高世武¹, 傅志伟¹, 陈云¹, 林兆里², 许莉萍¹, 郭晋隆^,1,*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

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

GAO Shi-Wu¹, FU Zhi-Wei¹, CHEN Yun¹, LIN Zhao-Li², XU Li-Ping¹, GUO Jin-Long^,1,*1 Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture and Rural Affairs / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
2 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

通讯作者: 郭晋隆, E-mail: jl.guo@163com

收稿日期:2019-06-10接受日期:2019-09-26网络出版日期:2019-10-14

基金资助:

本研究由国家自然科学基金项目.31871690 国家留学基金委留学基金项目资助.20163035

Received:2019-06-10Accepted:2019-09-26Online:2019-10-14

Fund supported:

This study was supported by the National Natural Science Foundation of China.31871690 the Foundation for China Scholarship Council.20163035

作者简介 About authors
E-mail:gaoshiwu2008@126.com。








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, Cd ²⁺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Cu ²⁺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。
关键词： 甘蔗;金属硫蛋白;重金属;实时荧光定量PCR

Abstract
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 Cu ²⁺ 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 Zn ²⁺ stress. ScMT2-1-5 was involved in the molecular responses of Cd ²⁺ 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.
Keywords：sugarcane (Saccharum officinarum L.);metallothionein;heavy metal;real-time qPCR


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本文引用格式
高世武, 傅志伟, 陈云, 林兆里, 许莉萍, 郭晋隆. 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[J]. 作物学报, 2020, 46(2): 166-178. doi:10.3724/SP.J.1006.2020.84086
GAO Shi-Wu, FU Zhi-Wei, CHEN Yun, LIN Zhao-Li, XU Li-Ping, GUO Jin-Long. Cloning and expression analysis of metallothionein family genes in response to heavy metal stress in sugarcane (Saccharum officinarum L.)[J]. Acta Agronomica Sinica, 2020, 46(2): 166-178. doi:10.3724/SP.J.1006.2020.84086


金属硫蛋白(metallothionein, MT)是广泛存在于生物中的一类富含半胱氨酸(cysteine, Cys)的低分子量蛋白^[1]。自1957年被首次报道以来, 已在动物、植物、以及微生物中发现含有MT^[2]。由于MT的Cys富集结构域含有的巯基具有螯合金属离子的能力, 其在参与细胞金属离子代谢、调节金属离子的定向运输以及螯合解除重金属离子毒害等方面起重要作用, 而引发研究者的广泛关注^[3,4]。植物中MT基因以基因家族的形式出现, 在植物体内主要参与金属离子运输、维持氧化还原平衡和调控基因表达等^[1], 而且还参与一系列的植物生理调节过程, 包括通过结合重金属离子而间接调控细胞内活性氧水平^[5,6]、调控细胞生长与增殖、调节酶和转录因子的活性^[7], 参与细胞凋亡过程甚至可能扮演细胞氧化还原传感器的角色^[8,9]。

根据MT中Cys残基的位置及排列方式, 前人将植物MT分为MT1、MT2、MT3和MT4四个类型^[1,10]。其中, MT1型金属硫蛋白在肽链的N端和C端分别含有3个保守的Cys-Xaa-Cys排列结构(Cys代表半胱氨酸, Xaa代表其他氨基酸), 被一个不含Cys的中间区域分开; 肽链的两端富含Cys, 不含芳香族氨基酸和疏水性氨基酸, 氨基酸个数大约在10~15; 而中间区域往往含有芳香族氨基酸和疏水性氨基酸而且保守性低, 氨基酸个数大约在30~45^[10]。MT2型金属硫蛋白氨基酸的排列方式与MT1型类似, N端和C端富含Cys且被一个含有大约40个氨基酸的中间区域分开; 但与MT1型不同的是, 在MT2型MT的前4个氨基酸位置具有其特有的Cys-Cys排列方式^[10]。MT3型金属硫蛋白在肽链的N端只含有4个Cys, 且前3个Cys按照保守的结构Cys-Gly-Asn-Cys-Asp-Cys排列, C端一般含有6个Cys, 且都是以Cys-Xaa-Cys的排列方式存在^[10]。上述3类MT在结构上具有很高的相似性: 在氨基酸序列的N端和C端各存在一个富含Cys的结构域, 即α和β结构域; 这2个结构域被一条不含Cys的中间区域分开。与其他3种类型的MT不同, MT4型MT含有3个富含Cys的结构域, 平均散布在整条肽链中, 由2条含10~15个氨基酸残基的中间区域隔开, 每个结构域中含有大约5~6个Cys^[10]。金属绑定结构域(Cys富集区)的排列方式变化多样, 表明了MT家族各成员功能的多样性^[11]。

研究者已相继从多种植物中分离鉴定出植物MT家族成员基因^[12]。目前, 已从大麦(Hordeum vulgare L.)中分离到10个MT成员基因^[13]; 从拟南芥(Arabidopsis thaliana L.)中分离到8个MT成员基因(不计剪接变体), 其中有3个MT1型基因 (AtMT1a、AtMT1b和AtMT1c)、2个MT2型基因(AtMT2a和AtMT2b)、1个MT3型基因(AtMT3-1)和2个MT4型基因(AtMT4a-1和AtMT4b-1)(http://www. arabidopsis.org/index.jsp)。此外, 前人还基于全基因组分析, 表明水稻(Oryza sativa L. subsp. japonica)中共有13个MT基因编码15个蛋白, 包括7个MT1型基因(OsMT1a、OsMT1b、OsMT1c、OsMT1d、OsMT1e、OsMT1f和OsMT1g)、4个MT2型基因(OsMT2a、OsMT2b、OsMT2c和OsMT2d)、1个MT3型基因和1个MT4型基因^[14]。甘蔗(Saccharum spp. hybrid)和拟南芥(A. thaliana L.)、水稻(Oryza sativa L.)是迄今少数几个含有4种类型MT的植物^[1]。基于DNA凝胶杂交技术^[15]和甘蔗EST数据库分析^[16]表明, 多倍体作物甘蔗基因组中含有典型的MT1型、MT2型和MT3型基因各8个, 含有MT4型基因1个, 但有核酸序列报道的目前仅有1个MT1型基因(MT1)^[15]、4个MT2型基因(ScMT2-1-1、ScMT2-1-2、ScMT2-1-3和ScMT2-1-4)^{[15,16,17,18]}和1个MT3型基因(MT3)^[15]。研究者对植物MT家族基因各成员在基因结构的差异和重金属结合等功能上有一定的认识, 但对同一物种MT家族各成员基因相互间的进化关系、在植物应答重金属等逆境胁迫中的协同作用及调控机理等研究鲜见报道。

甘蔗既是世界上最重要的糖料作物, 也是重要的生物能源作物, 具有重要的经济地位^[17,19]。同时, 甘蔗作为大田栽培的高生物量C4禾本科作物, 具有适应性广、抗逆性强、耐连作以及对镉等重金属具有良好的耐受和超富集能力, 是潜在的土壤重金属污染的植物修复物种^[15]。分离鉴定甘蔗应答重金属胁迫的关键基因是解析其功能并进一步利用的基础, 对筛选、培育和利用植物修复物种具有重要的现实和理论意义。现代甘蔗栽培种是异源多倍体和非整倍体作物, 遗传背景高度杂合^[20,21]。为了明确甘蔗MT基因家族各成员的组成, 深入研究和阐明MT各成员在应答重金属胁迫过程中的功能奠定基础, 本研究以染色体的倍性相对明确、栽培面积最大的甘蔗热带原种Badila (S. officinarum L. cv. Badila) (2n = 80)^[22]为材料, 克隆了MT1、MT2和MT3三个类型的金属硫蛋白基因, 分析了3类MT基因对CuCl₂、ZnSO₄或CdCl₂胁迫的响应特点, 结合不同重金属离子胁迫下甘蔗的表型、地上部和地下部富集重金属离子的水平, 初步探讨了甘蔗不同类型MT基因在甘蔗应答重金属胁迫中的功能。

1 材料与方法

1.1 植物材料处理

供试甘蔗材料为热带种原种Badila。挑选长势一致的组培苗, 按单株置72孔穴盘中, 28℃, 光12 h/暗12 h培养4周。用1/10 Hongland营养液培养, 每隔2 d更换一次培养液。待植株长至4~5片完全展开叶后, 开始胁迫试验。将组培苗分为9组, 其中6组为处理组, 分别采用含有250 μmol L^-1 CdCl₂、250 μmol L^-1 CuCl₂和1000 μmol L^-1 ZnSO₄的1/10 Hongland营养液培养6 h和48 h; 另外3组为对照组, 以1/10 Hongland营养液分别培养0、6和48 h。将上述处理后的甘蔗组培苗全株洗净, 分为地上部(根以上部分)和地下部(根部)取样, 转入下一步处理(重金属含量检测)或放置于液氮中速冻后转到-80℃冰箱保存备用(RNA提取)。实验设置3次生物学重复, 每个重复3株。

1.2 重金属含量检测

用双蒸水反复冲洗整个植株表面3~5次, 然后将各植株的地下部分别浸没在装有1.0 mmol L^-1 EDTA溶液的烧杯中, 室温条件下浸泡2 h, 以去除植物根部表面残留的重金属离子, 再用双蒸水冲洗甘蔗植株, 用滤纸把植株表面的水吸干, 分为地上部和地下部取样。用锡箔纸包好样品后在105℃杀青30 min, 在90℃下烘干至恒重, 称量其干物质量, 最后将所有处理过的样品放入干燥罐中保存备用。依据国家标准GB/T 5009.13-2003和GB/T 5009.14-2003, 采用火焰法测定铜和锌含量; 依据国家标准GB/T 5009.15-2003, 采用石墨炉法测定镉含量。

1.3 ScMTs基因的克隆与生物信息学分析

分别以已报道的甘蔗及其同源物种的MT1、MT2和MT3基因作为出发序列, 利用NCBI (http:// www.ncbi.nlm.nih.gov/)中的blastn工具搜索甘蔗EST数据库, 得到分别以甘蔗EST序列cf575433、ca231193和ca109790为代表的3个EST拼接群(Contig)。以上述EST拼接群的同源区段为模板, 应用Primer Premier 5.0软件分别设计克隆ScMT1、ScMT2-1-5和ScMT3基因所用的特异性引物(表1)。

