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百脉根钼转运蛋白基因LjMOT1的克隆与表达分析

发表时间:2017-02-16  浏览量:1136  下载量:265
全部作者: 巫飞飞,沈知临,纪 萍,高俊山
作者单位: 安徽农业大学生命科学学院
摘 要: 克隆百脉根钼转运蛋白基因LjMOT1,其全长 cDNA 为1 824 bp,包括1 407 bp 的开放阅读框(open reading frame,ORF)、79 bp的 5¢-非编码区(untranslated region,UTR)和323 bp的3¢-UTR,以及15 bp的polyA,推测LjMOT1基因编码468个氨基酸。氨基酸序列比较显示百脉根LjMOT1蛋白与大豆GmMOT1蛋白的同源性最高,达到 81.14 %. 实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)分析结果表明,百脉根根中LjMOT1的表达量随培养基中钼浓度的增加呈现出上升的趋势,而叶中在+Mo和 10×Mo 浓度下LjMOT1的表达量并没有明显的差异,但是含钼条件下叶中的LjMOT1表达量明显比-Mo条件下的高。构建真核表达载体pCambia1304-LjMOT1,在烟草上进行瞬时表达,通过激光共聚焦显微镜确定LjMOT1 定位于质膜上。这些结果为进一步鉴定LjMOT1的功能奠定了理论基础。
关 键 词: 分子生物学;百脉根;钼;LjMOT1;实时荧光定量PCR;亚细胞定位
Title: Cloning and expression analysis of a molybdenum transporter gene LjMOT1 from Lotus japonicus
Author: WU Feifei, SHEN Zhilin, JI Ping, GAO Junshan
Organization: School of Life Sciences, Anhui Agricultural University
Abstract: In this paper, a Mo transporter gene LjMOT1 was cloned from Lotus japonicus, and the full-length cDNA of LjMOT1 is 1 824 bp, which includes an 1 407 bp open reading frame (ORF), 79 bp 5¢-untranslated region (UTR) and 323 bp 3¢-UTR and 15 bp polyA. The LjMOT1 gene was predicted to encode 468 amino acids. The alignment of amino acid sequences showed the LjMOT1 protein had the highest identities (81.14%) with the GmMOT1 protein from soybean. The results of quantitative real-time PCR (qRT-PCR) illuminated that the expression level of LjMOT1 in roots appeared a rising trend with increasing Mo concentrations in the media. The expression level of LjMOT1 in leaves on +Mo and 10×Mo media had no significant difference, but they were obviously higher than those on -Mo medium. A eukaryotic expression vector pCambia1304-LjMOT1 was constructed, and was transiently expressed in tobacco. The result showed that the LjMOT1 protein was located on the plasma membrane by a laser scanning confocal microscope. These results provide theoretical basis for further identifying the function of LjMOT1.
Key words: molecular biology; Lotus japonicus; molybdenum; LjMOT1; quantitative real-time PCR; subcellular localization
发表期数: 2017年2月第3期
引用格式: 巫飞飞,沈知临,纪 萍,等. 百脉根钼转运蛋白基因LjMOT1的克隆与表达分析[J]. 中国科技论文在线精品论文,2017,10(3):248-257.
 
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2017-02-22 10:36:42
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