| 丹参SmRopGEF1基因的克隆、亚细胞定位与表达分析 |
| 投稿时间：2023-02-13 修订日期：2023-02-21 点此下载全文
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| 作者中文名 | 作者英文名 | 单位中文名 | 单位英文名 | E-Mail |
| 陈颖 |
chenying |
北京中医药大学中药学院 |
School of Chinese Pharmacy,Beijing University of Chinese Medicine |
yingchen2200@163.com |
| 满金辉 |
manjinhui |
北京中医药大学中药学院 |
School of Chinese Pharmacy,Beijing University of Chinese Medicine |
mjh19901218@163.com |
| 石玥 |
shiyue |
北京中医药大学生命科学学院 |
School of Life Sciences,Beijing University of Chinese Medicine |
shiny0420@163.com |
| 黄钰莹 |
huangyuying |
北京中医药大学中药学院 |
School of Chinese Pharmacy,Beijing University of Chinese Medicine |
hyy19960126@163.com |
| 张晓芹 |
zhangxiaoqin |
北京中医药大学中药学院 |
School of Chinese Pharmacy,Beijing University of Chinese Medicine |
zxqymz881018@163.com |
| 王馨 |
wangxin |
北京中医药大学中药学院 |
School of Chinese Pharmacy,Beijing University of Chinese Medicine |
nz18813062869@163.com |
| 刘珊瑚 |
liushanhu |
北京中医药大学中药学院 |
School of Chinese Pharmacy,Beijing University of Chinese Medicine |
shanhu20212024@163.com |
| 何高洁 |
hegaojie |
北京中医药大学中药学院 |
School of Chinese Pharmacy,Beijing University of Chinese Medicine |
hegao727@163.com |
| 安克露 |
ankelu |
北京中医药大学中药学院 |
School of Chinese Pharmacy,Beijing University of Chinese Medicine |
ankelu17883687673@163.com |
| 王晓晖 |
wangxiaohui |
北京中医药大学 北京中医药研究院 中药现代研究中心 |
School of Chinese Pharmacy,Beijing University of Chinese Medicine |
wangxhui2014@163.com |
| 魏胜利* |
weishengli |
北京中医药大学中药学院 |
School of Chinese Pharmacy,Beijing University of Chinese Medicine |
wsl7491@126.com |
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| 基金项目:北京市科学技术委员会基金资助项目；黄芩、瓜蒌等4种精准药材批次分子防伪技术研究项目 |
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| 中文摘要:目的: 为了研究丹参SmRopGEF1蛋白对逆境胁迫的应激反应和激素诱导下的表达水平 方法: 根据前期丹参转录组测序结果设计SmRopGEF1基因特异引物，利用RT-PCR扩增SmRopGEF1基因序列，使用生物信息学方法分析SmRopGEF1序列特征及进化关系，构建pET28a- SmRopGEF1原核表达载体并转化至大肠杆菌中诱导表达，构建35S启动的SmRopGEF1的绿色荧光蛋白原生质体瞬时表达载体，利用激光共聚焦显微镜观察SmRopGEF1的亚细胞定位。实时荧光定量PCR检测逆境胁迫和激素诱导下的基因表达量。 结果: 丹参SmRopGEF1基因开放阅读框 (ORF) 全长为1701bp，编码566个氨基酸，蛋白分子质量是62.4kD。生物信息学分析显示 SmRopGEF1 蛋白含有 1 个 PRONE保守结构域, 系统进化树分析表明 SmRopGEF1 蛋白与芡欧鼠尾草(Salvia hispanica)和一串红(Salvia splendens) RopGEF1 蛋白为高同源蛋白。成功构建pET28a-SmRopGEF1原核表达载体并在大肠杆菌 Rosetta(DE3) 菌株中表达、纯化出SmRopGEF1 重组蛋白。亚细胞定位的定位结果显示SmRopGEF1存在于植物细胞的细胞核中。q-PCR实验结果表明盐胁迫和冷胁迫能够显著提高SmRopGEF1的表达水平；干旱胁迫后丹参愈伤中SmRopGEF1表达量下降。MeJA诱导后SmRopGEF1表达量升高。结论: 实验首次克隆了丹参SmRopGEF1基因，其在抗逆胁迫中起到重要作用并且可能参与MeJA信号转导。该研究丰富了药用植物丹参防御反应机制的研究，为选育丹参抗逆优良品种，提高丹参的产量与品质提供了有利的科学支撑。 |
| 中文关键词:丹参 鸟嘌呤核苷酸交换因子 序列分析 基因表达 |
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| Cloning and subcellular location of the SmRopGEF1 gene from Salvia miltiorrhiza and expression analyses with different stressors |
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| Abstract:Objective: In order to study the response of Salvia miltiorrhiza SmRopGEF1 protein to abiotic stress and the expression level induced by hormone. Methods: A full length cDNA of SmRopGEF1 gene was cloned by the reverse transcription PCR from Salvia miltiorrhiza. SmRopGEF1 sequence characteristics and phylogenetic relationship was analyzed by Bioinformatics method. The prokaryotic expression vector pET28a-SmRopGEF1 was constructed and was induced expression in Escherichia coli Rosetta (DE3) cells. The SmRopGEF1 CDS fused to the pCAMBIA 1300 vector for fusion with GFP under the control of the 35S promoter to generate the SmRopGEF1- GFP construct, observing subcellular localization under a confocal laser scanning microscope. Analysis of abiotic stress and hormone-induced expression by quantitative reverse transcription-polymerase chain reaction GFP construct. Results: The open reading frame (ORF) of SmRopGEF1 gene was 1701 bp, encoding a protein of 566 amino acids with a calculated molecular mass of 27.4 kD. Bioinformatic analysis showed that SmRopGEF1 protein contains a conserved PRONE domain. The phylogenetic analysis indicated that SmRopGEF1 protein had the highest level of homology with the RopGEF1 protein from Salvia hispanica and Salvia splendens. The recombinant SmRopGEF1 protein was expressed in Escherichia coli Rosetta (DE3) cells using the prokaryotic expression vector pET28a-SmRopGEF1 and was purified. Subcellular localization analysis showed that SmRopGEF1 was localized in the nucleus. The expression level of SmRopGEF1 was significantly induced by salt and cold stresses. Furthermore, SmRopGEF1 expression decreased under drought stress. The expression level of SmRopGEF1 was elevated methyl jasmonate (MeJA) treatments. Conclusion: The SmRopGEF1 gene was first cloned from Salvia miltiorrhiza, which plays an important role in stress and may be involved in MeJA signal transduction. This study enriches the study of defense response mechanism of Salvia miltiorrhiza and provides favorable scientific support for breeding stress-resistant varieties of Salvia miltiorrhiza and improving the yield and quality of Salvia miltiorrhiza. |
| keywords:Salvia miltiorrhiza Bge. RopGEF sequence analysis gene expression pattern |
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