Table 1
表1
表1甘蔗金属硫蛋白基因的克隆引物
Table 1Primers for isolation of sugarcane metallothionein genes

基因 Gene	上游引物 Forward primer (5°-3°)	下游引物 Reverse primer (5°-3°)
ScMT1	TGTAACACGCCCTTTGGTAGAT	TGTCCTTATTGTTCAACCACCAG
ScMT2-1-5	CGAGGAGAGGAAGAGGACGACT	TTATTCGTTCCCATCAGTACCA
ScMT3	AAGAAGGCCTTGCGCAACAAA	ACGTACCGCGTCAGGTCCTCGT

新窗口打开|下载CSV

应用ORF Finder在线工具进行核酸及氨基酸序列组成分析、开放阅读框(open reading frame, ORF)的查找和翻译; 用DNAMAN5.2软件进行多序列比对分析; 用Clustal W软件对不同植物的金属硫蛋白的氨基酸序列进行多序列比对后, 借助Mega 7.0软件的邻接法(Neighbor-joining Method)构建系统进化树。应用IBS (illustrator for Biological Sequences) 1.0.3软件绘制基因结构; 氨基酸比对的序列分别来源于拟南芥(A. thaliana L.)、水稻(O. sativa L.)和甘蔗(Saccharum spp. hybrid)的MT1、MT2和MT3亚家族成员, 其中拟南芥(A. thaliana L.) 5个MT亚家族成员的名称/登录号信息为AtMT1A/AT1G07600.1、AtMT1C/ AT1G07610.1、AtMT2A/AT3G09390.1、AtMT2B/ AT5G02380.1和AtMT3-1/AT3G15353.1, 水稻(O. sativa L.) 6个MT亚家族成员的名称/登录号信息为OsMT1A /XP_015617237.1、OsMT1B/XP_015628821.1、OsMT2A/ XP_015645105.1、OsMT2B/XP_015619130.1、OsMT2C/XP_015638726.1、OsMT3A/Os01g0200700, 甘蔗(Saccharum spp. hybrid) 7个MT亚家族成员的名称/登录号信息为ScMT1/KJ504373、ScMT2-1-1/ scrufl3063a10.g、ScMT2-1-2/AAV50043、ScMT2-1-3/ AFJ44225、ScMT2-1-4/KJ504375、ScMT2-1-5/MH191346、ScMT3/KJ504374.1。

1.4 ScMTs基因表达模式分析

按照TRIzol Reagent (Invitrogen, 美国)说明书提取甘蔗材料总RNA; 按照RQ1RNase-Free DNase试剂说明书(Promega, 美国)逆转录前用DNA酶处理RNA样品; 按照PrimeScript 1st StrandcDNA Synthesis Kit (TaKaRa, 中国大连)说明书逆转录; 按照SYBR Green PCR Master Mix Kit (Rox) (Roche, 美国)说明书配制定量反应体系。

根据所克隆到的甘蔗MT家族基因的序列信息, 应用Primer Premier 5.0软件设计ScMTs的定量引物。采用甘蔗甘油醛-3-磷酸脱氢酶(glyceraldehyde-3- phosphate dehydrogenase, GAPDH)基因作内参基因^[23], 引物具体信息见表2。qRT-PCR条件为50℃ 2 min; 95℃ 10 min; 95℃ 15 s, 60℃ 1 min, 40个循环。待反应停止, 进行融解曲线分析。采用2^-ΔΔCT算法分析实验结果^[24]。在ABIPRISM7300 (Applied Biosystems, 美国)型实时荧光定量PCR仪上进行基因定量表达分析。

Table 2
表2
表2检测甘蔗金属硫蛋白家族基因表达的荧光定量PCR引物
Table 2qPCR primers for amplification of sugarcane metallothionein genes

基因 Gene	上游引物 Forward primer (5°-3°)	下游引物 Reverse primer (5°-3°)
ScMT1	TGTAACACGCCCTTTGGTAGAT	CTCCTCCAGGTCAGGGTACTTC
ScMT2-1-5	GAGGAAGAGGACGACTGCGAGG	TCATCGCCAC CTCCTCACTT
ScMT3	GCTTCCAGCATGTCGGGCAC	ACGGTCACCTCCTCCTCAACGA
GAPDH	CACGGCCACTGGAAGCA	TCCTCAGGGTTCCTGATGCC

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2 结果及分析

2.1 重金属胁迫下甘蔗幼苗的表型特征

甘蔗幼苗在250 μmol L^-1 Cd²⁺或250 μmol L^-1 Cu²⁺胁迫6 h和24 h时, 植株无明显症状, 但当胁迫48 h时, 植株叶片卷曲、叶尖干枯发黄、根系褐化(图1-B, D)。与Cd²⁺、Cu²⁺胁迫后的表现不同, 在所观测的时间点内, 1000 μmol L^-1 Zn²⁺胁迫下的甘蔗植株未表现出明显的受害症状, 但有部分幼苗叶片的颜色变紫(图1-C)。解除重金属胁迫48 h时间后, 甘蔗幼苗表型开始恢复正常(图略)。

图1

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图1不同重金属胁迫48 h时甘蔗幼苗表型

Fig. 1Phenotype of sugarcane seedlings treated with heavy metals for 48 h

A: CK; B; CdCl₂; C: ZnSO₄; D: CuCl₂.

2.2 甘蔗幼苗中重金属的累积特点

由图2可知, 甘蔗植株对重金属的富集量均随着胁迫时间的延长而明显上升, 其中, 地下部累积浓度远远超出地上部。甘蔗幼苗在Cd²⁺、Zn²⁺、Cu²⁺胁迫处理48 h后, 在地上部的累积量分别为(32.2±24.3) mg kg^-1、(225.0±3.6) mg kg^-1和(34.3± 15.9) mg kg^-1, 在地下部的最大累积量分别为(196.7±26.6) mg kg^-1、(1063.8 ±101.9) mg kg^-1和(448.2±43.5) mg kg^-1。

图2

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图2Cd²⁺、Zn²⁺、Cu²⁺胁迫下甘蔗幼苗地上部、地下部重金属积累量

图柱上不同的小写字母表示5%水平下差异的显著性。
Fig. 2Heavy metal contents in sugarcane shoots and roots under the stresses of Cd²⁺, Zn²⁺, or Cu²⁺

Bars superscripted by different letters are significantly different at the 5% probability level.

2.3 ScMTs基因克隆与生物信息学分析

应用RT-PCR技术, 从Badila中获得3个MT基因的cDNA序列(图3), 分别命名为ScMT1、ScMT2-1-5和ScMT3 (对应的GenBank登录号分别为KJ504373、MH191346和KJ504374)。其中, ScMT1的cDNA全长448 bp, 5°非编码区长67 bp, 3°非编码区长153 bp, ORF长228 bp, 共编码75个氨基酸; 推导蛋白ScMT1氨基酸序列的N端和C端均含有3个CXC排列(C表示半胱氨酸残基Cys, X表示除了Cys外的任意氨基酸), 排列方式符合植物MT1型的结构特征(图4-A)。ScMT2-1-5的cDNA全长461 bp, 5°非编码区长36 bp, 3°非编码区长179 bp, 开放阅读框长246 bp, 共编码81个氨基酸; 推导蛋白ScMT2-1-5氨基酸序列的N端含有CC、CXC和CXXC的排列结构, C端含有3个CXC排列, 符合植物MT2型的典型特征(图4-B)。ScMT3的cDNA全长416 bp, 5°非编码区长64 bp, 3°非编码区长154 bp, 开放阅读框长198 bp, 共编码65个氨基酸; 推导蛋白ScMT3氨基酸序列N端的前3个Cys以CXXCXC方式排列, C端的Cys以3个CXC方式排列, 该排列方式符合植物MT3型的序列特征(图4-C)。系统进化树分析结果也表明, 甘蔗ScMT1、ScMT2-1-5和ScMT3分别属于植物MT1、MT2和MT3型金属硫蛋白(图5)。

图3

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图3甘蔗金属硫蛋白基因ScMTs的PCR产物凝胶电泳图

Fig. 3Agarose electrophoresis analysis of the RT-PCR products of ScMTs

M: DNA ladder marker; 1: ScMT1; 2: ScMT2-1-5; 3: ScMT3.

图4

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图4ScMTs cDNA序列及其推导的氨基酸序列

A: ScMT1; B: ScMT2-1-5; C: ScMT3。*: 终止密码子; C显示的是Cys富集结构域中的半胱氨酸残基。
Fig. 4Nucleotide sequence and deduced amino acid sequence for ScMTs

A: ScMT1; B: ScMT2-1-5; C: ScMT3. *: Terminal codon; C shows the conservative cysteine residual of Cys-rich domain.

图5

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图5甘蔗、水稻和拟南芥MTs的系统进化树及外显子/内含子结构示意图

方块和条线分别代表外显子和内含子。
Fig. 5Phylogenetic relationships of MTs and a schematic diagram of the exon/intron structures of the MT genes from Saccharum spp., Oryza sativa L., and Arabidopsis thaliana L.

Boxes and lines indicate exons and introns, respectively.


