时晨晶;王世威;彭佳铭;许海玉;
SHI Chen-jing;WANG Shi-wei;PENG Jia-ming;XU Hai-yu;Tianjin University of Traditional Chinese Medicine;Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences;School of Traditional Chinese Medicine, Guangdong Pharmaceutical University;School of Pharmacy, Anhui University of Chinese Medicine;

• 1:天津中医药大学
• 2:中国中医科学院中药研究所
• 3:广东药科大学中药学院
• 4:安徽中医药大学药学院

摘要(Abstract)：

14-3-3蛋白是植物体内一类重要的调控蛋白，对植物的生长发育及生物和非生物胁迫都具有重要的调控作用。该研究利用酵母双杂交从药用植物丹参cDNA文库中筛选并克隆到1个编码14-3-3蛋白基因(GenBank登录号为OM683281),该基因开放阅读框(ORF)由780 bp组成，编码259个氨基酸。生物信息学分析预测该蛋白的分子式为C_(1287)H_(2046)N_(346)O_(422)S_9,相对分子质量29.4 kDa,不含信号肽，为非跨膜蛋白。通过基因同源序列比对及进化树分析，证明该基因确实属于14-3-3蛋白家族，与拟南芥、水稻中的14-3-3蛋白基因家族亲缘关系较近。将丹参14-3-3基因构建到原核表达载体pGEX-4T-1上，在大肠杆菌BL21中，成功表达出重组蛋白。实时荧光定量PCR检测，确定该基因在丹参根、茎、叶、花不同组织部位中表达，叶中表达量最高，茎中次之，花中的表达量最低。15%PEG模拟干旱、植物激素水杨酸、茉莉酸甲酯和乙烯利诱导丹参植株，14-3-3基因表达水平在诱导的早期出现峰值，表明该基因能够快速响应干旱等非生物胁迫和水杨酸、茉莉酸、乙烯等植物激素处理。为后续研究14-3-3蛋白调控丹参酮生物合成及响应生物和非生物胁迫的分子机制奠定基础。
14-3-3 proteins are important proteins in plants, as they regulate plant growth and development and the response to biotic or abiotic stresses. In this study, a 14-3-3 gene(GenBank accession: OM683281) was screened from the cDNA library of the medicinal species Salvia miltiorrhiza by yeast two-hybrid and cloned. The open reading frame(ORF) was 780 bp, encoding 259 amino a cids. Bioinformatics analysis predicted that the protein was a non-transmembrane protein with the molecular formula of C_(1287)H_(2046)N_(346)O_(422)S_9, relative molecular weight of 29.4 kDa, and no signal peptide. Homologous sequence alignment and phylogenetic tree analysis proved that the protein belonged to 14-3-3 family and had close genetic relationship with the 14-3-3 proteins from Arabidopsis thaliana, Oryza sativa, and Nicotiana tabacum. The 14-3-3 gene was ligated to the prokaryotic expression vector pGEX-4 T-1 and then transformed into Escherichia coli BL21 for the expression of recombinant protein. Real-time fluorescent quantitative PCR showed that the expression of this gene was different among roots, stems, leaves, and flowers of S. miltiorrhiza. To be specific, the highest expression was found in leaves, followed by stems, and the lowest expression was detected in flowers. S. miltiorrhiza plants were treated with 15% PEG(simulation of drought), and hormones salicylic acid, methyl jasmonate, and ethephon, respectively, and the expression of 14-3-3 gene peaked at the early stage of induction. Therefore, the gene can quickly respond to abiotic stresses such as drought and plant hormone treatments such as salicylic acid, jasmonic acid, and ethylene. This study lays the foundation for revealing the molecular mechanism of 14-3-3 protein regulating tanshinone biosynthesis and responding to biotic and abiotic stresses.

关键词(KeyWords)： 丹参;14-3-3蛋白;基因克隆;原核表达;诱导模式
Salvia miltiorrhiza;14-3-3 protein;gene cloning;prokaryotic expression;inducible expression pattern

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金重点项目(81830111)

作者(Authors): 时晨晶;王世威;彭佳铭;许海玉;
SHI Chen-jing;WANG Shi-wei;PENG Jia-ming;XU Hai-yu;Tianjin University of Traditional Chinese Medicine;Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences;School of Traditional Chinese Medicine, Guangdong Pharmaceutical University;School of Pharmacy, Anhui University of Chinese Medicine;

DOI: 10.19540/j.cnki.cjcmm.20220614.101

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