Abstract:

A novel cDNA sequence htMT2, which encodes a type 2 metallothionein like protein, was isolated from Helianthus tuberosus L. tuber cDNA library. The whole sequence is 509 bp, including an open reading frame (ORF) of 240 bp, a 5′UTR of 62 bp and a 3′UTR of 207 bp. Two genomic sequences covering the coding region of htMT2 were cloned by PCR reaction. Sequence analysis revealed that the genomic sequences htMTG-1 of 986 bp and htMTG-2 of 982 bp were both composed of three exons and two introns. The deduced protein consisted of 79 amino acid residues with a predicted molecular weight of 7.8 ku (kD). Amino terminal and carboxy terminal domains contained 8 and 7 cysteine residues respectively, separated by a central cysteine free spacer. Sequence alignment revealed that the predicted protein of htMT2 was homologous to type 2 metallothioneins (MTs) of plants. Southern blotting analysis indicated that htMT2 was encoded by a small multi gene family in H. tuberosus genome. Northern blotting analysis showed that htMT2 transcripts were detected in stems, leaves and leafstalks, but no transcripts were detected in roots. The expression level in stems was the highest among the above tissues. Transcripts in stems were significantly reduced by Cu2+ treatment. Judging from the homologies between the deduced HtMT2 and other type 2 plant metallothioneins as well as responses to metal ions, we believe that htMT2 encodes a new type 2 metallothionein.

菊芋类金属硫蛋白基因htMT2的克隆及其表达特征分析
常团结 陈 蕾 路子显 陈宛新 刘 翔 朱 祯*

（中国科学院遗传与发育生物学研究所，北京100101）

摘要： 从菊芋 (Helianthustuberosus L .)块茎cDNA文库中得到了一个新的植物类金属硫蛋白基因htMT2的cDNA序列 ,全长 5 0 9bp ,包括 2 4 0bp的开放阅读框、6 2bp的 5′端非翻译区、2 0 7bp的 3′端非翻译区。通过PCR获得了 2个htMT2编码区的部分基因组片段htMTG_1及htMTG_2 ,长度分别为 986bp和 982bp。分析表明两个基因组片段均包含3个外显子及 2个内含子 ,编码一个由 79个氨基酸残基组成的多肽 ,与从htMT2推测的多肽完全一致 ,该多肽具有植物类金属硫蛋白的典型结构特征 ,N端及C端结构域富含Cys ,分别具有 8个和 7个Cys残基 ,上述两个结构域被一个无Cys的中间区分开。Southern杂交结果表明 ,htMT2在菊芋基因组中以小基因家族的形式存在。Northern杂交结果表明htMT2在叶片、叶柄、茎及块茎中均有表达 ,在茎中有较高水平的表达 ,但在根中未检测到杂交信号。经Cu² + 处理后 ,htMT2在茎中的表达量显著降低。与其他 2型金属硫蛋白的序列同源性比较及htMT2对金属离子处理的反应均表明 ,htMT2是一种新的植物类金属硫蛋白基因。
关键词： 植物类金属硫蛋白；;RB9 序列；基因表达；金属离子处理；内含子

通讯作者。电话：010-64873490；E-mail:zhou @ genetics.ac.cn.

Key words: plant MT like protein, cDNA sequence, gene expression, metal ion treatment, intron