以gDNA为模板, 经PCR扩增, 获得甘蔗ScMT1、ScMT2-1-5和ScMT3基因的基因组DNA序列, 大小分别为615、1797和642 bp (图略)。比较cDNA基因和基因组DNA基因序列可知, ScMT1基因组DNA基因含有2个外显子, 由1个283 bp的内含子连接; ScMT2-1-5基因组DNA基因含有3个外显子, 由2个内含子(大小分别为483 bp和583 bp)连接; ScMT3基因组DNA基因含有2个外显子, 由1个内含子(259 bp)连接(图5)。上述内含子的结构均符合GT-AG法则。

2.4 ScMTs基因表达模式

ScMT1受Cd²⁺胁迫的诱导而上调表达(图6-A, B); 在Cd²⁺胁迫前期(6 h), 甘蔗地上部中ScMT1相对表达量的变化不明显, 但在胁迫后期(48 h)显著上调, 为对照的2.36倍(图6-B)。甘蔗地上部中ScMT1基因迅速正向响应Zn²⁺胁迫, 在6 h时显著上调表达为对照的1.86倍, 并在处理周期内维持高于对照的水平(图6-C, D); 地下部中ScMT1的相对表达水平在处理前期和后期均无显著变化(图6-D)。甘蔗地上部中ScMT1也迅速响应Cu²⁺胁迫且呈连续上调表达趋势, 在胁迫前期和后期, 该基因的表达水平分别为对照的1.51倍和2.19倍(图6-E, F)。在Cd²⁺或Cu²⁺胁迫下, 地下部中ScMT1的相对表达水平均呈“抑-扬”趋势, 在胁迫前期有所下调, 但在胁迫后期均显著上调, 分别为对照的1.74倍(图6-B)和3.27倍(图6-F)。

图6

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图6甘蔗ScMT1基因在不同重金属胁迫下的表达模式

A、C和E分别为ScMT1在CdCl₂、ZnSO₄和CuCl₂处理下的表达模式。B、D和F分别为ScMT1在CdCl₂、ZnSO₄和CuCl₂处理下和相应对照中相对表达量的比值。误差线为每组处理的标准误(n = 3)。图柱上不同的小写字母表示5%水平下差异的显著性。
Fig. 6Expression patterns of ScMT1 in sugarcane under different heavy metal stresses

A, C, and E represent the expression patterns of ScMT1 in response to CdCl₂, ZnSO₄, and CuCl₂, respectively. B, D, and F represent the ratio of ScMT1 expression level between treatment (CdCl₂, ZnSO₄, and CuCl₂, respectively) and their control groups. The error bars represent the standard errors of each treating group (n = 3). Bars superscripted by different letters are significantly different at the 5% probability level.


ScMT2-1-5的表达受Cd²⁺胁迫的诱导, 在甘蔗地上部和地下部均呈连续上调趋势(图7-A, B)。其中, ScMT2-1-5在地上部的相对表达量在处理6 h和48 h时分别为对照的1.84倍和2.05倍, 同时, 地下部该基因的表达水平分别为对照的1.64倍和4.46倍(图7-B)。在处理前期, ScMT2-1-5在甘蔗地上部和地下部均受Zn²⁺胁迫的诱导而上调表达, 分别为对照的2.75倍和2.29倍(图7-C, D); 其相对表达量在48 h时回落至低于对照的水平(图7-D)。在甘蔗地上部和地下部, ScMT2-1-5的相对表达水平在Cu²⁺胁迫前期变化不明显, 但在胁迫后期均显著上调, 分别为对照的1.62倍和3.69倍(图7-E, F)。

图7

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图7甘蔗ScMT2-1-5基因在不同重金属胁迫下的表达模式

A、C和E分别为ScMT2-1-5在CdCl₂、ZnSO₄和CuCl₂处理下的表达模式。B、D和F分别为ScMT2-1-5在CdCl₂、ZnSO₄和CuCl₂处理下和相应对照中相对表达量的比值。误差线为每组处理的标准误(n = 3)。图柱上不同的小写字母表示5%水平下差异的显著性。
Fig. 7Expression patterns of ScMT2-1-5 in sugarcane under different heavy metal stresses

A, C, and E represent the expression patterns of ScMT2-1-5 in response to CdCl₂, ZnSO₄, and CuCl₂, respectively. B, D, and F represent the ratio of ScMT2-1-5 expression level between treatment (CdCl₂, ZnSO₄, and CuCl₂, respectively) and their control groups. The error bars represent the standard errors of each treating group (n = 3). Bars superscripted by different letters are significantly different at the 5% probability level.


甘蔗地上部ScMT3受Cd²⁺胁迫诱导, 呈上调表达趋势, 在48 h时为对照的1.64倍; 甘蔗地下部ScMT3的相对表达量在Cd²⁺胁迫6 h时上调为对照的1.41倍, 但在48 h时下调为对照的0.47倍(图8-A, B)。在Zn²⁺处理下, 甘蔗地上部ScMT3的相对表达水平也呈现上调趋势, 在胁迫6 h时就上调至对照的1.98倍, 并在处理后期维持在高于对照1.66倍的水平; 与地上部相比, Zn²⁺处理对地下部中ScMT3基因表达的诱导作用更加显著, 其表达水平在胁迫6 h时就迅速上调至对照的10.09倍, 并在处理后期维持在高于对照4.38倍的水平(图8-C, D)。甘蔗地上部ScMT3的表达受Cu²⁺处理的诱导而呈现上调趋势, 在胁迫6 h时和48h时, 分别上调为对照的1.71倍和1.46倍; 甘蔗地下部ScMT3基因对Cu²⁺胁迫的响应迅速且强烈, 其相对表达量在处理6 h时就达到最高, 为对照的3.96倍, 随后回落为对照的1.24倍(图8-E, F)。

图8

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图8甘蔗ScMT3基因在不同重金属胁迫下的表达模式

A、C和E分别为ScMT3在CdCl₂、ZnSO₄和CuCl₂处理下的表达模式。B、D和F分别为ScMT3在CdCl₂、ZnSO₄和CuCl₂处理下和相应对照中相对表达量的比值。误差线为每组处理的标准误(n = 3)。图柱上不同的小写字母表示5%水平下差异的显著性。
Fig. 8Expression patterns of ScMT3 in sugarcane under different heavy metal stresses

A, C, and E represent the expression patterns of ScMT3 in response to CdCl₂, ZnSO₄, and CuCl₂, respectively. B, D and F represent the ratio of ScMT3 expression level between treatment (CdCl₂, ZnSO₄, and CuCl₂, respectively) and their control groups. The error bars represent the standard errors of each treating group (n = 3). Bars superscripted by different letters are significantly different at the 5% probability level.

3 讨论

现代甘蔗栽培种(Saccharum spp. hybrid)对重金属镉具有良好的耐受能力。甘蔗对Cd²⁺胁迫的临界浓度在土壤栽培条件下达100 mg kg^{-1 [25]}, 在水培条件下达56.21 mg L^{-1 [15]}, 分别高于镉超富集植物龙葵(Solanum nigrum L.)和青葙(Celosia argentea Linn.)的25 mg kg^{-1 [26]}和20 mg kg^{-1 [27]}。现代甘蔗栽培种对镉还具有一定的富集能力。在上述临界浓度下, 龙葵叶和根分别可富集到124.6 mg kg^-1和259.9 mg kg^{-1 [26]}, 青葙叶和根分别可富集到388 mg kg^-1和238 mg kg^{-1 [27]}, 而甘蔗地上部和地下部富集到的镉含量分别为451 mg kg^-1 和9621 mg kg^-1 (干重)^[15]。与前二者相比, 尽管甘蔗对镉的转运系数小于1, 但基于其高生物量及对镉的高度富集能力, 依然被Sereno 等^[15]认为是潜在的镉修复植物。此外, 甘蔗还对重金属铜具有一定的耐受与富集能力^[15]。

与现代甘蔗栽培种类似, 本研究中原始热带种Badila对镉、锌、铜等重金属也有一定的耐受与富集能力, 组培幼苗能在含有100 μmol L^-1 CdCl₂、500 μmol L^-1 ZnSO₄或100 μmol L^-1 CuCl₂的培养液中生长而不发生毒害症状(图略); 当Cd²⁺或Cu²⁺的处理浓度为250 μmol L^-1 (Cd²⁺或Cu²⁺的含量分别相当于28.10 mg L^-1和15.89 mg L^-1)时, 幼苗在胁迫48 h时表现出受胁迫的表型(图1), 但生物量未见显著下降(结果略); 同时幼苗地上部中上述3种重金属的累积量分别达(32.2±24.3) mg kg^-1、(225.0±3.6) mg kg^-1和(34.3±15.9) mg kg^-1, 在地下部的最大累积量分别为(196.7±26.6) mg kg^-1、(1063.8±101.9) mg kg^-1和(448.2±43.5) mg kg^-1 (图3)。上述3种重金属离子经由Badila根部吸收并运输和分配到地上部分, 进而引发了相应的表型变化, 这为后续深入研究金属胁迫下MT等功能蛋白对甘蔗吸收与耐受重金属的机理奠定了基础。

植物金属硫蛋白(MT)被认为是细胞内维持金属离子平衡的关键蛋白^[1]。MT基因各型亚家族成员的克隆及其在重金属胁迫下的表达特性研究在拟南芥^[28]、水稻^[14]和大麦^[13]等植物中已有较系统有报道。甘蔗是除了拟南芥、水稻以外的少数几个含有全部4类MT的植物^[1]。一般认为, MT4仅在发育的种子中表达^[1,29], 考虑到甘蔗是中日性植物, 开花结实需要特定的光温条件, 且生产上采用蔗茎进行无性繁殖, 因此本研究暂未关注甘蔗MT4类型的基因。MT2型基因在甘蔗中的报道最多, 例如克隆自甘蔗现代栽培种的ScMT2-1-1、ScMT2-1-2和ScMT2- 1-3 ^[15,16,17], 以及来源于Badila的ScMT2-1-4^[18]。Guo等^[17]将植物MT2型分为3个亚类, 本研究中ScMT2- 1-5与上述甘蔗MT2型基因的推导蛋白序列均属于MT2-1亚类, 序列一致性为94.46%。ScMT2-1-5与ScMT2-1-4一样, 均来源于Badila, 且二者的推导蛋白序列一致性为96.39%。其中, ScMT2-1-4比ScMT2-1-5多了2个氨基酸残基, 且还有1个氨基酸残基的不同, 这3个氨基酸残基位于不含Cys的中间区域(图略), 初步推测二者为等位基因, 但还需要通过克隆该基因上下游区段等方法做进一步确认。关于甘蔗MT1型和MT3型基因的报道较少, Sereno等^[15]应用RNA点杂交技术研究了甘蔗MT1 (GenBank登录号为CA287650)、MT2 (CA232620)和MT3 (CA109591) 3个类型的MT基因在Cu²⁺和Cd²⁺胁迫下的应答模式, 但上述基因均来源于甘蔗EST序列, 并无实体克隆的报道。本研究进一步对其推导的氨基酸序列分析发现, 该MT1 (GenBank登录号为CA287650) C端的Cys富集区结构域缺失, 而MT3 (CA109591)的N端和C端的Cys富集区结构域也缺失, 均不符合植物MT1型和MT3型的结构特征。本研究克隆的ScMT1、ScMT2-1-5和ScMT3基因, 其推导的氨基酸序列分别符合植物MT1型、MT2型和MT3型的结构特征(图3)。进一步比较拟南芥、水稻和甘蔗中上述3个类型的MT基因的结构, MT1型MT基因的外显子与内含子的构成在拟南芥、水稻和甘蔗中较为保守, 均只含有1个内含子; 拟南芥MT2型MT基因含有1个内含子, 而禾本科的水稻和甘蔗的MT2型MT基因含有2个内含子; 值得指出的是, 甘蔗MT3型MT基因仅含有1个内含子, 与拟南芥和水稻MT3型含有2个内含子的基因结构不同(图4)。

