CN101817879A - 金属硫蛋白及其编码基因与应用 - Google Patents
金属硫蛋白及其编码基因与应用 Download PDFInfo
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- CN101817879A CN101817879A CN200910078200A CN200910078200A CN101817879A CN 101817879 A CN101817879 A CN 101817879A CN 200910078200 A CN200910078200 A CN 200910078200A CN 200910078200 A CN200910078200 A CN 200910078200A CN 101817879 A CN101817879 A CN 101817879A
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Abstract
本发明公开了一种金属硫蛋白及其编码基因与应用。该蛋白质是序列表中序列2所示的氨基酸序列组成的蛋白质；该蛋白可用于培育抗旱能力提高和/或锌含量提高的植物。本发明还公开了一种培育抗旱能力提高和/或锌含量提高的植物的方法，是将所述基因导入植物中，得到抗旱能力提高和/或锌含量提高的转基因植物。本发明的金属硫蛋白及其编码基因对农业生产具有重要的意义。
Description
技术领域
本发明涉及金属硫蛋白及其编码基因与应用。
背景技术
金属硫蛋白(metallothioneins，MT)是一类低分子量(6000～7000Da)、高Cys含量，具有金属结合能力的多肽，广泛存在于生物界。它具有独特的氨基酸排列顺序，即多肽的N端和C端具有两个富含Cys的金属结合结构域。Cyc残基与许多金属离子以硫醇键形式结合形成金属硫的四面体单位。由于MT特有的氨基酸组成与分子结构，它在生物体内担负着重要的生理功能。目前对植物MT功能的研究还处于起步状态，尚无十分明确的结论。一般认为MT在金属离子的储存和运输、重金属的解毒、维持金属离子浓度稳态等方面起重要作用。另外，MT还与植物的生长发育、胚胎发生、果实成熟、衰老等过程有关。越来越多的研究表明MT是植物体内重要的氧自由基清除剂之一，能提高植物的抗氧化能力。
根据氨基酸尤其是Cys的排列方式，可以把植物中的MT分为四类。I型在根中表达量远远高于地上部分，II型则刚好相反，集中在叶中表达。III型主要在成熟果实中表达。而IV型迄今为止只在种子中发现，比如小麦胚芽和芝麻籽。水稻的MT家族包含11个成员，分别属于四类。除了被各种金属离子诱导外，其他一些刺激比如蔗糖饥饿、双氧水和盐胁迫也能提高水稻MT合成增加。初步研究表明，水稻MT参与了重金属代谢、种子发育和抗氧化过程。
水稻是一种重要的粮食作物，是世界上约1/3人口的主要粮食来源。现今世界范围内水稻的发展受到水资源匮乏以及地区性、季节性干旱的严重限制，增产潜力受到明显制约。中国作为发展中国家，飞速发展的工业化和城市化进程加剧了水资源危机。另外，由于农药的广泛使用，大量的汞、镉、铅等重金属造成土壤污染日趋严重。旱灾及各种污染影响了水稻的质量和产量。因此，对水稻抗旱、抗金属毒害等生理进行研究，以期提高其抗旱等抗逆能力，显得十分必要。
作物抗旱等非生物逆境研究是植物研究领域最具挑战性的工作之一。选择合适的研究材料是关键的第一步。陆稻原产于巴西，又称巴西旱稻。一般认为，旱稻是由水稻演变而来的适于旱地栽培的“土地生态型”，因而陆稻的抗旱性比水稻强。陆稻与水稻的亲缘性很近。比起远缘的双子叶植物比如拟南芥等，从陆稻研究中得到的功能基因应用于水稻抗旱实践更具效果。
发明内容
本发明的目的是提供一种金属硫蛋白及其编码基因与应用。
本发明所提供的金属硫蛋白，命名为OsMT1a，来源于巴西旱稻，是如下a)或b)的蛋白：
a)序列表中序列2所示的氨基酸序列组成的蛋白质；
b)在序列表中序列2的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸由a)衍生的蛋白质。
为了使a)的OsMT1a蛋白质便于纯化，可在由序列表中序列2所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1.标签的序列
| 标签 | 残基 | 序列 |
| Poly-Arg | 5-6(通常为5个) | RRRRR |
| Poly-His | 2-10(通常为6个) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
上述b)中的OsMT1a蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。上述b)中的OsMT1a蛋白质的编码基因可通过将序列表中序列1所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
