药学学报, 2022, 57(5): 1506-1515
引用本文:
石玥, 满金辉, 江伟业, 张靖晗, 张志飞, 尹光耀, 王馨, 黄钰莹, 张媛, 王晓晖, 魏胜利. 三七PnMYB1R1基因的克隆、亚细胞定位与不同胁迫下的表达分析[J]. 药学学报, 2022, 57(5): 1506-1515.
SHI Yue, MAN Jin-hui, JIANG Wei-ye, ZHANG Jing-han, ZHANG Zhi-fei, YIN Guang-yao, Wang Xin, HUANG Yu-ying, ZHANG Yuan, WANG Xiao-hui, WEI Sheng-li. Cloning and subcellular location of the PnMYB1R1 gene from Panax notoginseng and expression analysis with different stresses[J]. Acta Pharmaceutica Sinica, 2022, 57(5): 1506-1515.

三七PnMYB1R1基因的克隆、亚细胞定位与不同胁迫下的表达分析
石玥1#, 满金辉1#, 江伟业1, 张靖晗1, 张志飞1, 尹光耀1, 王馨1, 黄钰莹1, 张媛1,3, 王晓晖1,2*, 魏胜利1,3*
1. 北京中医药大学中药学院, 北京 102488;
2. 北京中医药大学中药学院中药现代研究中心, 北京 100029;
3. 中药材规范化生产教育部工程研究中心, 北京 100102
摘要:
MYB转录因子是植物中最大的转录因子家族之一,在植物信号转导、生长发育和植物防御反应中起到重要作用。本研究以三七(Panax notoginseng)为材料,设计特异引物,克隆了三七中1R-MYB转录因子PnMYB1R1基因,并进行氨基酸序列分析、原核表达和纯化、亚细胞定位、转录活性分析、组织特异性分析及非生物胁迫下的诱导表达分析。PnMYB1R1基因开放阅读框(ORF)长738 bp,编码245个氨基酸,蛋白分子质量27.0 kD。序列分析及系统进化树结果表明PnMYB1R1包含1个保守的R3结构域,属于1R-MYB型转录因子中的TRF-like类蛋白。构建原核表达载体pET28a-PnMYB1R1并在大肠杆菌BL21(DE3)菌株中成功表达PnMYB1R1重组蛋白,纯化得到可溶性重组蛋白。亚细胞定位实验结果表明PnMYB1R1定位于植物细胞的细胞核中。转录活性分析显示PnMYB1R1转录因子具有转录激活活性。实时荧光定量PCR结果表明PnMYB1R1基因在三七主根中表达量最高,茎、叶次之,须根中表达量最低。盐、低温及干旱胁迫均能够影响主根、须根、茎、叶中PnMYB1R1基因的表达水平,其中盐胁迫能够显著提高主根、须根、茎和叶中PnMYB1R1基因的表达水平。本研究为进一步揭示1R-MYB转录因子在三七中防御反应中作用奠定基础。
关键词:    三七      PnMYB1R1      原核表达      亚细胞定位      转录活性      表达分析     
Cloning and subcellular location of the PnMYB1R1 gene from Panax notoginseng and expression analysis with different stresses
SHI Yue1#, MAN Jin-hui1#, JIANG Wei-ye1, ZHANG Jing-han1, ZHANG Zhi-fei1, YIN Guang-yao1, Wang Xin1, HUANG Yu-ying1, ZHANG Yuan1,3, WANG Xiao-hui1,2*, WEI Sheng-li1,3*
1. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China;
2. Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China;
3. Engineering Research Center of Good Agricultural Practice for Chinese Crude Drugs of Ministry of Education, Beijing 100102, China
Abstract:
MYB transcription factors, one of the largest transcription factor families in plants, play an important role in signal transduction, plant growth and plant resistance. In this study a full-length cDNA of the PnMYB1R1 gene was cloned from Panax notoginseng. Sequence analysis, prokaryotic expression and purification, subcellular location, transcriptional activity analysis, tissue-specific analysis and expression analysis under different abiotic stresses was performed. The open reading frame (ORF) of PnMYB1R gene was 738 bp, encoding a protein of 245 amino acids with a predicted molecular mass (MW) of 27.0 kD. The sequence analysis and polygenetic analysis indicated that the PnMYB1R1 protein contains a conserved R3 domain, belonging to TRF-like protein in 1R-MYB-type transcription factors. The recombinant PnMYB1R1 protein was expressed in Escherichia coli BL21(DE3) cells using the prokaryotic expression vector pET28a-PnMYB1R1 and was purified. Subcellular localization analysis showed that PnMYB1R1 was localized in the nucleus. Transcriptional activity analysis indicated that the PnMYB1R1 transcription factor has transcriptional activation activity. Expression analysis indicated that PnMYB1R1 was primarily expressed in roots, followed by stems and leaves, and then rootlets. The expression level of PnMYB1R1 in root, stems, leaves and rootlets was influenced by salt, low temperature and drought treatment, while the abundance of PnMYB1R1 was significantly induced by salt stress in these tissues. These results provide valuable insights into the role of 1R-MYB transcription factors in plant defense.
Key words:    Panax notoginseng    PnMYB1R1    prokaryotic expression    subcellular localization    transcription activity    expression analysis   
收稿日期: 2021-11-11
DOI: 10.16438/j.0513-4870.2021-1617
基金项目: 北京市科学技术委员会基金资助项目(Z201100005420005).
通讯作者: 魏胜利,E-mail:wsl7491@126.com;王晓晖,E-mail:wangxhui2014@163.com
Email: wsl7491@126.com;wangxhui2014@163.com
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