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作物学报 ›› 2013, Vol. 39 ›› Issue (01): 76-83.doi: 10.3724/SP.J.1006.2013.00076

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆Gm TIP1;1基因的克隆与表达分析

张大勇1,胡国民3,易金鑫1,*,许玲1,Ali ZULFIQAR 4,刘晓庆1,袁玲玲2,徐照龙1,2,何晓兰1,黄益洪1,马鸿翔1   

  1. 1江苏省农业生物学重点实验室, 江苏省农业科学院农业生物技术研究所, 江苏南京 210014; 2南京农业大学农学院, 江苏南京 210095; 3扬州大学生物科学与技术学院, 江苏扬州225009, 4 Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad 38040, Pakistan
  • 收稿日期:2012-02-21 修回日期:2012-09-05 出版日期:2013-01-12 网络出版日期:2012-11-14
  • 通讯作者: 易金鑫, E-mail: yij@jaas.ac.cn, Tel: 025-84391105
  • 基金资助:

    本研究由国家自然科学基金项目(31101166, 30971798), 江苏省农业科技自主创新资金[CX(11)2052, CX (10)433], 江苏省自然科学基金(BK2010474)和国家科技支撑计划项目(2011BAD35B06)资助。

Isolation and Expression Analysis of Gm TIP1;1 in Soybean (Glycine max L.)

ZHANG Da-Yong1,HU Guo-Min3,YI Jin-Xin1,*,XU Ling1,Ali ZULFIQAR4,LIU Xiao-Qing1,YUAN Ling-Ling2,XU Zhao-Long1,2, HE Xiao-Lan1, HUANG Yi-Hong1,MA Hong-Xiang1   

  1. 1 Provincial Key Laboratory of Agrobiology, Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210094, China; 2 College of Agriculture, Nanjing Agricultural University, Nanjing 210095, China; 3College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China; 4 Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad 38040, Pakistan
  • Received:2012-02-21 Revised:2012-09-05 Published:2013-01-12 Published online:2012-11-14
  • Contact: 易金鑫, E-mail: yij@jaas.ac.cn, Tel: 025-84391105

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摘要：

利用RT-PCR方法从大豆根部组织获得Glyma03g34310.1开放阅读框(ORF)全长, 经测序验证、Blast比对与同源性分析发现该序列编码的蛋白质与其他植物的TIP1;1蛋白具有较高的相似性, 故命名为GmTIP1;1基因(GenBank登录号为AK285481), 该基因ORF长753 bp, 编码1个包含250个氨基酸的蛋白, 在ORF内部第381个核苷酸处含有1个94 bp的内含子, 符合↓GT--AG↓的剪接方式; 系统进化树分析发现Gm TIP1;1聚类到豆科植物分支, 其他不同科的植物也有规律地聚到了不同分支, 推测该蛋白氨基酸序列可以作为植物分类的依据; 半定量RT-PCR结果表明该基因在大豆的不同器官、不同器官的不同发育阶段均具较高且同等的表达水平, 暗示该基因在植物的整个发育进程中均具重要作用; 在盐胁迫的不同时间点其表达量有下降的趋势, 但仍然保持较高的表达水平; 以pYES2为酵母表达载体, 转化酿酒酵母INVSc1菌株, 获得重组酵母INVSc1 (pYES2-GmTIP1;1), 转化菌株在盐胁迫下的存活率明显高于对照INVSc1 (pYES2), 而在干旱胁迫下则没有显著差异, 表明该基因的表达能有效地提高酵母的耐盐性。

关键词: Gm TIP1;1, 组织表达, 诱导表达, 酵母表达

Abstract:

The full length open reading frame (ORF) of gene named Glyma03g34310.1 was amplified from the soybean root tissues by reverse transcriptase polymerase chain reaction (RT-PCR) method. Sequencing, Blast and homology analyses showed that the amino acids encoded by this gene had the higher similarity with the TIP1;1 from other species, so designated Gm TIP1;1 (GenBank accession number: AK285481), its ORF was 753 bp, encoded a polypeptide with 250 amino acids, and contained a 94 bp intron at the site of the 381th nucleotide complying with the ↓GT--AG↓ mode of splicing. Gm TIP1;1 had two copies in the soybean genome, with the other one of Glyma19g37000.1. Multiple ali MEGA5.05gnment using protein indicated that GmTIP1;1 contained six conserved transmembrane domains and two higher conserved NPA motifs. Phylogenetic tree analysis showed that Gm TIP1;1 was indeed grouped into the legumes clade and several different clades which belonged to the different plant coleus were regularly separated based on the TIP1;1 protein sequences, which implied that TIP1;1 sequence probably could be regard as a proof of plant taxonomy. Semi-quantity RT-PCR analysis demonstrated that the Gm TIP1;1 gene constitutively expressed in soybean organs including root, stem, leaf, flower and pod, and the expression levels were no obvious difference in different tissues at the different developmental stages, which implied Gm TIP1;1 gene plays important roles in plant growth. The expression of Gm TIP1;1 appeared a declined  trend at the different time points under the treatment of salt solution (200 mmol L–1 NaCl), although still showing the abundant transcript. In addition, the recombinant plasmid pYES2-Gm TIP1;1 was constructed by inserting the Gm TIP1;1 gene into the yeast expression vector pYES2. The recombinant plasmid pYES2-Gm TIP1;1 was transformed into yeast Saccharomyces cerevisiae INVScl, then treated with salt and drought stresses, respectively. The results showed that the survival rate of the recombinant yeast INVScl (pYES2-Gm TIP1;1) was higher than that of the control strain under the salinity condition, but no difference under the drought. These results indicated that the heterologous expression of Gm TIP1;1 could effectively improve the tolerance of yeast to salinity stress.

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