铁皮石斛锌铁调控转运蛋白基因的克隆与表达分析(1)
[摘要]锌铁调控转运蛋白(ZRT， IRT-like protein， ZIP)在植物生长发育过程中起重要的调控作用。研究采用实时定量PCR(QPCR)和RACE技术，首次从珍稀濒危兰科药用铁皮石斛Dendrobium officinale中克隆得到1个ZIP基因cDNA全长，命名为DoZIP1，提交GenBank获得注册号KJ946203。生物信息学分析显示，该基因开放阅读框1 056 bp，编码1条由351个氨基酸组成的肽链，相对分子质量37.57 kDa，等电点6.09。推定的DoZIP1蛋白具有保守的锌铁调控转运相关蛋白结构域，二级结构包含α螺旋50.71%、延伸链11.11%、β转角1.99%与随机卷曲36.18%，具有信号肽和8个跨膜域，预测定位在质膜。该蛋白质氨基酸序列与拟南芥、苜蓿、水稻等物种ZIP蛋白相似性较高，系统进化分析表明其与AtZIP10，OsZIP3蛋白的亲缘关系较近，聚在一个分支。QPCR分析表明，DoZIP1基因转录本在根中相对表达量较高，为茎中的4.19倍;叶次之，为茎的1.12倍。DoZIP1基因的分子特征为下一步研究其在铁皮石斛生长发育过程的生物学功能奠定基础。
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[关键词]铁皮石斛;锌铁调控转运蛋白;基因克隆;序列分析;实时定量PCR
[收稿日期]2014-08-20
[基金项目]国家自然科学基金项目(31070300， 31101608， 31170314);陕西省科技计划青年科技新星项目(2012KJXX-44)
[通信作者]*郭顺星，博士，研究员，研究方向为药用植物菌根生物学，E-mail： sxguo1986@163.com
[作者简介]张岗，博士，副教授，研究方向为药用植物分子生物学，E-mail： Jay_gumling2003@aliyun.com
Cloning and expression analysis of a zinc-regulated transporters (ZRT)，iron-regulated transporter (IRT)-like protein encoding gene in
, 百拇医药
Dendrobium officinale
ZHANG Gang1，2， LI Yi-min1， LI Biao2， ZHANG Da-wei2， GUO Shun-xing2*
(1. College of Pharmacy and Shaanxi Provincial Key Laboratory for Chinese Medicine Basis & New Drugs
Research， Shaanxi University of Chinese Medicine， Xi′an 712046， China;
2. Institute of Medicinal Plant Development， Chinese Academy of Medical Sciences-Peking Union
, 百拇医药
Medical College， Beijing 100193， China)
[Abstract]The zinc-regulated transporters (ZRT)， iron-regulated transporter (IRT)-like protein (ZIP) plays an important role in the growth and development of plant. In this study， a full length cDNA of ZIP encoding gene， designed as DoZIP1 (GenBank accession KJ946203)， was identified from Dendrobium officinale using RT-PCR and RACE. Bioinformatics analysis showed that DoZIP1 consisted of a 1 056 bp open reading frame (ORF) encoded a 351-aa protein with a molecular weight of 37.57 kDa and an isoelectric point (pI) of 6.09. The deduced DoZIP1 protein contained the conserved ZIP domain， and its secondary structure was composed of 50.71% alpha helix， 11.11% extended strand， 36.18% random coil， and beta turn 1.99%. DoZIP1 protein exhibited a signal peptide and eight transmembrane domains， presumably locating in cell membrane. The amino acid sequence had high homology with ZIP proteins from Arabidopsis， alfalfa and rice. A phylogenetic tree analysis demonstrated that DoZIP1 was closely related to AtZIP10 and OsZIP3， and they were clustered into one clade. Real time quantitative PCR analysis demonstrated that the transcription level of DoZIP1 in D. officinale roots was the highest (4.19 fold higher than that of stems)， followed by that of leaves (1.12 fold). Molecular characters of DoZIP1 will be useful for further functional determination of the gene involving in the growth and development of D. officinale., 百拇医药(张岗 李依民 李标 张大为 郭顺星)
, http://www.100md.com
[关键词]铁皮石斛;锌铁调控转运蛋白;基因克隆;序列分析;实时定量PCR
[收稿日期]2014-08-20
[基金项目]国家自然科学基金项目(31070300， 31101608， 31170314);陕西省科技计划青年科技新星项目(2012KJXX-44)
[通信作者]*郭顺星，博士，研究员，研究方向为药用植物菌根生物学，E-mail： sxguo1986@163.com
[作者简介]张岗，博士，副教授，研究方向为药用植物分子生物学，E-mail： Jay_gumling2003@aliyun.com
Cloning and expression analysis of a zinc-regulated transporters (ZRT)，iron-regulated transporter (IRT)-like protein encoding gene in
, 百拇医药
Dendrobium officinale
ZHANG Gang1，2， LI Yi-min1， LI Biao2， ZHANG Da-wei2， GUO Shun-xing2*
(1. College of Pharmacy and Shaanxi Provincial Key Laboratory for Chinese Medicine Basis & New Drugs
Research， Shaanxi University of Chinese Medicine， Xi′an 712046， China;
2. Institute of Medicinal Plant Development， Chinese Academy of Medical Sciences-Peking Union
, 百拇医药
Medical College， Beijing 100193， China)
[Abstract]The zinc-regulated transporters (ZRT)， iron-regulated transporter (IRT)-like protein (ZIP) plays an important role in the growth and development of plant. In this study， a full length cDNA of ZIP encoding gene， designed as DoZIP1 (GenBank accession KJ946203)， was identified from Dendrobium officinale using RT-PCR and RACE. Bioinformatics analysis showed that DoZIP1 consisted of a 1 056 bp open reading frame (ORF) encoded a 351-aa protein with a molecular weight of 37.57 kDa and an isoelectric point (pI) of 6.09. The deduced DoZIP1 protein contained the conserved ZIP domain， and its secondary structure was composed of 50.71% alpha helix， 11.11% extended strand， 36.18% random coil， and beta turn 1.99%. DoZIP1 protein exhibited a signal peptide and eight transmembrane domains， presumably locating in cell membrane. The amino acid sequence had high homology with ZIP proteins from Arabidopsis， alfalfa and rice. A phylogenetic tree analysis demonstrated that DoZIP1 was closely related to AtZIP10 and OsZIP3， and they were clustered into one clade. Real time quantitative PCR analysis demonstrated that the transcription level of DoZIP1 in D. officinale roots was the highest (4.19 fold higher than that of stems)， followed by that of leaves (1.12 fold). Molecular characters of DoZIP1 will be useful for further functional determination of the gene involving in the growth and development of D. officinale., 百拇医药(张岗 李依民 李标 张大为 郭顺星)