MT1型基因成员在镉、锌或铜的富集和耐受方面的功能已分别在拟南芥^[28,30]和水稻^[31]等模式植物中得到实验证据的支持。柽柳(Tamarix sp.) MT1基因也被证明有助于提高转基因烟草植株对Cd²⁺的抗性^[32]。不同植物的MT1基因对重金属胁迫的应答模式有类似的特点。大麦叶片中HvMT1a受到不同浓度Zn²⁺胁迫的诱导, 在胁迫48 h时均显著上调表达; 而不同浓度的Cd²⁺胁迫均抑制了该基因的表达; 此外, HvMT1a对Cu²⁺胁迫的响应存在剂量效应, 低浓度Cu²⁺胁迫(50 μmol L^-1 CuSO₄)下HvMT1a的表达水平变化不大, 而高浓度Cu²⁺胁迫(500 μmol L^-1 CuSO₄) 48 h时诱导HvMT1a的表达^[13]。与HvMT1a类似, 水稻幼苗中OsMT1a基因受低浓度Zn²⁺ (10 μmol L^-1)胁迫的诱导显著上调表达, 而对低浓度Cu²⁺ (10 μmol L^-1)胁迫无明显响应^[31]。本研究中, ScMT1对Cd²⁺胁迫的响应相对滞后, 在甘蔗地上部和地下部的表达水平在胁迫前期变化均不明显(图5-A), 随着甘蔗地上部和地下部镉的富集量从15.5 mg kg^-1和93.7 mg kg^-1分别上升到32.2 mg kg^-1和196.7 mg kg^-1 (图2), 甘蔗地上部和地下部ScMT1分别显著上调表达为对照的2.37倍和1.75倍(图5-B)。上述结果表明ScMT1对Cd²⁺胁迫的响应存在域值或延后效应, 提示了ScMT1可能并不直接与Cd²⁺螯合, 但参与了胁迫后期过量Cd²⁺毒害引起的生理生化与分子响应。甘蔗地上部和地下部富集到的锌含量均随处理时间的延长呈连续上升的趋势, 且甘蔗地下部富集到的锌含量显著高于地上部(图2)。Zn²⁺胁迫下, ScMT1在甘蔗幼苗地上部的相对表达量总体上维持高于对照的水平, 但ScMT1在甘蔗幼苗地下部对Zn²⁺胁迫的响应却不敏感(图5-D), 提示了该基因在根中并不参与甘蔗对过量Zn²⁺胁迫的生理及分子响应, 而仅在地上部发挥其功能。ScMT1在Cu²⁺胁迫下的应答模式总体上与拟南芥AtMT1a类似, 后者也是在根和叶均受到Cu²⁺胁迫的诱导而上调表达^[29], 而甘蔗地上部和地下部ScMT1对Cu²⁺胁迫的应答模式还是有所区别(图5-E, F): 地上部ScMT1对Cu²⁺胁迫的响应呈连续上调趋势(图5-F); 而地下部ScMT1对Cu²⁺胁迫的正向响应则相对滞后(图5-F)。这种滞后表现为甘蔗地下部富集到307.3 mg kg^-1的铜时, ScMT1相对表达水平并不高于对照(图5-F), 而当铜含量达到448.2 mg kg^-1时, 显著上调表达为对照的3.27倍(图5-F)。大麦HvMT1a^[13]和水稻OsMT1a^[31]对Cu²⁺胁迫的响应也存在相类似的剂量效应。ScMT1在地上部对Cu²⁺胁迫相对敏感, 而在地下部的响应存在域值或延后效应, 提示ScMT1参与甘蔗对过量Cu²⁺胁迫的分子响应, 但在地上部和地下部的机制有所不同。

与MT1类成员主要参与植物对重金属的富集有所区别, 已有的证据表明MT2类成员主要在提高植物对镉等重金属的耐受能力方面发挥功能。过表达AtMT2b提高了烟草对Cd²⁺的耐受能力, 但单独过表达AtMT2b对Cd²⁺和Zn²⁺从根部向地上部运输方面的促进作用不显著^[33], AtMT2b在Cu²⁺转运或累积到拟南芥地上部方面也不起直接的作用^[28]。过表达甘蔗ScMT2-1-3提高了重组大肠杆菌对Cd²⁺和Cu²⁺的耐受性^[17], 也提示了甘蔗MT2基因有类似的功能。不同植物的MT2基因对重金属胁迫的响应各有特点, 二色补血草(Limonium bicolor (Bunge) O. Kuntze) LbMT2基因的表达受Cd²⁺和Cu²⁺诱导, 在地上部和地下部总体上均上调表达^[34]; 东南景天(Sedum alfredii Hance) SaMT2基因的表达受Cd²⁺和Zn²⁺诱导, 在地上部和地下部均上调表达^[35]; 而AtMT2对Cu²⁺胁迫的响应具有组织特异性, Cu²⁺(25 μmol L^-1 CuSO₄)能诱导拟南芥AtMT2a和AtMT2b分别在叶和根中高表达^[29]。上述MT2基因对不同重金属胁迫的差异应答, 体现了MT2基因在不同植物适应重金属等氧化胁迫的进化过程中出现了功能分化。值得指出的是, 甘蔗是高度杂合的多倍体或非整倍体作物, 染色体组中ScMT2基因的等位基因数量较多, 对同一种重金属胁迫的应答趋势也不尽相同。ScMT2-1-3在甘蔗FN39组培苗(全株)中的表达受到Cu²⁺的促进, 然而却受Cd²⁺的抑制^[17]; ScMT2-1-4在地上部受Cd²⁺胁迫的诱导呈先抑后扬的模式, 但在地下部受Cd²⁺的抑制而稳定下调; 其在地上部和地下部响应Zn²⁺胁迫而呈先扬后抑的表达趋势; 并在地上部和地下部受Cu²⁺胁迫的诱导总体上有所上调^[18]。本研究中ScMT2-1-5对Zn²⁺和Cu²⁺胁迫下的应答模式与ScMT2-1-4类似(图6-D, F), 但其在地上部和地下部的表达受Cd²⁺胁迫的诱导均呈连续上调趋势(图6-B), 表明ScMT2-1-5在甘蔗耐受Cd²⁺胁迫过程中可能起相对主要的作用; 其与等位基因ScMT2-1-4在甘蔗对Zn²⁺和Cu²⁺响应过程中可能起正向协同作用, 而在甘蔗地下部响应Cd²⁺重金属胁迫的过程中, 二者的功能及调控方式可能有所分化。ScMT2-1-4和ScMT2-1-5对同一种重金属胁迫的差异应答现象, 为了解等位基因在高度杂合的多倍体或非整倍体——甘蔗中的复杂功能及相应的调控机制提供了进一步的证据与思路。

有证据表明, 植物MT3在应答Cd²⁺胁迫方面并不起主要作用。天蓝遏蓝菜(Thlaspi caerulescens J. & C. Presl) TcMT3基因在地上部的表达水平高于地下部, Cd²⁺胁迫(100 μmol L^-1)对该基因在地上部和地下部的表达影响不显著^[36], 而50 μmol L^-1 Cu²⁺胁迫诱导该基因在地上部显著上调表达。与TcMT3类似, 水稻OsMT3基因受到Cu²⁺胁迫的诱导在叶片中表达量显著上调^[37]。体外Cd²⁺、Zn²⁺或Cu²⁺胁迫下的酵母异源互补等实验也表明, 大麦(Hordeum vulgare L.) MT3是维持重金属锌和铜在体内平衡的看家蛋白^[38]。与TcMT3和OsMT3仅在地上部上调表达不同, ScMT3在地上部和地下部受Cu²⁺胁迫的诱导均上调表达, 并且在地下部上调幅度达3.96倍, 高于其在地上部的上调水平(图7-F)。ScMT3与OsMT3分别在地下部与地上部正向响应Cu²⁺等重金属胁迫或许与二者基因结构(内含子)的差异(图4)有关。同时, ScMT3还受Zn²⁺胁迫的诱导上调表达, 并且在地下部的表达水平高于对照10倍, 也远远高于其在地上部的上调幅度。此外, 尽管甘蔗地上部及地下部也富集了相当水平的镉(图2), ScMT3受Cd²⁺胁迫的诱导也有所上调(图7-B), 但上调表达的幅度明显低于其在Zn²⁺和Cu²⁺胁迫时的上调表达水平(图7-D, F)。上述结果提示了ScMT3主要在甘蔗地下部响应Zn²⁺和Cu²⁺胁迫时发挥更积极的作用。

尽管近年来已经有不少关于植物MT功能的研究, 但植物MT家族成员丰富, 要阐明MT各成员的功能及之间的协同作用, 仍然需用更多的证据^[11]。

4 结论

从甘蔗热带种Badila中克隆获得金属硫蛋白家族3个成员基因ScMT1、ScMT2-1-5和ScMT3, 它们分别归属于MT1、MT2和MT3型植物MT亚家族。在营养液培养条件下Badila幼苗对重金属Cd²⁺、Zn²⁺和Cu²⁺有一定的耐受和富集能力, 上述重金属离子经由根部吸收并运输、分配到地上部, 引发了相应的表型和分子响应。ScMT1、ScMT2-1-5和ScMT3参与上述分子响应过程, 且在甘蔗不同组织中对Cd²⁺、Zn²⁺或Cu²⁺表现出协同或互补的应答特性, 提示了上述MT成员基因在甘蔗应对重金属伤害及其代谢机理等方面的功能分化, 和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。

参考文献 View Option 原文顺序
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Among the heavy metal-binding ligands in plant cells the phytochelatins (PCs) and metallothioneins (MTs) are the best characterized. PCs and MTs are different classes of cysteine-rich, heavy metal-binding protein molecules. PCs are enzymatically synthesized peptides, whereas MTs are gene-encoded polypeptides. Recently, genes encoding the enzyme PC synthase have been identified in plants and other species while the completion of the Arabidopsis genome sequence has allowed the identification of the entire suite of MT genes in a higher plant. Recent advances in understanding the regulation of PC biosynthesis and MT gene expression and the possible roles of PCs and MTs in heavy metal detoxification and homeostasis are reviewed.

