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  

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

作　　者：高世武[1] 傅志伟[1] 陈云[1] 林兆里[2] 许莉萍[1] 郭晋隆[1] GAO Shi-Wu;FU Zhi-Wei;CHEN Yun;LIN Zhao-Li;XU Li-Ping;GUO Jin-Long(Key Laboratory of Sugarcane Biology and Genetic Breeding(Fujian),Ministry of Agriculture and Rural Affairs/Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China;Sugarcane and Products Quality Supervisory Inspection and Test Center of Ministry of Agriculture and Rural Affairs/Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China)

机构地区：[1]福建农林大学/农业农村部福建甘蔗生物学与遗传育种重点实验室,福建福州350002 [2]福建农林大学/农业农村部甘蔗及制品质量监督检验测试中心,福建福州350002

出　　处：《作物学报》2020年第2期166-178,共13页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31871690);国家留学基金委留学基金项目(20163035)资助~~

摘　　要：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Metallothioneins(MTs)are cysteine-rich,low-molecular-weight proteins.Plant MTs play important roles in detoxification 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 con

关 键 词：甘蔗 金属硫蛋白 重金属 实时荧光定量PCR 

分 类 号：R73[医药卫生—肿瘤]