所述蛋白的编码基因也属于本发明的保护范围。
所述蛋白的编码基因是如下1)或2)或3)：
1)其核苷酸序列是序列表中序列1；
2)在严格条件下与1)限定的DNA片段杂交且编码金属硫蛋白的DNA分子；
3)与1)或2)的基因具有90％以上的同源性，且编码金属硫蛋白的DNA分子。
所述步骤3)中的基因，与1)的基因最好有95％以上的同源性。
上述严格条件可为在6×SSC，0.5％ SDS的溶液中，在68℃下杂交，然后用2×SSC，0.1％ SDS和1×SSC，0.1％ SDS各洗膜一次。
扩增OsMT1a基因全长或任一片段的引物对也属于本发明的保护范围。
含有上述OsMT1a基因的重组载体、转基因细胞系和重组菌也属于本发明的保护范围。
可用现有的植物表达载体构建含有OsMT1a基因的重组表达载体。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等，如pCAMBIA3301、pCAMBIA1300、pBI121、pBin19、pCAMBIA2301、pCAMBIA1301-UbiN或其它衍生植物表达载体。携带有本发明的OsMT1a基因的植物表达载体可通过Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、显微注射、电导、农杆菌介导等常规生物学方法转化到植物细胞或组织中。
使用OsMT1a基因构建重组植物表达载体时，在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子，如花椰菜花叶病毒(CaMV)35S启动子、泛生素基因Ubiquitin启动子(pUbi)等，它们可单独使用或与其它的植物启动子结合使用；此外，使用本发明的OsMT1a基因构建植物表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。
为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用植物表达载体进行加工，如加入可在植物中表达可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、具有抗性的抗生素标记物(庆大霉素标记物、卡那霉素标记物等)或是抗化学试剂标记基因(如抗除莠剂基因)等。
本发明的另一个目的是提供一种培育抗旱能力提高和/或锌含量提高的植物的方法。
本发明所提供的培育抗旱能力提高和/或锌含量提高的植物的方法，是将所述的OsMT1a基因导入植物中，得到抗旱能力提高和/或锌含量提高的转基因植物。
所述植物可以为各种单子叶或双子叶植物，如棉花、小麦或者水稻等。
本发明通过SSH(抑制消减杂交)的方法从盐胁迫的巴西旱稻中克隆得到一个I型的金属硫蛋白OsMT1a。转OsMT1a基因酵母和水稻中的微量元素锌的累积提高，并且转OsMT1a基因水稻的抗氧化及耐逆性能提高。本发明的金属硫蛋白OsMT1a可用来培育抗旱能力提高和/或锌含量提高的植物，对农业生产具有重要的意义。
附图说明
图1为Northern杂交分析日本晴中OsMT1a的表达。
图2为金属处理的巴西旱稻中OsMT1a的表达。
图3为非生物胁迫处理的巴西旱稻中OsMT1a的表达。
图4为300mM甘露醇处理的野生型日本晴(WT)和转OsMT1a基因水稻表型变化及失水率测定。
图5为CAT、APX和POD活性测定结果。
图6为转OsMT1a基因株系中Ossiz的表达。
具体实施方式
下述实施例中如无特殊说明所用方法均为常规方法，所用试剂均可从商业途径获得。下述实施例中如无特殊说明所述百分含量均为质量百分含量。
实施例1、金属硫蛋白(OsMT1a)基因的应用
1)OsMT1a基因的克隆
以盐处理和未处理的巴西旱稻为材料，利用消减抑制杂交的方法，直接从旱稻中分离了94个盐诱导基因。其中一个基因的cDNA全长222bp，编码74个氨基酸，在N端和C端分别有六个半胱氨酸位于保守的位置，还有大约40氨基酸的spacer区，其中包含芳香族氨基酸酪氨酸和苯丙氨酸，这是I型植物MT蛋白的典型特征，将其命名为OsMT1a。OsMT1a蛋白与其他单子叶植物I型MT蛋白都有较高的相似性，比如玉米(73.7％)，大麦(67.6％)。
分别取日本晴植株根、叶、花以及幼苗部分，按文献(Chomczynski，P.and Sacchi，N.(1987)Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem，162，156-159.)方法提取RNA，以OsMT1a全长cDNA为探针进行Northern杂交分析。
Northern杂交结果如图1所示，表明OsMT1a在水稻的根、花、叶、幼苗均有表达，其中在根中表达量最高。
图1中，“1”代表花、“2”代表叶、“3”代表幼苗、“4”代表根。
