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Isolation of a gene for metallothionein-like protein of higherplant

高等植物金属硫蛋白样蛋白基因的分离

基本信息

批准号：
02454058
负责人：
CHINO Mitsuo
金额：
$ 1.15万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454058/
关键词：
Metallothionein Copper Soybean Arabidopsis Gene cloning Promoter Zink Cystein 抗体遺伝子免疫組織化学

项目摘要

Using a synthetic oligonucleotide that corresponded to the consensus nucleotide sequence of the N-terminal region of mammalian metallothionein as probe, we isolated a cDNA clone from a soybean library. The clone had an ORF that encode a protein of 79 amino acids which showed significant homology to both N- and C-terminal regions of manunalian and Neurospora crassa metallothioneins.The localization of metallothionein (MT) in the seeds and roots of soybean was investigated by immunohistochemistry. The germinating seeds at 2 hr, 1, 2, 3, 4 and 6 d including 1-mo root tips of soybean (c. v. Toyosuzu) with and without heavy metals (Cu 400 mug L^<-1> or Zn 3 mug ml^<-1>) treatment were used to demonstrate the localization of MT by the indirect immunoperoxidase technique using polyclonal rabbit antirat MT conjugated to ascaris as a primary antibody. Metallothionein was localized in the proliferating regions such as the embryo in seeds, and root and shoot apices of both the control and heavy metals-treated plants. The intensity of MT staining in the proliferating regions generally increased as the soybean seeds germinate. Starting at about 1 day after germination, MT was found in the veins and vascular bundles suggesting its translocation to other organs. Similar observation hold true in the case of plants treated with heavy metals. This means that heavy metals treatment had no effect on MT localization. However, the heavy metalstreated plants showed higher concentration of MT over the control with respect to the growth stage of soybean seeds. These indicate that MT found in soybean plays a physiological role in heavy metal transport, detoxification and cell division in a similar manner to mammalian MT.

以合成的与哺乳动物金属硫蛋白N-末端一致的核苷酸序列相对应的寡核苷酸为探针，从大豆文库中克隆了一个cDNA克隆。该克隆具有一个编码区，编码79个氨基酸，与粗毛脉孢菌和甘露糖金属硫蛋白的N-末端和C-末端有显著的同源性。以含1个月的大豆根尖的萌发种子为材料，分别在2小时、1、2、3、4和6d分别用含重金属(Cu400mug L)和不含重金属(Cu400mug L)或含锌3mugml1&gt；-1&gt；的大豆根尖，用间接免疫过氧化物酶技术，以兔抗鼠蛔虫MT为一抗，对MT进行了定位。金属硫蛋白定位于对照和重金属处理植物的种子胚胎、根尖和顶端等增殖区。随着大豆种子萌发，增殖区MT染色强度普遍增强。萌发后1天左右，在叶脉和维管束中发现MT，表明MT向其他器官转移。类似的观察结果也适用于用重金属处理过的植物。这意味着重金属处理对MT的定位没有影响。然而，在大豆种子的生长阶段，重金属处理的植株表现出比对照更高的MT浓度。这些结果表明，在大豆中发现的MT在重金属的运输、解毒和细胞分裂中起着与哺乳动物MT相似的生理作用。