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金属硫蛋白是一类分子量较小、富含Cys的金属结合蛋白，广泛分布于生物界。近年来从植物中克隆到许多编码金属硫蛋白的基因，并在研究基因表达模式、组织表达特异性以及基因结构，如启动子、内含子在染色体上的定位等方面取得了一定进展，但对其功能的研究还处于起步阶段。很多实验表明，植物金属硫蛋白可以通过其大量的Cys残基螯合重金属并清除活性氧，使植物避免氧化损伤。文章介绍了植物金属硫蛋白的分类、特征、基因结构及其在植物重金属解毒中的作用。






Quan X Q, Zhang H T, Shan L, Bi Y P . Advances in plant metallothionein and its heavy metal detoxification mechanisms
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金属硫蛋白是一类分子量较小、富含Cys的金属结合蛋白，广泛分布于生物界。近年来从植物中克隆到许多编码金属硫蛋白的基因，并在研究基因表达模式、组织表达特异性以及基因结构，如启动子、内含子在染色体上的定位等方面取得了一定进展，但对其功能的研究还处于起步阶段。很多实验表明，植物金属硫蛋白可以通过其大量的Cys残基螯合重金属并清除活性氧，使植物避免氧化损伤。文章介绍了植物金属硫蛋白的分类、特征、基因结构及其在植物重金属解毒中的作用。






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Metallothioneins are cysteine-rich proteins, which function as (i) metal carriers in basal cell metabolism and (ii) protective metal chelators in conditions of metal excess. Metallothioneins have been characterized from different eukaryotic model and cultivable species. Presently, they are categorized in 15 families but evolutionary relationships between these metallothionein families remain unresolved. Several cysteine-rich protein encoding genes that conferred Cd-tolerance in Cd-sensitive yeast mutants have previously been isolated from soil eukaryotic metatranscriptomes. They were called CRPs for "cysteine-rich proteins". These proteins, of unknown taxonomic origins, share conserved cysteine motifs and could be considered as metallothioneins. In the present work, we analyzed these CRPs with respect to their amino acid sequence features and their metal-binding abilities towards Cd, Zn and Cu metal ions. Sequence analysis revealed that they share common features with different known metallothionein families, but also exhibit unique specific features. Noticeably, CRPs display two separate cysteine-rich domains which, when expressed separately in yeast, confer Cd-tolerance. The N-terminal domain contains some conserved atypical Cys motifs, such as one CCC and two CXCC ones. Five CRPs were expressed and purified as recombinant proteins and their metal-binding characteristics were studied. All these CRPs chelated Cd(II), Zn(II) and Cu(I), although displaying a better capacity for Zn(II) coordination. All CRPs are able to confer Cd-tolerance, and four of them confer Zn-tolerance in the Zn-sensitive zrc1Δ yeast mutant. We designated these CRPs as environmental metallothioneins belonging to a new formerly undescribed metallothionein family.

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Metallothioneins are small, ubiquitous Cys-rich proteins known to be involved in reactive oxygen species (ROS) scavenging and metal homeostasis. We found that the expression of a metallothionein gene (OsMT2b) was synergically down-regulated by OsRac1 and rice (Oryza sativa) blast-derived elicitors. Transgenic plants overexpressing OsMT2b showed increased susceptibility to bacterial blight and blast fungus. OsMT2b-overexpressing cells showed reduced elicitor-induced hydrogen peroxide production. In contrast, homozygous OsMT2b::Tos17-inserted mutant and OsMT2b-RNAi-silenced transgenic cells showed significantly higher elicitor-induced hydrogen peroxide production than the wild-type cells. In vitro assay showed that recombinant OsMT2b protein possessed superoxide- and hydroxyl radical-scavenging activities. Taken together, these results showed that OsMT2b is an ROS scavenger and its expression is down-regulated by OsRac1, thus potentiating ROS, which function as signals in resistance response. The results suggest that OsRac1 plays a dual role as an inducer of ROS production and a suppressor of ROS scavenging.

[7] Haq F, Mahoney M, Koropatnick J . Signaling events for metallothionein induction
Mutat Res, 2003,533:211-226.
DOI:10.1016/j.mrfmmm.2003.07.014URLPMID:14643422 [本文引用: 1]
Metallothioneins (MTs) are low molecular weight, cysteine-rich metal-binding proteins found in a wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species. They are the single most abundant group of intracellular zinc-binding proteins in eukaryotic cells [Prog. Food Nutr. Sci. 14 (1990) 193]: 5-10% of zinc in human hepatocytes is bound to MTs [Methods Enzymol. 205 (1991) 613; FEBS Lett. 39 (1974) 229]. A critical physiological role for MTs has been suggested to encompass control of zinc availability to proteins requiring zinc for activity, but has not been definitively described. Nevertheless, a hallmark of MT gene regulation is their pattern of expression in different cell types and tissues, and their inducibility by a host of chemical and physical agents acting directly or indirectly on multiple cis-acting motifs in the regulatory regions of MT genes [Prog. Nucleic Acid. Res. Mol. Biol., 66 (2001) 357]. The pattern of physical and chemical events regulating basal MT gene transcription, and induction or repression of MT gene activity, provides valuable insight into the potential functions of MTs. In this review, we describe the transcriptional regulatory processes involved in MT gene expression.

[8] Fabisiak J P, Borisenko G G, Liu S X, Tyurin V A, Pitt B R, Kagan V E . Redox sensor function of metallothioneins
Methods Enzymol, 2001,353:268-281.
DOI:10.1016/s0076-6879(02)53055-xURLPMID:12078502 [本文引用: 1]
In summary, the redox conversions of MT cysteines are likely to be the principal mechanisms for regulation of metal binding and release by this protein. Oxidative and/or nitrosative challenges can serve to promote metal ion release from MT to render their delivery to specific target proteins. It is tempting to consider the potential roles of MTs as redox sensors because of their high sensitivity to cysteine modification, as well as their potential to amplify signals by releasing multiple metal ions. In other words, MTs may act early in a biological signaling cascade that triggers metal-dependent biochemical and cellular responses. Alternatively, uncontrolled release of metals by excessive oxidative stress may contribute to metal toxicity. Because oxidative and nitrosative signaling is ubiquitous within cells, the physiological function of MT demands that efficient recycling of modified cysteines be operative. Little is known regarding the potential mechanisms for the regeneration of MT after oxidative/nitrosative modification, but they may involve endogenous dithiols, such as thioredoxin, and pharmacologically relevant dithiols, such as dihydrolipoate.

[9] Va?ák M, Hasler D W . Metallothioneins: new functional and structural insights
Curr Opin Chem Biol, 2000,4:177-183.
DOI:10.1016/s1367-5931(00)00082-xURLPMID:10742189 [本文引用: 1]
In the past few years, tissue-specific mammalian metallothioneins (i. e. metallothionein-3 and metallothionein-4) have been discovered that possess distinct functional properties. Other recent developments include an insight into the role of the widely expressed mammalian metallothionein-1 and metallothionein-2 isoforms in zinc homeostasis and apoptosis. The three-dimensional structure of the evolutionary distant sea urchin Cd(7)-metallothionein A, which contains two metal-thiolate clusters, supports previous conclusions regarding the functional importance of this structural motif. Despite correlated efforts involving techniques of structural biochemistry and molecular biology, the primary function of metallothioneins remains enigmatic.

[10] Freisinger E . Structural features specific to plant metallothioneins
J Biol Inorg Chem, 2011,16:1035-1045.
DOI:10.1007/s00775-011-0801-zURLPMID:21688177 [本文引用: 5]
The metallothionein (MT) superfamily combines a large variety of small cysteine-rich proteins from nearly all phyla of life that have the ability to coordinate various transition metal ions, including Zn(II), Cd(II), and Cu(I). The members of the plant MT family are characterized by great sequence diversity, requiring further subdivision into four subfamilies. Very peculiar and not well understood is the presence of rather long cysteine-free amino acid linkers between the cysteine-rich regions. In light of the distinct differences in sequence to MTs from other families, it seems obvious to assume that these differences will also be manifested on the structural level. This was already impressively demonstrated with the elucidation of the three-dimensional structure of the wheat E(c)-1 MT, which revealed two metal cluster arrangements previously unprecedented for any MT. However, as this structure is so far the only one available for the plant MT family, other sources of information are in high demand. In this review the focus is thus set on any structural features known, deduced, or assumed for the plant MT proteins. This includes the determination of secondary structural elements by circular dichroism, IR, and Raman spectroscopy, the analysis of the influence of the long linker regions, and the evaluation of the spatial arrangement of the sequence separated cysteine-rich regions with the aid of, e.g., limited proteolytic digestion. In addition, special attention is paid to the contents of divalent metal ions as the metal ion to cysteine ratios are important for predicting and understanding possible metal-thiolate cluster structures.

[11] Grennan A K . Metallothioneins, a diverse protein family
Plant Physiol, 2011,155:1750-1751.
DOI:10.1104/pp.111.900407URLPMID:21459979 [本文引用: 2]

[12] Leszczyszyn O I, Imam H T, Blindauer C A . Diversity and distribution of plant metallothioneins: a review of structure, properties and functions
Metallomics, 2013,5:1146-1169.
DOI:10.1039/c3mt00072aURL [本文引用: 1]
More than 30 years have passed since the discovery of the first plant metallothionein in wheat embryos, from which the emergence of a uniquely diverse metallothionein family with a fascinating array of structural nuances and molecular properties has been witnessed. Metallothioneins are not only constitutively expressed, but the production of different types of plant metallothionein is also stimulated by a myriad of endogenous and exogenous agents in both a temporally and spatially regulated manner. This ubiquitous, yet discrete expression of metallothioneins not only signifies their importance for plant survival and development, but also suggests a functional divergence for the individual plant metallothionein subfamilies. Understanding why one type of plant metallothionein has more advantageous structural and metal binding attributes over another for a given biological process is a crucial piece in the puzzle of assigning physiological functions to these proteins. In this review, we discuss how in vivo and in vitro studies have advanced our understanding of the structure-property-function relationship for the plant metallothionein family. In particular, we highlight the progress that has been made for the Type 4 plant metallothioneins.

