甘蔗热带种金属硫蛋白家族基因的克隆及响应重金属胁迫的表达分析

金属硫蛋白是一类富含巯基的低分子量蛋白,在植物的重金属解毒及细胞氧化还原调控等方面起重要的作用。本研究以甘蔗热带种Badila组培苗为材料,分别测定了其在CdCl2、ZnSO4和CuCl2水溶液培养条件下地上部和地下部的重金属含量,结果显示其对上述3种重金属有较强的耐受与富集能力。继而克隆了ScMT1(登录号为KJ504373)、ScMT2-1-5(登录号为MH191346)和ScMT3(登录号为KJ5043704)3个金属硫蛋白家族基因,它们分别属于植物MT亚家族中的MT1、MT2和MT3型基因。ScMT1含有1个内含子和2个外显子,开放阅读框(Open Reading Frame,ORF)长228 bp,编码75个氨基酸;ScMT2-1-5含有2个内含子和3个外显子,ORF长246 bp,编码81个氨基酸;ScMT3含有1个内含子和2个外显子,ORF长198 bp,编码65个氨基酸。RT-qPCR显示,Cd2+胁迫下,在甘蔗地上部和地下部,ScMT2-1-5均连续显著上调表达,而ScMT1的上调应答出现延迟。ScMT3在地上部的上调应答出现延迟,在地下部呈“扬-抑”趋势,提示甘蔗响应Cd^2+胁迫过程中ScMT2-1-5起更积极的作用,ScMT1参与胁迫后期的分子响应,而ScMT3不起主导作用。Cu^2+胁迫下,地上部ScMT1连续显著上调表达,ScMT2-1-5和ScMT3呈总体上调的表达趋势;地下部,ScMT1和ScMT2-1-5的上调表答均出现延迟,仅在胁迫后期显著上调表达,而ScMT3仅在胁迫前期显著上调表达。该结果提示了ScMT1、ScMT2-1-5和ScMT3在Cu2+胁迫响应过程中的协作关系,三者共同参与了地上部的胁迫响应,其中ScMT1起更积极的作用;此外三者还先后参与了地下部对Cu^2+胁迫的分子响应。Zn^2+胁迫下,ScMT1和ScMT3分别仅在地上部和地下部显著上调表达;ScMT2-1-5在地上部和地下部均呈“扬-抑”的应答趋势;提示了在甘蔗响应Cd^2+胁迫应答过程中ScMT1和ScMT3分别在地上部和地下部起主要作用,ScMT2-1-5参与了胁迫前期的分子响应。ScMT1、ScMT2-1-5和ScMT3在甘蔗不同组织中及在重金属(Cd^2+、Zn^2+或Cu^2+)不同累积水平下呈现出相似或互补的应答特性,提示上述甘蔗MT家族不同成员在重金属解毒及细胞氧化还原调控等方面产生了功能分化,且三者在应对过量Cd^2+、Zn^2+或Cu^2+对甘蔗组织造成伤害的过程中存在时空上的协同作用。该研究为深入理解多倍体植物甘蔗中MT家族各成员基因在重金属耐受过程中的协同作用机制奠定了基础。

Cloning and expression analysis of metallothionein family genes in response to heavy metal stress in sugarcane (Saccharum officinarum L.)

Metallothioneins(MTs)are cysteine-rich,low-molecular-weight proteins.Plant MTs play important roles in detoxi?cation and cellular redox regulation.In this study,hydroponic experiments of sugarcane(Saccharum officinarum L.cv.Badila)were carried out to study the effects of CdCl2,ZnSO4,and CuCl2 treatments on plantlet growth.And then three kinds of heavy metal content in both shoots and roots of sugarcane were detected,showing an enrichment and tolerance ability to Cd^2+,Zn^2+,and Cu^2+ in Badila seedlings.Three metallothionein genes,termed as ScMT1(accession number:KJ504373),ScMT2-1-5(accession number:MH191346),and ScMT3(accession number:KJ5043704),were isolated from Badila.ScMT1 contained an open reading frame(ORF)of 228 bp encoding 75 amino acids residues.The first(52 bp)and second(176 bp)extrons of ScMT1 were separated by an intron(283 bp).ScMT2-1-5 contained an ORF of 246 bp encoding 81 amino acids residues.The first(64 bp),second(87 bp),and third(101 bp)extrons of ScMT2-1-5 were separated by two introns(483 bp and 853 bp).ScMT3 contained an ORF of 198 bp encoding 65 amino acids residues.The first(50 bp)and second(148 bp)extrons of ScMT3 were separated by an intron(259 bp).The deduce protein ScMT1,ScMT2-1-5,and ScMT3 were categorized into the subfamily of plant type1,type2,and type3 MTs,respectively.The expression profiles of the three genes under different heavy-metal stresses were investigated by real-time quantitative PCR(qRT-PCR)analysis.When treated with Cd^2+,the expression of ScMT2-1-5 was continuously and significantly up-regulated in both shoots and roots,while that of ScMT1 showed a delayed up-regulation pattern.The expression of ScMT3 showed a delayed up-regulation pattern in shoots and a trend of first raising and then suppressing in roots.The results suggested that ScMT2-1-5 might play a more active role in response to Cd^2+ in sugarcane while ScMT3 might not,and ScMT1 is involved in the molecular responses of Cd^2+ stress at the later stage.When treated with Cu^2+,the expression of ScMT1 was continuously and significantly up-regulated,and that of ScMT2-1-5 and ScMT3 general up-regulated in shoots.As in the roots,the expression of both ScMT1 and ScMT2-1-5 showed a delayed up-regulation pattern which was significantly up-regulated at the later stage,while that of ScMT3 significantly up-regulated at the early stage.It revealed that ScMT1 cooperated with ScMT2-1-5 and ScMT3 is involved in the positive response to Cu2+stress in roots,and might play a more positive role than ScMT2-1-5 and ScMT3,and the three genes are successively involved in the molecular responses of Cu^2+ stress in roots.When treated with Zn^2+,the expression of ScMT1 and ScMT3 increased only in shoots and roots,respectively.The expression of ScMT2-1-5 was up-regulated at the early stage and then suppressed in both shoots and roots.It suggests that ScMT1 and ScMT3 mainly function in shoots and roots,respectively,when exposed to Zn2+stress.ScMT2-1-5 was involved in the molecular responses of Cd2+stress at the early stage.ScMT1,ScMT2-1-5,and ScMT3 showed similar or complementary expression patterns in different tissues of sugarcane when exposed to heavy metals,which revealed the functional diversity of sugarcane MTs in detoxi?cation and cellular redox regulation,and their spatiotemporal coordination in mitigating or even preventing tissue injury caused by excessive heavy metals.The results provide a basic information for further research on mechanisms in the synergistic enhancement of tolerance to heavy metals for MTs family genes in the highly polyploid sugarcane.