大豆耐铝毒候选基因GmSTOP1的克隆与表达分析

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

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At STOP1(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Gm STOP1。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Gm STOP1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Gm STOP1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Gm STOP1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Gm STOP1定位于细胞核,说明Gm STOP1蛋白可能在细胞核中发挥其功能。Gm STOP1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25μmol L–1 Al Cl3溶液处理大豆幼苗,Gm STOP1基因在根中上调表达,24 h达到最高相对表达量,约为对照(0μmol L–1 Al Cl3)的9.2倍,表明该基因的表达受铝离子的诱导。此外,ABA、Na Cl和PEG等胁迫也能诱导大豆根和叶中Gm STOP1基因的上调表达。由此推测Gm STOP1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。

Aluminum toxicity is one of the major factors that limits the growth and production of crops in acid soils. AtSTOP1 transcription factor 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 STOPl-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-rieh 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 STOPl-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.