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作物学报 ›› 2015, Vol. 41 ›› Issue (12): 1802-1809.doi: 10.3724/SP.J.1006.2015.01802

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆耐铝毒候选基因GmSTOP1的克隆与表达分析

丛亚辉，王婷婷，柳聚阁，王宁，高萌萌，李艳*，盖钧镒*   

  1. 南京农业大学作物遗传与种质创新国家重点实验室 / 国家大豆改良中心 / 农业部大豆生物学与遗传育种重点实验室，江苏南京 210095
  • 收稿日期:2015-04-01 修回日期:2015-07-20 出版日期:2015-12-12 网络出版日期:2015-08-28
  • 通讯作者: 李艳, E-mail: yanli1@njau.edu.cn; 盖钧镒, E-mail: sir@njau.edu.cn
  • 基金资助:

    本研究由国家自然科学基金项目(31371645), 国家重点基础研究发展计划(973计划)项目(2011CB1093), 教育部长江学者和创新团队发展计划项目(PCSIRT13073), 教育部新世纪优秀人才支持计划项目(NCET-12-0891), 农业部大豆生物学与遗传育种创新团队项目和江苏省双创计划项目资助。

Cloning and Expression Analysis of Tolerance to Aluminum-toxicity Candidate Gene GmSTOP1 in Soybean

CONG Ya-Hui,WANG Ting-Ting,LIU Ju-Ge,WANG Ning,GAO Meng-Meng,LI Yan*,GAI Jun-Yi*   

  1. National Key Laboratory of Crop Genetics and Germplasm Enhancement / National Center for Soybean Improvement / Key Laboratory for Biology and Genetic Improvement of Soybean (General), Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
  • Received:2015-04-01 Revised:2015-07-20 Published:2015-12-12 Published online:2015-08-28
  • Contact: 李艳, E-mail: yanli1@njau.edu.cn; 盖钧镒, E-mail: sir@njau.edu.cn
  • Supported by:

    This research was supported by the National Natural Science Foundation of China (31371645), the National Key Basic Research Project (2011CB1093), Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (PCSIRT13073), Program for New Century Excellent Talents in University of Ministry of Education of China (NCET-12-0891), Program for Soybean Biology and Genetic Breeding Innovative Research Team of Ministry of Agriculture of China, and Program for High-level Innovative and Entrepreneurial Talents in Jiangsu Province.

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摘要：

酸性土壤中的铝毒害是限制作物生长和产量的主要因素之一。拟南芥中的AtSTOP1 (Arabidopsis thaliana sensitive to proton rhizotoxicity 1)是一个调控多种铝毒耐受机制相关基因表达的转录因子，在拟南芥耐铝毒中发挥重要作用。为研究大豆中STOP1-like基因的表达特性，本研究利用RT-PCR从耐铝毒大豆品种科丰1号中克隆了一个位于第16染色体的STOP1-like基因，命名为GmSTOP1。该基因的编码区(coding DNA sequence, CDS)序列长度为1566 bp，编码521个氨基酸。在GmSTOP1起始密码子上游1500 bp的核苷酸序列区间预测到多种顺式作用元件，包括与激素、热、逆境响应等相关的应答元件，如ABRE、HSE、TC-rich重复序列等。蛋白质结构预测表明GmSTOP1不具有跨膜结构和信号肽，含有4个保守的Cys-2-His-2锌指蛋白结构域。系统进化分析显示GmSTOP1与菜豆(Phaseolus vulgaris)中的STOP1-like蛋白亲缘关系较近。亚细胞定位结果显示GmSTOP1定位于细胞核，说明GmSTOP1蛋白可能在细胞核中发挥其功能。GmSTOP1基因在种子中的相对表达量最高，在根、茎尖分生组织、茎、叶、花、荚等多种组织中也均有表达。用25 μmol L–1 AlCl3溶液处理大豆幼苗，GmSTOP1基因在根中上调表达，24 h达到最高相对表达量，约为对照(0 μmol L–1 AlCl3)的9.2倍，表明该基因的表达受铝离子的诱导。此外，ABA、NaCl和PEG等胁迫也能诱导大豆根和叶中GmSTOP1基因的上调表达。由此推测GmSTOP1基因可能参与大豆对铝毒、高盐和渗透等非生物胁迫的应答过程。

关键词: 酸性土壤, 铝毒, 大豆, STOP1亚细胞定位, 荧光定量PCR

Abstract:

Aluminum toxicity is one of the major factors that limits the growth and production of crops in acid soils. AtSTOP1 transcription factors can regulate the expression of genes related to aluminum-toxicity tolerance mechanisms, which plays an important role in aluminum-toxicity tolerance in Arabidopsis. To study the expression features of the STOP1-like gene in soybean, we cloned a STOP1 gene located on chromosome 16 from the aluminum-toxicity tolerant soybean cultivar (Kefeng-1) using RT-PCR, and designated as GmSTOP1. The length of GmSTOP1 coding DNA sequence was 1566 bp, which encoded 521 amino acid residues. Diverse cis-acting promoter elements involved in hormone, heat and stress responses were discovered in the 1500 bp upstream region of GmSTOP1, such as ABRE, HSE, TC-rich repeats, and other elements. Protein structure prediction showed that it did not have any signal-peptide or transmembrane region, but contained four conservative Cys-2-His-2 zinc-finger domains. Phylogenetic analysis demonstrated that GmSTOP1 was similar to the putative STOP1-like protein from Phaseolus vulgaris. Results of subcellular localization showed that GmSTOP1 protein is located in the cell nucleus. The transcripts of GmSTOP1 were detected in all organs tested including root, shoot apical meristem, stem, leaf, flower, pod and seed, with the highest level in seed. GmSTOP1 was up-regulated in soybean roots by 25 μmol L–1 AlCl3 treatment, and reached the highest relative expression level at 24 hours, which was about 9.2 times of the level in control (0 μmol L–1 AlCl3). In addition, Real-time PCR analysis showed that the expression of GmSTOP1 in soybean leaf and root was also up-regulated by ABA, NaCl, and PEG, respectively. These results indicated that GmSTOP1 might participate in soybean response to abiotic stresses including aluminum-toxicity, high salinity and osmosis stress, which provides the basis for further studying the functions of GmSTOP1.

Key words: Acid soil, Aluminum toxicity, Soybean, STOP1, Subcellular location, Real-time PCR

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