巴西旱稻种子在37℃浸种两天，露白后铺在放有两层纱布的平皿上培养，培养条件为28℃，持续光照强度为2500lux，光周期为16/8h。培养两周后取幼苗进行处理。
金属处理：分别用10μM ZnCl2、CuCl2、MnCl2和FeCl3溶液处理24小时。
非生物胁迫处理：分别用150mM NaCl、7％ PEG6000和10μM ABA溶液处理24小时。
检测金属处理1、6、12和24小时和非生物胁迫处理0、0.5、6和24小时OsMT1a的表达。
提取各个处理不同时间的巴西旱稻的总RNA，用Promega公司的MMLV逆转录酶反转录后利用实时定量荧光PCR技术，根据厂家(Bio-Rad)提供的使用方法，在PCR体系中加入SYBR green I荧光染料，在荧光定量PCR仪(Bio-Rad)上检测OsMT1a的表达模式。用水稻ACTIN1基因表达水平做内参。扩增OsMT1a的引物分别为：上游引物5’-GAAGATGTCTTGCAGCTGTGGAT-3’，下游引物5’-AGATGGTAGATGCAGGCAGGC-3’。
同时，以OsMT1a全长cDNA为探针进行Northern杂交分析。
金属处理的实时定量荧光PCR结果如图2所示，表明10μM的Zn能诱导OsMT1a的表达上调，同样浓度的Mn、Fe、Cu不能诱导OsMT1a的表达上调。
图2中，A为10μM Mn2+、Fe3+、Cu2+处理0～24小时OsMT1a表达变化；B为10μM Zn2+处理0～24小时OsMT1a表达变化。
非生物胁迫处理的实时定量荧光PCR结果如图3所示，OsMT1a受到渗透胁迫、盐胁迫以及ABA的诱导表达，其中渗透胁迫的诱导最为明显。
设计正反向引物，利用PCR方法从水稻基因组DNA中扩增出OsMT1a基因，并在正反向引物末端分别添加限制性酶EcoRI和BamHI的酶切位点。
正向引物为5’-GAATTCGAAGATGTCTTGCAGCTGTG-3；
反向引物为5’-GGATCCGCAGGCAGGCATCTTA-3’。
PCR产物回收后连接入pMD18-T(TAKARA，大连)中进行测序，测序结果表明，PCR产物的核苷酸序列如序列表中序列1所示，编码序列表中序列2所示的蛋白。
2)培育抗旱和/或锌含量提高的水稻
用EcoRI与BamHI双酶切PCR产物，与同样双酶切的双元表达载体pCAMBIA2300-pUbiquitin-OCS(购自Cambia，澳大利亚，https://www.cambia.org/daisy/cambia/home.html)连接，得重组表达载体pCAMBIA2300-pUbiquitin-OsMT1a-OCS。
将载体pCAMBIA2300-pUbiquitin-OsMT1a-OCS通过农杆菌AGL1(购自ATCC(American Type Culture Collection))导入到水稻日本晴愈伤组织中。转化筛选方法参考如下文献：易自力，曹守云，王力，何锶洁，储成才，唐祚舜，周朴华，田文忠(2001)提高农杆菌转化水稻频率的研究，遗传学报，28(4)：352-358)。获得抗性筛选阳性转OsMT1a基因水稻T0代株系共10株。
分别取营养生长同一时期的野生型日本晴(WT)6株和转OsMT1a基因植株(L6和L7各6株)叶片及收割后去掉谷壳的种子进行金属含量测定，实验重复3次，对测定结果进行统计学分析。
测定方法如下：
材料洗净后，经过烘箱80℃彻底干燥24小时后，磨碎，进行微波消解。每200mg加9ml HNO3、2ml H2O2在180℃消解20min，冷却后开盖，转移至25mL容量瓶中加超纯水定容，摇匀。利用Inductively Coupled Plasma-Optical Emission Spectroscopy(ICP-OES)电感耦合等离子发生光谱(Optima-2000DV，Perkin Elemer，USA)进行相关重金属含量的测定。叶片及种子中金属含量的测定结果如表1所示。
表1.叶片及种子中金属的含量
表1中金属含量的单位为(μg/g干重)，**代表通过t检验得出转基因株系和野生型有显著性差异的置信概率为99％。
表1表明，两个转OsMT1a基因株系L6和L7中，Mn和Zn的积累都有所提高，而Zn的提高是显著性的，比野生型分别提高了45.38％和54.15％。在种子中也有相同的趋势，Zn的积累最为明显，L6和L7分别比野生型提高44.43％和53.93％。因此，OsMT1a基因可用于提高作物中的微量元素锌的累积。
将生长两周的野生型日本晴(WT)10株和转OsMT1a基因(L6和L7各10株)水稻幼苗转移至含有300mM甘露醇的MS培养基中培养10天，观察对比表型变化，实验重复3次。
表型变化结果如图4A所示，对水稻幼苗进行300mM甘露醇处理两周后，可以看到野生型叶片极度卷曲、发黄、萎缩，几近枯死；而转OsMT1a基因(L6和L7)表现较好的生长状态，叶片微卷、叶尖发黄，主体仍颜色鲜绿。
