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中国大鲵Sox19基因全长cDNA序列的克隆及组织表达分析 [中文引用][英文引用]

Cloning and tissue expression analysis of the full length cDNA encoding the Sox19 gene in Chinese giant salamander (Andrias davidianus)

作者:刘静  何青  刘丽丽  李成磊  王勤 
作者(英文):LIU Jing  HE Qing  LIU Lili  LI Chenglei  WANG Qin 
单位(英文):College of Life Sciences  Sichuan Agricultural University  Ya'an 625014  China 
关键词(英文):Andrias davidianus  Sox19 gene  gene clone  gene tissue distribution 
分类号:S917
出版年·卷·期(页码):2015·22·第6期(1142-1149)
DOI: 10.3724/SP.J.1118.2015.14527
-----摘要:-------------------------------------------------------------------------------------------

采用逆转录聚合酶链反应(RT-PCR)及cDNA末端快速扩增(RACE)技术克隆了中国大鲵(<i>Andrias davidianus</i>)<i> Sox19</i>基因全长cDNA序列, 并进行各组织间的表达分析.中国大鲵<i>Sox19</i>基因全长1290 bp, ORF长858 bp, 编码285个氨基酸, 位于39~109位的HMG-box是其主要功能区.氨基酸序列分析表明, 其与舌齿鲈(<i>Dicentrarchus labrax</i>)、斑马鱼(<i>Danio rerio</i>)及红鳍东方鲀(<i>Takifugu rubripes</i>)的相似度分别为64%、58%、55%.采用邻接法对不同物种的<i>Sox19</i>编码区序列构建分子系统树发现: 中国大鲵<i>Sox19</i>基因属于较早从鱼类分化出来的基因, 表明<i>Sox19</i>基因从中国大鲵到鱼类的进化速度比较快.荧光定量PCR结果显示, <i>Sox19</i>在所有检测的组织中均有表达, 且在心脏中的表达量最高, 卵巢中次之, 肾脏中的表达量略高于肠道.中国大鲵<i>Sox19</i>基因的发现打破了<i>Sox19</i>基因一直被认为是鱼类所特有基因的观点, 填补了两栖类有关此基因的空白, 为更进一步的了解<i>Sox19</i>基因在中国大鲵中的重要功能奠定了基础, 同时<i>Sox19</i>基因还可作为研究中国大鲵早期胚胎神经系统发育情况的分子标记, 为其他两栖类动物的研究提供借鉴.

-----英文摘要:---------------------------------------------------------------------------------------

The <i>Sox</i> family of transcription factors is involved in a variety of developmental processes, including sex determination and gonadal differentiation. The high-mobility-group (HMG) domains of all <i>Sox</i> proteins have highly conserved primary structures, and all appear to be capable of binding to the same target DNA sequence of AACAA(A/T)G. The <i>Sox</i> gene family is divided into 10 subgroups (designated A-J) based on sequence similarity and gene organization. <i>Sox19 </i>is a member of the group B <i>Sox </i>genes, though it differs from other members in possessing an intron. The <i>Sox</i> B group is further divided into the B1 subgroup, which encodes transcriptional activators, and the B2 subgroup, which encodes repressors. Based on sequence and phylogenetic analyses, <i>Sox19</i> has been assigned to the B1 subgroup. Interestingly, the <i>Sox19</i> gene has always been considered fish specific but the full length cDNA encoding <i>Sox19</i> has only been cloned in <i>Danio rerio</i>, <i>Takifugu rubripes</i>, and <i>Dicentrarchus labrax</i>, and little is known about the expression patterns in other species. In this study, we successfully cloned the full-length <i>Sox19 </i>cDNA sequence of the Chinese giant salamander (<i>Andrias davidianus</i>), which is the largest extant amphibian, and used quantitative real-time polymerase chain reaction (PCR) analysis to detect <i>Sox19 </i>gene expression in different tissues. We cloned a 1290-bp <i>Sox19 </i>gene cDNA, including 270 bp of the 5'-untranslated region (UTR) sequence, 858 bp of the open reading frame (ORF), and 162 bp of the 3'-UTR sequence. The ORF encoded 285 amino acids, of which a 71 amino acid protein was from the N-region and a 39-109 amino acid region was the highly conserved HMG-box. Bioinformatics software was applied to analyze the <i>A. davidianus Sox19 </i>protein, which is hydrophilic. A subcellular localization analysis showed that 96% of the protein was present in the nucleus, possibly without the transmembrane region or signal peptide, 24.91% was in the α-helix, 3.16% was in the extended strand, 2.11% was in the β-corner, and 69.82% was in the random coil. The tertiary structural prediction revealed a α-helical structure as the functional site. The <i>A. davidianus Sox19</i> protein encoded 285 amino acids, which is less than that in fish. Therefore, fish have more similar "AAAA" polymers than those of the <i>A. davidianus </i>(150 amino acids), but <i>A. davidianus</i> has an additional "QNLV" at the C-terminal end, suggesting that the <i>Sox19</i> gene may have lost the lactamine oligomer during evolution and mutated to a more elaborate and complex transcriptional regulatory region. The amino acid sequence analysis revealed that the putative <i>A. davidianusSox19</i> protein had 55%, 58%, and 64% identity with <i>T. rubripes</i>, <i>D. rerio</i>, and <i>D. labrax</i>, respectively. The phylogenetic analysis further demonstrated that <i>A. davidianus </i>split off earlier based on the <i>Sox19 </i>gene data, revealing a more primitive evolutionary status. The <i>Sox19 </i>gene could have evolved through ancient fish-specific duplication of <i>Sox3</i>, although <i>Sox19</i> is expressed in the developing lens and central nervous system. We analyzed <i>Sox19</i> expression in the gonad, heart, kidney, and bowel of adult <i>A. davidianus</i> to confer the functions of the protein by fluorescence quantitative PCR. The results show the highest <i>Sox19 </i>gene expression level in the heart, suggesting that <i>Sox19 </i>helps maintain heart function. The high expression level in the gonads shows that <i>Sox19</i> may be involved in gonadal development and differentiation. <i>Sox19 </i>was also expressed in the kidney and intestine, indicating that <i>Sox19 </i>plays a role maintaining kidney and bowel functions in A<i>. davidianus</i>. The <i>Sox19</i> gene expression levels in <i>A. davidianus </i>were not the same as those in the fish species investigated. Our results provide insight into the role of the <i>Sox19</i> gene in <i>A. davidianus </i>and further our understanding of <i>Sox19 </i>gene expression in amphibians. The <i>Sox19 </i>gene could be the earliest molecular marker of embryogenesis in <i>A. davidianus</i>.

-----参考文献:---------------------------------------------------------------------------------------

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若需在您的论文中引用此文,请按以下格式著录参考文献:
中文著录格式: 刘静,,何青,,刘丽丽,,李成磊,,王勤.中国大鲵Sox19基因全长cDNA序列的克隆及组织表达分析.中国水产科学.2015;22(6):1142-1149.
英文著录格式: LIU,Jing,,HE,Qing,,LIU,Lili,,LI,Chenglei,,WANG,Qin.Cloning and tissue expression analysis of the full length cDNA encoding the Sox19 gene in Chinese giant salamander (Andrias davidianus).No Title Settings.2015;22(6):1142-1149.

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