丹参蛋白激酶SmSnRK2.4的克隆及表达分析

Cloning and expression analysis of protein kinase SmSnRK2.4 from Salvia miltiorrhiza

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 non-fermenting 1-related protein kinase 2（SnRK2） plays a key role in abiotic stress signaling in plants.In this study,we cloned a Sm SnRK2.4 gene belonging to subclass I of SnRK2 from Salvia miltiorrhiza by screening its transcriptome database.The Sm SnRK2.4 gene contains 8 introns and 9 exons,with a 1 068 bp open reading frame encoding a polypeptide of 355 amino acids,the predicted molecular mass of which is 40.63 k Da.Prokaryotic expression of Sm SnRK2.4 protein using p MAL-c2 X as the expression vector displayed that the recombinant protein of Sm SnRK2.4 gene in E.coli was consistent with the predicted size.A 3 000 bp promoter sequence of Sm SnRK2.4 contained some stress-responsive elements and hormone-responsive elements.Quantitative real-time PCR analysis revealed that the expression of Sm SnRK2.4 in root was much higher than that in stem and leaf,Sm SnRK2.4 was strongly induced by PEG stress,weakly induced by ABA stress.This research provided a basis for further study of the Sm SnRK2.4 gene playing the role in accumulate mechanism of secondary metabolites in S.miltiorrhiza under drought.