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蔗糖非酵解型蛋白激酶家族2(SnRK2)在逆境植物信号转导途径中起着重要作用。该研究根据丹参转录组数据从丹参c DNA中克隆得到一个SnRK2家族基因,命名为Sm SnRK2.4,属于Ⅰ类SnRK2。Sm SnRK2.4基因包含8个内含子,9个外显子,其开放阅读框1 068 bp,编码355个氨基酸,推测其蛋白相对分子质量为40.63 k Da,将其构建到原核表达载体p MAL-c2X上,原核诱导表达出与预测蛋白大小一致的目的蛋白。依据丹参基因组序列,Plant CARE分析Sm SnRK2.4起始密码子ATG上游3 0 0 0 bp的启动子系列,结果显示该序列包含I-box,GA-m otif,H SE,LTR,TC-rich repeats等逆境胁迫响应元件,以及GARE-m otif,P-box,ABRE,CGTCA-m otif等激素响应元件。实时荧光定量PCR分析表明Sm SnRK2.4在丹参根中的含量较高,在茎中和叶中含量相当,ABA和PEG胁迫响应分析表明,Sm SnRK2.4对PEG渗透胁迫响应显著,对ABA胁迫响应微弱。该研究为进一步探讨Sm SnRK2.4基因在丹参干旱胁迫下次生代谢产物积累机制中的作用奠定了基础。
Sucrose untukable protein kinase family 2 (SnRK2) plays an important role in stress signal transduction pathways. In this study, a SnRK2 family gene was cloned from Salvia miltiorrhiza cDNA according to Salvia miltiorrhiza transcriptome data and named Sm SnRK2.4, belonging to class I SnRK2. The Sm SnRK2.4 gene contains 8 introns and 9 exons. The ORF of Sm SnRK2.4 gene is 1 068 bp in open reading frame and encodes a protein of 355 amino acids. The relative molecular mass of Sm SnRK2.4 gene is 40.63 kDa. The Sm SnRK2.4 gene was cloned into prokaryotic expression vector p MAL-c2X, prokaryotic expression of the predicted protein and the size of the target protein. According to the genome sequence of Salvia miltiorrhiza, Plant CARE was used to analyze the promoter sequence of 300 bp upstream of the ATG of Sm SnRK2.4. The results showed that the sequence contained I-box, GA-m otif, H SE, LTR, TC-rich repeats and other stress response components, as well as GARE-m otif, P-box, ABRE, CGTCA-m otif and other hormone response elements. Real-time quantitative PCR analysis showed that Sm SnRK2.4 content in Salvia miltiorrhiza roots was higher than that in the stems and leaves, and ABA and PEG stress response analysis showed that Sm SnRK2.4 had a significant response to PEG osmotic stress. The response is weak. This study laid the foundation for the further study on the role of Sm SnRK2.4 gene in the mechanism of accumulation of secondary metabolites in Danshen under drought stress.