丹参超氧化物歧化酶SmMSD2基因的克隆与表达分析

Cloning and expression analysis of superoxide dismutase SmMSD2 gene from Salvia miltiorrhiza

超氧化物歧化酶(superoxide dismutase,SOD)是清除生物体内超氧阴离子自由基(O_(2)^(·-))的关键酶,在植物生长发育和逆境胁迫中发挥重要的作用。本研究基于丹参基因组和转录组数据,共挖掘出9条SOD基因,分别为5个Cu/Zn-SOD、2个Fe-SOD和2个Mn-SOD,并对9条基因进行了生物信息学和基因表达模式分析。结合蛋白质组学和转录组数据,从丹参中克隆得到SmMSD2的全长cDNA,根据氨基酸多重序列比对和系统进化树分析,SmMSD2属于超氧化物歧化酶的锰离子亚族,且C端含有保守的金属结合结构域“DVWEHAYY”。构建原核表达重组载体pMAL-c2X-SmMSD2,成功诱导表达出重组蛋白,并对其进行了酶学性质分析。时空表达分析表明,该基因在各个组织中均有表达,为组成表达型基因。实时荧光定量PCR结果显示模拟干旱(15%PEG6000)、脱落酸(abscisic acid,ABA)和吲哚-3-乙酸(indole-3-acetic acid,IAA)均能够诱导丹参SmMSD2基因表达,推测SmMSD2可能参与丹参响应干旱等非生物胁迫,以及植物激素ABA和IAA的信号传导途径,为进一步阐明超氧化物歧化酶参与丹参逆境响应及有效活性成分的积累奠定了基础。

Superoxide dismutase(SOD)is a key enzyme that scavenge superoxide anion free radical(O_(2)^(·-))in vivo,and plays an important role in plant growth and development and stress.In this study,according to the genome and transcriptome data of Salvia miltiorrhizae,9 SOD genes were identified and the expression patterns of SOD family genes were further analyzed,including 5 Cu/Zn-SOD,2 Fe-SOD and 2 Mn-SOD.On the basis of proteomic analysis,combined with transcriptome data,one full-length cDNA of Mn-SOD gene,namely SmMSD2 was cloned from Salvia miltiorrhizae.The results of amino acid sequence alignment and phylogenetic analysis showed that SmMSD2 protein belongs to the manganese superoxide dismutase(Mn-SOD)subfamily,and SmMSD2 protein shares high sequence identity with the Mn-SOD proteins of various plants that all contain a C-terminal conserved metal-binding domain"DVWEHAYY".The prokaryotic expression vector pMAL-c2X-SmMSD2 was constructed and transformed into E.coli BL21 expressing strain,and the target recombinant protein was successfully induced and its enzymatic properties were analyzed.Spatiotemporal expression analysis showed that SmMSD2 gene was expressed in all tissues,indicating that SmMSD2 gene was constitutively expressed at a stable level.Real-time quantitative PCR indicated that drought(15%PEG6000),abscisic acid(ABA)and indole-3-acetic acid(IAA)could induce the expression of SmMSD2 gene,suggesting that SmMSD2 may be involved in the response of Salvia miltiorrhizae to abiotic stress such as drought,as well as the signaling pathways of phytohormone ABA and IAA.These results lay the foundation for further elucidating the involvement of superoxide dismutase in the stress response and accumulation of active components of Salvia miltiorrhiza.