Abstract：
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.
摘要：
14-3-3蛋白质是植物中的重要蛋白质,因为它们调节植物的生长和发育以及对生物或非生物胁迫的反应。在这项研究中,通过酵母双杂交从药用物种丹参的cDNA文库中筛选并克隆了14-3-3基因（GenBank登录号：OM683281）。开放阅读框(ORF)为780bp,编码259个氨基的cids。生物信息学分析预测该蛋白为非跨膜蛋白,分子式为C_(1287)H_(2046)N_(346)O_(422)S_9,相对分子质量为29.4kDa,没有信号肽.同源序列比对和系统发育树分析证明，该蛋白属于14-3-3家族，与拟南芥14-3-3蛋白具有密切的亲缘关系，水稻,还有烟草.将14-3-3基因连接到原核表达载体pGEX-4T-1，然后转化到大肠杆菌BL21中表达重组蛋白。实时荧光定量PCR结果表明,该基因在不同根中的表达量不同,茎,叶子,和丹参的花。具体而言,最高的表达是在叶子中发现的，其次是茎，在花中检测到最低的表达。用15%PEG(模拟干旱)处理丹参植物，和激素水杨酸，茉莉酸甲酯,还有乙烯利,分别,14-3-3基因的表达在诱导早期达到高峰。因此,该基因可以快速响应非生物胁迫，如干旱和植物激素处理，如水杨酸，茉莉酸,和乙烯。本研究为揭示14-3-3蛋白调控丹参酮生物合成以及响应生物和非生物胁迫的分子机制奠定了基础。