取在温室中于大小体积相等的花盆中生长两个月的野生型日本晴(WT)5株和转OsMT1a基因(L6和L7各5株)约1cM长样段迅速称取0.5g置于室内桌面，室温下每隔5-10分钟称重一次，直至一个小时。利用以下公式计算失水率，实验重复3次：
失水率结果如图4B所示，转OsMT1a基因(L6和L7)的叶片一小时后失水率滞后于野生型3.75％。
上述实验结果表明OsMT1a过表达株系比野生型具有更好的抗旱性。
用EcoRI与BamHI双酶切PCR产物，与同样双酶切的p181AINE载体(p181AINE载体构建方法参照文献Vernet，T.，Dignard，D.and Thomas，D.V.(1987)，A family of yeast expression vectors containing the phage f1 intergenic region，Gene52，225-233；Riesmeier JW，Willmitzer L，Frommer WB(1992)，Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast.EMBO J 11：4705-4713；Daram P，Brunner S，Persson BL，Amrhein N，Bucher M.(1998)，Functional analysis and cell-specific expression of a phosphate transporter from tomato.Planta 206：225-233.)(中国科学院遗传与发育生物学研究所)连接，获得重组载体p181AINE::OsMT1a，将p181AINE::OsMT1a和p181AINE分别转化酵母BY4741(MATA his3 leu2 met15ura3)(购自Euroscarf，目录号Y00000)。在SD/Leu(-)缺陷固体培养基上于30℃生长2-3天，长出的克隆为带p181AINE-leu标签的阳性克隆。再以5’-GAAGATGTCTTGCAGCTGTGGAT-3’和5’-AGATGGTAGATGCAGGCAGGC-3’为引物进行PCR扩增，扩增出253bp的片段鉴定转p181AINE::OsMT1a阳性克隆。
每升SD/leu(-)缺陷液体培养基：YNB酵母氮源(无氨基酸、硫酸铵)1.7g、硫酸铵5g、营养缺陷混合物(Drop-out Mix)1.4g、葡萄糖20g、L-Trp、L-His和Uracil各20mg，加水至1L灭菌。每升SD/leu(-)缺陷液体培养基中加入20g琼脂粉得到SD/leu(-)缺陷固体培养基。其中YNB、Drop-out Mix、L-Trp、L-His、Uracil均购自sigma公司，货号分别为Y1251、Y2001、T0254、H8000、U0750。
将p181AINE::OsMT1a阳性克隆与空载体p181AINE阳性克隆于SD/Leu-缺陷液体培养基中30℃培养到指数生长期，然后加入1mM ZnCl2溶液，经过10小时处理后收菌、干燥后进行金属含量测定，每个样品取三次独立重复实验的测定结果并进行统计学分析。金属含量的测定结果如表2所示。
表2.酵母中金属的含量
表2中星号**代表通过t检验得出转基因酵母和对照有显著性差异的置信概率为99％，表中金属含量的单位为(μg/g干重)。
表2的结果表明，过量表达OsMT1a的酵母细胞在非Zn处理的普通状态下，能够比对照多吸收19.2％的Zn；而在1mM Zn处理条件下，能比对照多积累1.4倍的Zn，显著地提高对Zn的吸收。OsMT1a蛋白可能参与了水稻体内Zn离子的代谢过程。
3)转OsMT1a基因植株抗氧化酶活测定
生长两周的野生型日本晴(WT)10株和转OsMT1a基因(L6和L7各10株)水稻幼苗用1mM双氧水处理5小时，分别取0.5g叶片，加入预冷的酶提取液(100mMTris HCl(pH7.0)，20％甘油，1％PVP)，研磨至匀浆；4℃，10000g离心30分钟；上清为粗酶提取液。
测定APX时，在上述提取液中加1mM AsA。
CAT、APX和POD活性参照Rahnama和Ebrahimzadeh(2005)的方法测定(Rahnama H and Ebrahimzadeh H(2005)，The effect of NaCl on antioxidant enzyme activities in potato seedlings，Biol Plant 49，pp.93-97.)。CAT活性用ΔA240mg-1proteinmin-1表示；APX活性用ΔA265mg-1proteinmin-1表示；POD活性用ΔA530mg-1proteinmin-1表示。不同波长下的吸光度变化用美卡西斯OPTIZEN-2120UV分光光度计连续测定。蛋白质含量测定参照(Bradford，M.M.(1976)A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal.Biochem.72，248-254.)的方法。每个样品取三次独立重复实验的测定结果，并进行统计学分析。