[13] Schiller M, Hegelund J N, Pedas P, Kichey T, Laursen K H, Husted S, Schjoerring J K . Barley metallothioneins differ in ontogenetic pattern and response to metals
Plant Cell Environ, 2014,37:353-367.
DOI:10.1111/pce.12158URL [本文引用: 4]
The barley genome encodes a family of 10 metallothioneins (MTs) that have not previously been subject to extensive gene expression profiling. We show here that expression of MT1a, MT2b1, MT2b2 and MT3 in barley leaves increased more than 50-fold during the first 10d after germination. Concurrently, the root-specific gene MT1b1 was 1000-fold up-regulated. Immunolocalizations provided the first evidence for accumulation of MT1a and MT2a proteins in planta, with correlation to transcript levels. In developing grains, MT2a and MT4 expression increased 4- and 300-fold over a 28-day-period after pollination. However, among the MT grain transcripts MT2c was the most abundant, whereas MT4 was the least abundant. Excess Cu up-regulated three out of the six MTs expressed in leaves of young barley plants. In contrast, most MTs were down-regulated by excess Zn or Cd. Zn starvation led to up-regulation of MT1a, whereas Cu starvation up-regulated MT2a, which has two copper-responsive elements in the promoter. Arabidopsis lines constitutively overexpressing barley MT2a showed increased sensitivity to excess Cd and Zn but no Cu-induced response. We suggest that barley MTs are differentially involved in intracellular homeostasis of essential metal ions and that a subset of barley MTs is specifically involved in Cu detoxification.

[14] Kumar G, Kushwaha H R, Panjabi-Sabharwal V, Kumari S, Joshi R, Karan R, Mittal S, Pareek S L, Pareek A . Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging
BMC Plant Biol, 2012,12:107.
DOI:10.1186/1471-2229-12-107URLPMID:22780875 [本文引用: 2]
Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented.

[15] Sereno M L, Almeida R S, Nishimura D S, Figueira A . Response of sugarcane to increasing concentrations of copper and cadmium and expression of metallothionein genes
J Plant Physiol, 2007,164:1499-1515.
DOI:10.1016/j.jplph.2006.09.007URLPMID:17175063 [本文引用: 11]
Sugarcane (Saccharum spp.) offers the potential to be a phytoremediator species due to its outstanding biomass production, but its prospective metal accumulation and tolerance have not been fully characterized. Sugarcane plantlets were able to tolerate up to 100microM of copper in nutrient solution for 33 days, with no significant reduction in fresh weight, while accumulating 45mgCukg(-1) shoot dry weight. Higher levels of copper in solution (250 and 500microM) were lethal. Sugarcane displayed tolerance to 500microM Cd without symptoms of toxicity, accumulating 451mgCdkg(-1) shoot dry weight after 33 days, indicating its potential as Cd phytoremediator. DNA gel blot analyses detected 8 fragments using a metallothionein (MT) Type I probe, while 10 were revealed for the MT Type II and 8 for MT Type III. The number of genes for each type of MT in sugarcane might be similar to the ones identified in rice considering the interspecific origin of sugarcane cultivars. MT Type I gene appeared to present the highest level of constitutive expression, mainly in roots, followed by MT Type II, corroborating the expression pattern described based on large-scale expressed sequence tags sequencing. MT Type II and III genes were more expressed in shoots, where MT I was also importantly expressed. Increasing Cu concentration had little or no effect in modulating MT genes expression, while an apparent minor modulation of some of the MT genes could be detected in Cd treatments. However, the level of response was too small to explain the tolerance and/or accumulation of Cd in sugarcane tissues. Thus, cadmium tolerance and accumulation in sugarcane might derive from other mechanisms, although MT may be involved in oxidative responses to high levels of Cd. Sugarcane can be considered a potential candidate to be tested in Cd phytoremediation.

[16] Figueira A, Kido E A, Almeida R S . Identifying sugarcane expressed sequences associated with nutrient transporters and peptide metal chelators
Genet Mol Biol, 2001,24:207-220.
DOI:10.1590/S1415-47572001000100028URL [本文引用: 3]

[17] Guo J, Xu L, Su Y, Wang H, Gao S, Xu J, Que Y . ScMT2-1-3, a metallothionein gene of sugarcane, plays an important role in the regulation of heavy metal tolerance/accumulation
Biomed Res Int, 2013. doi: http://zwxb.chinacrops.org/article/2020/0496-3490/10.1155/2013/904769.
URLPMID:31886774 [本文引用: 6]
[This corrects the article DOI: 10.1155/2018/1095459.].

[18] 陈云, 王竹青, 傅志伟, 杨玉婷, 郭晋隆, 苏亚春, 许莉萍 . 甘蔗金属硫蛋白基因(ScMT2-1-4)的克隆及表达分析
基因组学与应用生物学, 2015,34:357-364.
[本文引用: 3]

Chen Y, Wang Z Q, Fu Z W, Yang Y T, Guo J L, Su Y C, Xu L P . Cloning and expression analysis of metallothionein gene ScMT2-1-4 in Sugarcane
Genom Appl Biol, 2015,34:357-364 (in Chinese with English abstract).
[本文引用: 3]

[19] Verma A K, Agarwal A K, Dubey R S, Solomon S, Singh S B . Sugar partitioning in sprouting lateral bud and shoot development of sugarcane
Plant Physiol Biochem, 2013,62:111-115.
DOI:10.1016/j.plaphy.2012.10.021URLPMID:23208305 [本文引用: 1]
Sugarcane is a leading energy crop of the world due to its ability to capitulate high sucrose. To understand the mechanism associated with shoot establishment from the lateral bud of sugarcane setts, at the time of germination, we established shoots and these shoots were incubated in total darkness and dark/light regime. The concentration of sugars (sucrose and hexoses) and activities of sugar metabolizing enzymes were measured from 0 to 21 days with 7 days intervals during shoot establishment using sugarcane cultivar CoS 97264. A decrease in sucrose concentration and increase in hexoses level was observed in intermodal tissues whereas in the shoots, the level of both sucrose and hexoses increased continuously during shoot establishment. During 0-21 days shooting period, the dry mass of internodes declined by 20 and 25% in plants incubated in dark/light and darkness respectively. All invertases, soluble acid invertase, neutral invertase and cell wall bound invertase were expressed with almost similar pattern in both the intermodal tissues and the shoots. The activity of enzyme sucrose synthase, measured within first 10 days of shooting in both types of tissues, appeared to be higher particularly in sugar breakdown direction. In the shoots, slight increase in sucrose synthase activity in sucrose synthesis direction was observed throughout shooting period in the shoots. The results suggest that sucrose is the main substrate used during shoot establishment and that shoot establishment period is characterized by increased activities of invertases and sucrose synthase and increased level of hexoses in the shoots.

[20] D’hont A, Grivet L, Feldmann P, Rao S, Berding N, Glaszmann J C . Characterisation of the double genome structure of modern sugarcane cultivars (Saccharum spp.) by molecular cytogenetics
Mol Gen Genet, 1996,250:405-413.
DOI:10.1007/bf02174028URLPMID:8602157 [本文引用: 1]
Cultivated sugarcane clones (Saccharum spp., 2n=100 to 130) are derived from complex interspecific hybridizations between the species S. officinarum and S. spontaneum. Using comparative genomic DNA in situ hybridization, we demonstrated that it is possible to distinguish the chromosomes contributed by these two species in an interspecific F1 hybrid and a cultivated clone, R570. In the interspecific F1 studied, we observed n + n transmission of the parental chromosomes instead of the peculiar 2n + n transmission usually described in such crosses. Among the chromosomes of cultivar R570 (2n = 107-115) about 10% were identified as originating from S. spontaneum and about 10% were identified as recombinant chromosomes between the two species S. officinarum and S. spontaneum. This demonstrated for the first time the occurrence of recombination between the chromosomes of these two species. The rDNA sites were located by in situ hybridization in these two species and the cultivar R570. This supported different basic chromosome numbers and chromosome structural differences between the two species and provided a first bridge between physical and genetical mapping in sugarcane.

[21] Edmé S J, Comstock J C, Miller J D, Tai P Y P . Determination of DNA content and genome size in sugarcane
J Am Soc Sugar Cane Technol, 2005,25:1-16.
DOI:10.1038/s41467-018-05051-5URLPMID:29980662 [本文引用: 1]
Sugarcane (Saccharum spp.) is a major crop for sugar and bioenergy production. Its highly polyploid, aneuploid, heterozygous, and interspecific genome poses major challenges for producing a reference sequence. We exploited colinearity with sorghum to produce a BAC-based monoploid genome sequence of sugarcane. A minimum tiling path of 4660 sugarcane BAC that best covers the gene-rich part of the sorghum genome was selected based on whole-genome profiling, sequenced, and assembled in a 382-Mb single tiling path of a high-quality sequence. A total of 25,316 protein-coding gene models are predicted, 17% of which display no colinearity with their sorghum orthologs. We show that the two species, S. officinarum and S. spontaneum, involved in modern cultivars differ by their transposable elements and by a few large chromosomal rearrangements, explaining their distinct genome size and distinct basic chromosome numbers while also suggesting that polyploidization arose in both lineages after their divergence.

[22] Zhang J, Nagai C, Yu Q, Pan Y B, Ayala-Silva T, Schnell R J, Comstock J C, Arumuganathan A K, Ming R . Genome size variation in threeSaccharum species
Euphytica, 2012,185:511-519.
DOI:10.1007/s10681-012-0664-6URL [本文引用: 1]
species are autopolyploids with ploidy level ranging from 5 to 16, and are considered the most complex genomes among crop plants. In present study, the genome sizes of 28 accessions, 15 accessions, 28 accessions, and 12 hybrids spp. were analyzed using flow cytometry. The estimated genome sizes of accessions ranged from 7.50 to 8.55 Gb with an average size of 7.88 Gb. In , the estimated genome sizes ranged from 7.65 to 11.78, reflecting the variation of ploidy level. In , the estimated genome sizes varied widely, with a range from 3.36 to 12.64 Gb, also due to variation of ploidy level. The average monoploid genome size of was 985 Mb, and that of was 843 Mb. The results also showed that genome sizes were correlated with chromosome numbers, and based which, that the unknown chromosome numbers of some accessions could be predicted. The estimated genome sizes of germplasm also helped identify some mislabeled accessions and yielded information critical for sugarcane breeding and genome sequencing programs.

