Effects of sulfide stress on blood ${{\bf {SO}} _4^{2-}} $ concentration and SULT1B1-12 gene expression in Sinonovacula constricta
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摘要: 作为典型的埋栖型滩涂贝类，缢蛏（Sinonovacula constricta）常暴露在富含硫化物的环境中，并表现出较强的硫化物耐受能力。胞质磺基转移酶1B1（SULT1B1）位于硫代谢途径下游，是催化磺化反应的关键酶，在甲状腺激素（THs）等内源性物质的生物转化过程中发挥重要作用。为研究ScSULT1B1-12基因在缢蛏耐硫中的作用，本研究采用生物信息学方法分析了其序列特征，并结合血液中
${\rm {SO}} _4^{2-} $ 浓度变化，开展其组织表达及不同浓度（50 μmol/L、150 μmol/L、300 μmol/L）硫化物胁迫72 h后的表达特征研究。结果表明，ScSULT1B1-12基因全长cDNA为1 100 bp，含有 897 bp的开放阅读框，编码298个氨基酸。序列分析表明，ScSULT1B1-12含有4个催化活性位点（56K、104N、106H和134A）、1个N端的PAPS结合域（YPKSGTXW）、1个C端的PAPS结合和二聚化域（RKGXXGDWKNXFTVXXE），表明其在结构上具有催化磺化反应的能力。组织分布表明，ScSULT1B1-12基因在鳃中高表达，其次为闭壳肌和肝胰腺。硫化物胁迫后缢蛏血液中${\rm {SO}} _4^{2-} $ 浓度呈下降趋势，ScSULT1B1-12基因的表达模式也在波动中呈下降趋势，表明硫酸盐可进一步被活化生成磺化反应的供体，而ScSULT1B1-12介导的磺化反应受抑制后可使缢蛏体内THs保持在一定水平，以加强其代谢机能和免疫功能，使机体适应高硫化物的不良环境。-
关键词:
- 缢蛏 /
- ScSULT1B1-12 /
- 硫化物胁迫 /
- 时间表达 /
- 硫酸根
Abstract: As a typical dwelled tidal shellfish, Sinonovacula constricta is often exposed to sulfide-rich environment and shows strong sulfide tolerance. The cytosolic sulfotransferase 1B1 (SULT1B1) is located at downstream of the sulfur metabolism pathway, while it is a key enzyme catalyzing the sulfation reaction and plays an important role in the biotransformation of endogenous substances such as thyroid hormones (THs). In order to study the role of ScSULT1B1-12 in sulfur resistance, the sequence characteristics were analyzed by bioinformatics method. Combined with the changes of blood${\rm {SO}} _4^{2-} $ concentration, the spatial expression and temporal expression profiles during 72 h sulfide stress (50 μmol/L, 150 μmol/L, 300 μmol/L) were studied. The full-length cDNA of ScSULT1B1-12 gene was 1 100 bp, containing an open reading frame of 897 bp, and encoding 298 amino acids. Sequence analysis showed that ScSULT1B1-12 contains four catalytic active sites (56K, 104N, 106H, and 134A), one PAPS binding domain (YPKSGTXW) at N terminal, and one PAPS binding and dimerization domain (RKGXXGDWKNXFTVXXE) at C terminal, indicating that it was structurally able to catalyze the sulfation reaction. Spatial expression showed that ScSULT1B1-12 was highly expressed in gills, followed by the adductor muscle and hepatopancreas. Blood${\rm {SO}} _4^{2-} $ concentration decreased, and the expression patterns of ScSULT1B1-12 also declined with fluctuation after sulfide stress, indicating that sulfate can be further transformed to sulfated donors, and ScSULT1B1-12-mediated sulfation may be inhibited to keep THs at a certain level in S. constricta, in order to strengthen the metabolic and immune functions, and make the organism adapt the adverse environment of high sulfide.-
Key words:
- Sinonovacula constricta /
- ScSULT1B1-12 /
- sulfide stress /
- temporal expression /
- sulfate ion
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图 1 硫化物胁迫下缢蛏血液
$ {{\rm {SO}}_4^{2-}} $ 浓度的变化同类型字母中不同字母表示同一胁迫浓度下不同时间点间的数据差异显著（p<0.05）
Fig. 1 Changes in
$ {{\rm {SO}}_4^{2-}} $ concentration of Sinonovacula constricta under sulfide stressDifferent letters of the same type represented significant changes among different time at the same exposure concentration (p<0.05)
图 2 ScSULT1B1-12基因全长cDNA序列及其推导的氨基酸序列
红色加粗字体为起始密码子、终止密码子；阴影部分为蛋白的保守结构域，加粗字体和下划线标注表示PAPS结合域；黄色加粗字体和下划线标注表示PAPS结合和二聚化域；蓝色加粗字体为氨基酸活性位点；左侧数字为核苷酸和氨基酸的位置
Fig. 2 Full-length cDNA sequence of the ScSULT1B1-12 gene and its deduced amino acid sequences
Red bold font indicated the initiation codon and stop codon, the shaded part indicated the conserved domain of the protein, the bold font and underlined mark indicated the PAPS binding domains; the yellow bold font and underlined mark indicated PAPS binding and dimerization domains; the blue bold font showed the catalytic active sites; the numbers on the left indicate the positions of nucleotide and amino acid
图 3 11种动物SULT1B1氨基酸序列的多重比较
绿色箭头表示催化活性位点；红框表示PAPS结合域；蓝框表示PAPS结合和二聚化域；所使用序列的物种名及基因登录号见附表S1
Fig. 3 Multiple alignments of the amino acid sequences of SULT1B1 in eleven animals
The green arrows indicated the catalytic active site; the red box indicated the PAPS binding domain; the blue box indicated the PAPS binding and dimerization domain; the species name and the corresponding accession numbers of the sequences used are shown in appendix Table S1
图 4 邻接法构建的ScSULT1B1-12系统进化树
所使用序列的基因登录号见附表S1
Fig. 4 The ScSULT1B1-12 phylogenetic tree constructed by neighbor-joining method
Accession numbers of the sequences used in construction of tree are shown in appendix Table S1
图 5 ScSULT1B1-12基因在缢蛏不同组织中的表达
不同字母表示组织间的数据差异显著（p<0.05）
Fig. 5 The expression of ScSULT1B1-12 gene in different tissues of Sinonovacula constricta
Different letters represented significant changes among different tissues (p<0.05)
图 6 硫化物胁迫下ScSULT1B1-12基因在缢蛏鳃（M）和肝胰腺（m）中的表达特征
同类型字母中不同字母表示同一胁迫浓度下不同时间点间的数据差异显著（p<0.05）
Fig. 6 The expression characteristics of the ScSULT1B1-12 gene in gill (M) and hepatopancreas (m) of Sinonovacula constricta under sulfide stress
Different letters of the same type represented significant changes among different time at the same exposure concentration (p<0.05)
表 1 实验所用引物及其序列
Tab. 1 Primers and their sequences used in the experiment
引物名称 序列（5' -3'） SULT1B1-12-F CAAATCCGAATGGAAAGGCGG SULT1B1-12-R CAACAGAATCTGTATGTGAAG RS9-F TGAAGTCTGGCGTGTCAAGT RS9-R CGTCTCAAAAGGGCATTACC 
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表S1 物种拉丁文学名及其NCBI登录号对照表.doc
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