1mM双氧水处理5小时前后水稻叶片内抗氧化物酶CAT、POD和APX的酶活变化如图5所示，说明转OsMT1a基因植株的CAT，POD，APX酶活相比野生型都有不同程度的提高；L7的CAT酶活比WT提高81％，POD酶活比WT提高32％，APX酶活比WT提高17％。
上述实验结果表明，过表达OsMT1a能提高体内抗氧化系统的效率。
图5中星号*和**代表通过t检验得出转OsMT1a基因株系与野生型之间存在显著性差异的置信概率分别为95％和99％。
4)OsMT1a提高植物抗逆性的机理
为了获得准确的耐逆性机理信息，选取了报道已知受到干旱胁迫诱导的三个锌指转录因子ZF1(Accession No.AF332876)、WRKY71(Accession No.NM_001052629)(Takatsuji，H.(1999).Zinc-finger proteins：the classical zinc finger emerges in contemporary plant science.Plant Mol.Biol.39，1073-1078；Segal，D.J.，Stege，J.T.，and Barbas，C.F.，III(2003).Zinc fingers and a green thumb：manipulating gene expression in plants.Curr.Opin.Plant Biol.6，163-168)和Ossiz(Accession No.AK108249)(Wu，Y.R.，Wang，Q.Y.，Ma，Y.M.，and Chu，C.C.(2005).Isolation and expression analysis of salt up-regulated ESTs in upland rice using PCR-based subtractive suppression hybridization method.Plant Sci 168，847-853.)，比较它们在野生型与转基因水稻中的表达量差别。
提取生长两周的野生型日本晴(WT)和转OsMT1a基因(L6和L7)水稻幼苗的总RNA，反转录后进行real-time PCR分析。
引物分别为：
ZF1：上游引物5’-TTGTGAATTGCGGTGGAAGC-3’；
下游引物5’-GGCTTCTTGAAGGCGAGGG-3’。
WRKY71：上游引物5’-CGAGGAGTGCAAGCCCAAGAT-3’；
下游引物5’-AATCCTTGGTCGGCGAGAGCT-3’。
Ossiz：上游引物5’-GCACCATGGCGAGCCGAGAG-3’；
下游引物5’-AGGATCCCGGGTGCTTCTACATCACAAGC-3’。
转OsMT1a基因株系中三个锌指转录因子Ossiz、ZF1、WRKY71的相对表达量如图6A所示，在OsMT1a过表达植株中几个锌指转录因子基因表达上调；CCCH家族的Ossiz，C2H2家族的ZF1，WRKY家族的WRKY71分别上调了约10倍、5倍和3倍。
选择Ossiz上调最明显的OsMT1a过表达植株进行Northern杂交。以OsMT1a和Ossiz全长cDNA为双探针进行Northern杂交分析。
图6B所示是Northern检测转OsMT1a基因株系中Ossiz的表达结果。在过表达OsMT1a的植株中检测到了Ossiz的积累。这说明Ossiz位于OsMT1a的下游。而且Ossiz同OsMT1a一样，也能受到Zn的诱导。
图6C所示是10μM Zn2+处理0～24小时Ossiz表达变化。
上述结果表明，金属硫蛋白OsMT1a一方面可能通过感应Zn浓度变化来响应外界胁迫，通过提高体内抗氧化系统的效率来清除自由基，并提高植物的抗旱功能。此外在对外界胁迫的应答过程中，OsMT1a能够向一些锌指的转录因子，比如Ossiz直接或间接地提供Zn离子，协同作用来传导抗逆信号，调节下游基因表达，最终提高植物抗逆性。
序列表
<110>中国科学院遗传与发育生物学研究所
<120>金属硫蛋白及其编码基因与应用
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<212>DNA
<213>稻属陆稻(Oryza glaberrima)
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atgtcttgca gctgtggatc tagctgcagc tgcggctcaa actgctcctg cggaaagaag 60
taccctgacc tggaagagaa gagcagcagc accaaggcca ccgtcgtgct gggtgtggcg 120
ccggagaaga aggcgcagca gtttgaggcg gccgcagagt ccggcgagac cgcccatggc 180