[23] Iskandar H M, Simpson R S, Casu R E, Bonnett G D, Maclean D J, Manners J M . Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane
Plant Mol Biol Rep, 2004,22:325-337.
DOI:10.1007/BF02772676URL [本文引用: 1]
A protocol for reverse transcription followed by real-time quantitative PCR (RT-qPCR) analysis of tissue-specific and genotype-variable gene expression in sugarcane (Saccharum sp.) was developed. A key requirement for this analysis was the identification of a housekeeping gene with transcript levels that were relatively stable across tissues and genotypes, suitable for use as a reference. Primers for β-actin, β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes and 25S ribosomal RNA were designed and tested by RT-qPCR, and formation of product in the reactions was measured with the SYBR green I dye system. Ribosomal RNA was the most sensitive and consistent as a reference gene. Determination of the expression levels of β-actin, β-tubulin, and GAPDH transcripts relative to that of 25S rRNA showed that GAPDH had the most consistent mRNA expression of protein-coding genes across different tissues. GAPDH also showed low variation in expression in maturing stem internodes when compared across 2 cultivars and 3 otherSaccharum species. GAPDH therefore appears to be a suitable “housekeeping gene” in addition to 25S rRNA as a reference for measuring the relative expression of other genes in sugarcane. With use of GAPDH as a reference, the relative expression of the sugarcane sugar transporter genePst2a was assessed in a range of tissues. The result obtained was similar to our previously published Northern blot analysis. The protocol described here, using GAPDH as a reference gene, is recommended for studying the expression of other genes of interest in diverse tissues and genotypes of sugarcane.

[24] Livak, K J, Schmittgen T D . Analysis of relative gene expression data using real-time quantitative PCR and the 2 ^-ΔΔCT method
Methods, 2001,25:402-408.
DOI:10.1006/meth.2001.1262URLPMID:11846609 [本文引用: 1]
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.

[25] 夏会龙, 程文伟, 池小雅 . 镉胁迫对甘蔗生长及生理性状的影响
中国土壤与肥料, 2009, ( 1):42-45.
URL [本文引用: 1]
通过盆栽模拟试验研究了甘蔗长期受高浓度重金属镉(Cd)胁迫的生理响应.结果表明,甘蔗生物量、茎径和茎节长随着土壤中Cd浓度的增加而降低,Cd浓度越高,抑制效应越明显;叶片中叶绿素a、叶绿素b、叶绿素a/b值、过氧化氢酶(CAT)活力及蔗茎中可溶性糖含量与土壤中Cd浓度呈一阶指数衰减关系,相关系数分别为0.998、0.999、0.971、0.997和0.996.叶片细胞质膜透性与土壤中Cd浓度呈S函数关系,相关系数为0.999;甘蔗能在Cd浓度为1000 mg/kg的土壤中存活.可见,甘蔗对土壤中Cd胁迫有较好的耐性.
Xia H L, Cheng W E, Chi X Y . Effects of Cd stress on the growth and physiological properties of sugarcane
Soil Fert Sci China, 2009, ( 1):42-45 (in Chinese with English abstract).
URL [本文引用: 1]
通过盆栽模拟试验研究了甘蔗长期受高浓度重金属镉(Cd)胁迫的生理响应.结果表明,甘蔗生物量、茎径和茎节长随着土壤中Cd浓度的增加而降低,Cd浓度越高,抑制效应越明显;叶片中叶绿素a、叶绿素b、叶绿素a/b值、过氧化氢酶(CAT)活力及蔗茎中可溶性糖含量与土壤中Cd浓度呈一阶指数衰减关系,相关系数分别为0.998、0.999、0.971、0.997和0.996.叶片细胞质膜透性与土壤中Cd浓度呈S函数关系,相关系数为0.999;甘蔗能在Cd浓度为1000 mg/kg的土壤中存活.可见,甘蔗对土壤中Cd胁迫有较好的耐性.

[26] 魏树和, 周启星, 王新 . 超积累植物龙葵及其对镉的富集特征
环境科学, 2005,26(3):167-171.
[本文引用: 2]

Wei S F, Zhou Q X, Wang X . Cadmium-hyperaccumulator Solanum nigrum L. and its accumulating characteristics
Environ Sci, 2005,26(3):167-171 (in Chinese with English abstract).
[本文引用: 2]

[27] 姚诗音, 刘杰, 王怡璇, 朱园芳, 丰顺 . 青葙对镉的超富集特征及累积动态研究
农业环境科学学报, 2017,36:1470-1476.
[本文引用: 2]

Yao S Y, Liu J, Wang Y X, Zhu Y F, Feng S . Cd hyperaccumulation and accumulative kinetics ofCelosia argentea Linn. for phytoremediation of Cd-contaminated soil
J Agro-Environt Sci, 2017,36:1470-1476 (in Chinese with English abstract).
[本文引用: 2]

[28] Guo W J, Meetam M, Goldsbrough P B . Examining the specific contributions of individualArabidopsis metallothioneins to copper distribution and metal tolerance
Plant Physiol, 2008,146:1697-1706.
DOI:10.1104/pp.108.115782URLPMID:18287486 [本文引用: 3]
Metallothioneins (MTs) are small cysteine-rich proteins found in various eukaryotes. Plant MTs are classified into four types based on the arrangement of cysteine residues. To determine whether all four types of plant MTs function as metal chelators, six Arabidopsis (Arabidopsis thaliana) MTs (MT1a, MT2a, MT2b, MT3, MT4a, and MT4b) were expressed in the copper (Cu)- and zinc (Zn)-sensitive yeast mutants, Deltacup1 and Deltazrc1 Deltacot1, respectively. All four types of Arabidopsis MTs provided similar levels of Cu tolerance and accumulation to the Deltacup1 mutant. The type-4 MTs (MT4a and MT4b) conferred greater Zn tolerance and higher accumulation of Zn than other MTs to the Deltazrc1 Deltacot1 mutant. To examine the functions of MTs in plants, we studied Arabidopsis plants that lack MT1a and MT2b, two MTs that are expressed in phloem. The lack of MT1a, but not MT2b, led to a 30% decrease in Cu accumulation in roots of plants exposed to 30 mum CuSO(4). Ectopic expression of MT1a RNA in the mt1a-2 mt2b-1 mutant restored Cu accumulation in roots. The mt1a-2 mt2b-1 mutant had normal metal tolerance. However, when MT deficiency was combined with phytochelatin deficiency, growth of the mt1a-2 mt2b-1 cad1-3 triple mutant was more sensitive to Cu and cadmium compared to the cad1-3 mutant. Together these results provide direct evidence for functional contributions of MTs to plant metal homeostasis. MT1a, in particular, plays a role in Cu homeostasis in the roots under elevated Cu. Moreover, MTs and phytochelatins function cooperatively to protect plants from Cu and cadmium toxicity.

[29] Guo W J Bundithya W, Goldsbrough P B . Characterization of theArabidopsis metallothionein gene family: tissue-specific expression and induction during senescence and in response to copper
New Phytol, 2003,159:369-381.
DOI:10.1046/j.1469-8137.2003.00813.xURL [本文引用: 3]

[30] Zimeri A M, Dhankher O P, McCaig B, Meagher R B . The plant MT1 metallothioneins are stabilized by binding cadmiums and are required for cadmium tolerance and accumulation
Plant Mol Biol, 2005,58:839-855.
DOI:10.1007/s11103-005-8268-3URL [本文引用: 1]
The small Arabidopsis genome contains nine metallothionein-like (MT) sequences with classic, cysteine-rich domains separated by spacer sequences, quite unlike the small conserved MT families found vertebrate genomes. Phylogenetic analysis revealed four ancient and divergent classes of plant MTs that predate the monocot–dicot divergence. A distinct cysteine spacing pattern suggested differential metal ion specificity for each class. The in vivo stability of representatives of the four classes of plant MT proteins and a mouse MT2 control expressed in E. coli were enhanced by cadmium (Cd). Particular MTs were also stabilized by arsenic (As), copper (Cu), and or zinc (Zn). To understand why plants have such a diversity of MT sequences, the Arabidopsis MT1 class, comprised of three genes, MT1a, MT1b, and MT1c, was characterized in more detail in plants. MT1 family transcripts were knocked down to less than 5–10% of wild-type levels in Arabidopsis by expression of a RNA interference (RNAi) construct. The MT1 knockdown plant lines were all hypersensitive to Cd and accumulated several fold lower levels of As, Cd, and Zn than wildtype, while Cu and Fe levels were unaffected. The ancient class of MT1 protein sequences may be preserved in plant genomes, because it has distinct metal-binding properties, confers tolerance to cadmium, and can assist with zinc homeostasis.

[31] Yang Z, Wu Y R, Li Y, Ling H Q, Chu C C . OsMT1a, a type 1 metallothionein, plays the pivotal role in zinc homeostasis and drought tolerance in rice
Plant Mol Biol, 2009,70:219-229.
DOI:10.1007/s11103-009-9466-1URL [本文引用: 3]
Metallothioneins (MTs) are small, cysteine-rich, metal-binding proteins that may be involved in metal homeostasis and detoxification in both plants and animals. OsMT1a, encoding a type 1 metallothionein, was isolated via suppression subtractive hybridization from Brazilian upland rice (Oryza sativa L. cv. Iapar 9). Expression analysis revealed that OsMT1a predominantly expressed in the roots, and was induced by dehydration. Interestingly, the OsMT1a expression was also induced specifically by Zn²⁺ treatment. Both transgenic plants and yeasts harboring OsMT1a accumulated more Zn²⁺ than wild type controls, suggesting OsMT1a is most likely to be involved in zinc homeostasis. Transgenic rice plants overexpressing OsMT1a demonstrated enhanced tolerance to drought. The examination of antioxidant enzyme activities demonstrated that catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) were significantly elevated in transgenic plants. Furthermore, the transcripts of several Zn²⁺-induced CCCH zinc finger transcription factors accumulated in OsMT1a transgenic plants, suggesting that OsMT1a not only participates directly in ROS scavenging pathway but also regulates expression of the zinc finger transcription factors via the alteration of Zn²⁺ homeostasis, which leads to improved plant stress tolerance.

