木和短小蛇根草的STR蛋白聚为一类,其亲缘关系最近;亚细胞共定位实验表明UrSTR蛋白定位于液泡膜上;SDS-PAGE结果表明pET-28a-UrSTR重组蛋白成功表达且大小与预期相符;启动子顺式作用元件分析表明其含有光响应、胁迫响应和激素响应有关的多种调控元件;启动子活性分析UrSTR基因启动子具有转录活性。成功克隆了UrSTR基因及其启动子序列并对其进行生物信息学分析和启动子活性分析;后期需要优化UrSTR原核表达体系,为进一步纯化UrSTR蛋白研究其结构和功能奠定基础。

     

    Abstract: On the basis of the Uncaria transcriptome, specific primers were designed for UrSTR. The full-length cDNA of UrSTR (GeneBank:OL310251) was 1 541 bp, encoding 345 amino acid residues, and the promoter region sequence of UrSTR (GeneBank:OL310252) was 1 179 bp. Phylogenetic tree is revealed that UrSTR had a closest relationship with STR from Ophiorrhiza pumila and Ophiorrhiza japonica. Localization of UrSTR protein is revealed located in the vacuole membrane. Plant-care analysis indicated that the promoter region sequence of UrSTR, covering multiple light, stress and hormone-response cis-regulatory elements, and verified transcriptional activity. The results of SDS-PAGE show that pET-28a-UrSTR recombinant protein was successfully expressed, and the size was anticipated. The UrSTR prokaryotic expression system needs to be optimized in the later stage. The research lays the foundation for further purification to study its structure and functional characterization of the UrSTR protein.

     

/

返回文章
返回