tgcagctgcg gttccagctg caggtgcaac ccttgcaact gt 222
<210>2
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<212>PRT
<213>稻属陆稻(Oryza glaberrima)
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Met Ser Cys Ser Cys Gly Ser Ser Cys Ser Cys Gly Ser Asn Cys Ser
1 5 10 15
Cys Gly Lys Lys Tyr Pro Asp Leu Glu Glu Lys Ser Ser Ser Thr Lys
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Ala Thr Val Val Leu Gly Val Ala Pro Glu Lys Lys Ala Gln Gln Phe
35 40 45
Glu Ala Ala Ala Glu Ser Gly Glu Thr Ala His Gly Cys Ser Cys Gly
50 55 60
Ser Ser Cys Arg Cys Asn Pro Cys Asn Cys
65 70
Claims (8)
1.一种蛋白质，是如下a)或b)的蛋白：
a)序列表中序列2所示的氨基酸序列组成的蛋白质；
b)在序列表中序列2的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸由a)衍生的蛋白质。
2.权利要求1所述蛋白的编码基因。
3.根据权利要求2所述的基因，其特征在于：所述基因是如下1)或2)或3)：
1)其核苷酸序列是序列表中序列1；
2)在严格条件下与1)限定的DNA片段杂交且编码金属硫蛋白的DNA分子；
3)与1)或2)的基因具有90％以上的同源性，且编码金属硫蛋白的DNA分子。
4.含有权利要求2或3所述基因的重组表达载体。
5.含有权利要求2或3所述基因的转基因细胞系或重组菌。
6.一种培育抗旱能力提高和/或锌含量提高的植物的方法，是将权利要求2或3所述基因导入植物中，得到抗旱能力提高和/或锌含量提高的转基因植物。
7.扩增权利要求2或3所述基因的全长及其任意片段的引物对。
8.权利要求1所述蛋白、权利要求2或3所述基因、权利要求4所述重组表达载体、权利要求5所述转基因细胞系或重组菌在培育抗旱能力提高和/或锌含量提高的植物中的应用。
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| CN105543243A (zh) * | 2016-01-29 | 2016-05-04 | 中国科学院华南植物园 | 四个水稻金属硫蛋白基因的新应用 |
| CN108893480A (zh) * | 2018-07-06 | 2018-11-27 | 浙江海洋大学 | 毛蚶金属硫蛋白酶--一种新型海洋生物污染检测标记物 |
| CN110438131A (zh) * | 2019-07-23 | 2019-11-12 | 江西农业大学 | 黄瓜金属硫蛋白基因CsMT4的原核表达载体及其应用 |
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| CN102533788B (zh) * | 2012-03-19 | 2013-10-16 | 复旦大学 | 可提高水稻生殖期抗旱性的eadt1基因，编码序列和应用 |
| CN103937810B (zh) * | 2013-01-22 | 2017-05-31 | 中国科学院沈阳应用生态研究所 | 超富集植物龙葵金属硫蛋白基因（mt‑l3）序列及其克隆方法 |
| CN117551658B (zh) * | 2023-10-26 | 2024-09-20 | 六盘水师范学院 | 猕猴桃AcMT3a基因及其应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543243A (zh) * | 2016-01-29 | 2016-05-04 | 中国科学院华南植物园 | 四个水稻金属硫蛋白基因的新应用 |
| CN108893480A (zh) * | 2018-07-06 | 2018-11-27 | 浙江海洋大学 | 毛蚶金属硫蛋白酶--一种新型海洋生物污染检测标记物 |
| CN110438131A (zh) * | 2019-07-23 | 2019-11-12 | 江西农业大学 | 黄瓜金属硫蛋白基因CsMT4的原核表达载体及其应用 |
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