[32] 姜廷波, 陈虹, 唐鑫华, 丁宝建, 王玉成, 李凤娟, 李绍臣 . 转金属硫蛋白基因(MT1)烟草抗Cd ²+胁迫的生理特性分析
作物学报, 2007,33:1902-1905.
URL [本文引用: 1]
将柽柳（Tamarix sp.）金属硫蛋白基因（MT₁，GenBank登录号：AB298390）插入植物表达载体pBI121，取代花椰菜病毒35S启动子下游的gus基因，利用农杆菌（Agrobactrium tumefaciens）介导法将MT1导入烟草（Nicotiana tobacum）基因组。对具有卡那霉素抗性，且经PCR-Southern检测和Northern杂交表现阳性的转基因株系进行抗Cd²⁺分析表明，金属硫蛋白基因的过量表达提高了转基因植株的抗Cd²⁺能力，在含200 µmol L^-1和400 µmol L^-1 Cd²⁺的MS培养基上，转基因植株的株高和鲜重均明显优于非转基因株系；在生理性状上表现为转基因植株MDA含量及POD活性明显低于非转基因株系，叶绿素含量和SOD活性比非转基因株系明显增加。

Jiang T B, Chen H, Tang X H, Ding B J, Wang Y C, Li F J, Li S C . Analysis of physiologic characteristics for Cd ²+ tolerance on transgenic tobacco expressing metallothionein gene ( MT1)
Acta Agron Sin, 2007,33:1902-1905 (in Chinese with English abstract).
URL [本文引用: 1]
将柽柳（Tamarix sp.）金属硫蛋白基因（MT₁，GenBank登录号：AB298390）插入植物表达载体pBI121，取代花椰菜病毒35S启动子下游的gus基因，利用农杆菌（Agrobactrium tumefaciens）介导法将MT1导入烟草（Nicotiana tobacum）基因组。对具有卡那霉素抗性，且经PCR-Southern检测和Northern杂交表现阳性的转基因株系进行抗Cd²⁺分析表明，金属硫蛋白基因的过量表达提高了转基因植株的抗Cd²⁺能力，在含200 µmol L^-1和400 µmol L^-1 Cd²⁺的MS培养基上，转基因植株的株高和鲜重均明显优于非转基因株系；在生理性状上表现为转基因植株MDA含量及POD活性明显低于非转基因株系，叶绿素含量和SOD活性比非转基因株系明显增加。

[33] Grispen V M J, Hakvoort H W J, Bliek T, Verkleij J A C, Schat H . Combined expression of theArabidopsis metallothionein MT2b and the heavy metal transporting ATPase HMA4 enhances cadmium tolerance and the root to shoot translocation of cadmium and zinc in tobacco
Environ Exp Bot, 2011,72:71-76.
DOI:10.1016/j.envexpbot.2010.01.005URL [本文引用: 1]
We expressed the AtMt2b and AtHMA4 genes under the 35S cauliflower mosaic virus promoter simultaneously in Nicotiana tabacum (SR1), using leaf disc transformation. A single AtMT2b tobacco T2 line was used for re-transformation with AtHMA4 to obtain the double transformant. Cadmium (Cd) and zinc (Zn) tolerance, uptake and translocation were measured in the double transformant, and compared to untransformed ('wild type') tobacco and single gene transformants. The double transformant exhibited enhanced Cd-tolerance, enhanced Cd and Zn root to shoot transport, but unaltered Zn tolerance and Cd and Zn uptake, compared with wild type.
The single transformant lines did not show significant phenotypes. Our results suggest that the phenotypes of the double transformant are due to synergistic interaction between the transgenes. Except for Cd tolerance, the phenotypes were moderate for Cd and Zn root to shoot transport, which may be due to use of the 35S promotor, resulting in incorrect tissue-specificity. (C) 2010 Elsevier B.V.

[34] 班巧英, 刘桂丰, 王玉成, 张大伟, 蒋丽丽 . 一个新的二色补血草金属硫蛋白基因LbMT2的克隆及其表达分析
遗传, 2008,30:1075-1082.
DOI:10.3724/SP.J.1005.2008.01075URL [本文引用: 1]
从二色补血草cDNA文库中分离出一个新的金属硫蛋白基因LbMT2全长cDNA序列。该基因全长518 bp, 其中5′非翻译区(UTR)64 bp, 3′非翻译区205 bp, 开放阅读框(ORF)249 bp, 编码由82个氨基酸组成的蛋白质, 编码蛋白的分子量为8.1 kDa, 理论等电点(pI)为4.72。利用实时定量PCR方法研究了二色补血草在CuSO4、CdCl2、NaCl、低温和PEG胁迫下不同时间该基因的表达模式。结果表明, CuSO4、CdCl2、NaCl和低温处理均能诱导LbMT2基因在二色补血草的根和叶中的表达, 而PEG处理则抑制了LbMT2在根和叶中的表达。构建LbMT2基因的原核表达载体pGEX-LbMT2, 通过IPTG诱导在大肠杆菌(Escherichia coli) BL21中融合表达, SDS-PAGE 电泳获得35 kDa 的蛋白条带, 大小与预期相符。
Ban Q Y, Liu G F, Wang Y C, Zhang D W, Jiang L L . Cloning and expression of a novel metallothionein gene LbMT2 from Limonium bicolor
Hereditas(Beijing), 2008,30:1075-1082 (in Chinese with English abstract).
DOI:10.3724/SP.J.1005.2008.01075URL [本文引用: 1]
从二色补血草cDNA文库中分离出一个新的金属硫蛋白基因LbMT2全长cDNA序列。该基因全长518 bp, 其中5′非翻译区(UTR)64 bp, 3′非翻译区205 bp, 开放阅读框(ORF)249 bp, 编码由82个氨基酸组成的蛋白质, 编码蛋白的分子量为8.1 kDa, 理论等电点(pI)为4.72。利用实时定量PCR方法研究了二色补血草在CuSO4、CdCl2、NaCl、低温和PEG胁迫下不同时间该基因的表达模式。结果表明, CuSO4、CdCl2、NaCl和低温处理均能诱导LbMT2基因在二色补血草的根和叶中的表达, 而PEG处理则抑制了LbMT2在根和叶中的表达。构建LbMT2基因的原核表达载体pGEX-LbMT2, 通过IPTG诱导在大肠杆菌(Escherichia coli) BL21中融合表达, SDS-PAGE 电泳获得35 kDa 的蛋白条带, 大小与预期相符。

[35] Zhang J, Zhang M, Tian S, Lu L, Shohag M J I, Yang X . Metallothionein 2 (SaMT2) fromSedum alfrediihance confers increased Cd tolerance and acumulation in yeast and tobacco
PLoS One, 2014,9:e102750.
DOI:10.1371/journal.pone.0102750URLPMID:25032704 [本文引用: 1]
Metallothioneins are cysteine-rich metal-binding proteins. In the present study, SaMT2, a type 2 metallothionein gene, was isolated from Cd/Zn co-hyperaccumulator Sedum alfredii Hance. SaMT2 encodes a putative peptide of 79 amino acid residues including two cysteine-rich domains. The transcript level of SaMT2 was higher in shoots than in roots of S. alfredii, and was significantly induced by Cd and Zn treatments. Yeast expression assay showed SaMT2 significantly enhanced Cd tolerance and accumulation in yeast. Ectopic expression of SaMT2 in tobacco enhanced Cd and Zn tolerance and accumulation in both shoots and roots of the transgenic plants. The transgenic plants had higher antioxidant enzyme activities and accumulated less H2O2 than wild-type plants under Cd and Zn treatment. Thus, SaMT2 could significantly enhance Cd and Zn tolerance and accumulation in transgenic tobacco plants by chelating metals and improving antioxidant system.

[36] Roosens N H, Bernard C, Leplae R, Verbruggen N . Evidence for copper homeostasis function of metallothionein (MT3) in the hyperaccumulator Thlaspi caerulescens
FEBS Lett, 2004,577:9-16.
DOI:10.1016/j.febslet.2004.08.084URLPMID:15527754 [本文引用: 1]
Metallothioneins chelate metals and consequently may be a control point of metal homeostasis. Homologous to type 3 metallothioneins, TcMT3 cDNA was identified in the Cd/Zn hyperaccumulator, Thlaspi caerulescens. TcMT3 amino acid sequence showed modifications in the Cys positions when compared with its Arabidopsis orthologue. A structural model established that the MT3 carboxyterminal domain is similar to the beta domain of animal metallothioneins and predicts a smaller cavity to chelate metals for A. thaliana than for T. caerulescens. Functional testing in yeast and Northern blot analysis added further evidence for adaptative variations of MT3 for the maintenance of Cu homeostasis in a metal hyperaccumulator.

[37] Zhu G H, Zhang J J, Chen J S, Peng X X . Characterization of a rice metallothionein type 3 gene with different expression profiles under various nitrogen forms
Biol Planta, 2008,52:668-673.
DOI:10.1007/s10535-008-0129-xURL [本文引用: 1]

[38] Hegelund J N, Schiller M, Kichey T, Hansen T H, Pedas P, Husted S, Schjoerring J K . Barley metallothioneins: MT3 and MT4 are localized in the grain aleurone layer and show differential Zinc binding
Plant Physiol, 2012,159:1125-1137.
DOI:10.1104/pp.112.197798URL [本文引用: 1]
Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins believed to play a role in cytosolic zinc (Zn) and copper (Cu) homeostasis. However, evidence for the functional properties of MTs has been hampered by methodological problems in the isolation and characterization of the proteins. Here, we document that barley (Hordeum vulgare) MT3 and MT4 proteins exist in planta and that they differ in tissue localization as well as in metal coordination chemistry. Combined transcriptional and histological analyses showed temporal and spatial correlations between transcript levels and protein abundance during grain development. MT3 was present in tissues of both maternal and filial origin throughout grain filling. In contrast, MT4 was confined to the embryo and aleurone layer, where it appeared during tissue specialization and remained until maturity. Using state-of-the-art speciation analysis by size-exclusion chromatography inductively coupled plasma mass spectrometry and electrospray ionization time-of-flight mass spectrometry on recombinant MT3 and MT4, their specificity and capacity for metal ion binding were quantified, showing a strong preferential Zn binding relative to Cu and cadmium (Cd) in MT4, which was not the case for MT3. When complementary DNAs from barley MTs were expressed in Cu- or Cd-sensitive yeast mutants, MT3 provided a much stronger complementation than did MT4. We conclude that MT3 may play a housekeeping role in metal homeostasis, while MT4 may function in Zn storage in developing and mature grains. The localization of MT4 and its discrimination against Cd make it an ideal candidate for future biofortification strategies directed toward increasing food and feed Zn concentrations.