Molecular cloning and mRNA expression pattern of Sox10 in Paramisgurnus dabryanus

A number of genetic studies have established that Sox10 involved in a wide range of developmental processes including sex differentiation and neurogenesis in vertebrates. A Sox10 homologue was cloned from brain of Paramisgurnus dabryanus by using homologous cloning and RACE method, designated as PdSox10. The full-length cDNA of PdSox10 contains a 312 bp 5′ UTR, a 1,476 bp open reading frame (ORF) encoding 492 amino acids and a 262 bp 3′ UTR (Accession no.: JQ217143). The overall topology of the phylogenetic tree shows that the PdSox10 fits within the Sox10 clade. During embryogenesis, PdSox10 gene seemed to be de novo synthesized in the embryos from gastrulae stage. From the somitogenesis stage and thereafter, distinct expression of PdSox10 was observed in the medial neural tube, extending from the hindbrain through the posterior trunk. In adult, PdSox10 mRNA was detected primarily in the gonads, as well as in brain and heart by RT-PCR. In situ hybridization on gonadal sections further demonstrated that PdSox10 is expressed especially in premature germ cells, in early perinucleolus stage oocytes and cortical–alveolar stage oocytes in ovaries and in spermatogonia and spermatocytes in testes. These preliminary findings suggested that PdSox10 is highly conserved during vertebrate evolution and involved in a wide range of developmental processes including neurogenesis and sex differentiation in vertebrates.     

鳜胰岛素样生长因子-ⅠcDNA全长克隆及组织表达分析

采用RT-PCR、cDNA末端快速扩增法(RACE)等技术克隆了鳜(Siniperca chuatsi)肝组织胰岛素样生长因子-I(IGF-I)cDNA全长序列.结果表明,鳜IGF-I cDNA全长1 784 bp,包括5'端非翻译区233bp,3'端非翻译区990 bp和开放阅读框561 bp,共编码186个氨基酸;...     

Cloning,characterization and function analysis of <Emphasis Type="Italic">DAX1</Emphasis> in Chinese loach (<Emphasis Type="Italic">Paramisgurnus dabryanus</Emphasis>)

The mechanisms of sex determination and differentiation have not been elucidated in most fish species. In this study, the full-length cDNAs of DAX1 was cloned and characterized in aquaculture fish Chinese loach (Paramisgurnus dabryanus), designated as Pd-DAX1. The cDNA sequence of Pd-DAX1 was 1261 bp, including 795 bp open reading frame (ORF) encoding 264 amino acids. Pd-DAX1 shares highly identical sequence with DAX1 homologues from different species. The expression profiles of Pd-DAX1 in different developmental stages and diverse adult tissues were analyzed by quantitative real-time RT-PCR and in situ hybridization (ISH). Pd-DAX1 was continuously expressed during embryogenesis, with the extensive distribution in the development of the central nervous system. Tissue distribution analysis revealed that Pd-DAX1 expressed widely in adult tissues, with the highest expression level found in testis, moderate level in ovary, showing a sex-dimorphic expression pattern. Pd-DAX1 mainly located in spermatogonia cells, spermatocytes, primary oocytes and previtellogenic oocyte cells, implying that Pd-DAX1 may involve in gametogenesis. These preliminary findings suggest that Pd-DAX1 gene is highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, central nervous system development and gonad development.     

cDNA cloning, tissues, embryos and larvae expression analysis of Sox10 in half-smooth tongue-sole, Cynoglossus semilaevis

A half-smooth tongue-sole, Cynoglossus semilaevis Sox10 (Accession no.: EU070763) was isolated from brain of tongue sole by using homologous cloning and RACE method. The complete cDNA of the tongue sole Sox10 contains a 35 bp 5′UTR, a 1338 bp open reading frame (ORF) encoding 445 amino acids and a 1155 bp 3′UTR. A condensed phylogenetic tree was constructed based on the amino acid sequences of tongue sole Sox10 and other well-defined vertebrate Sox. The overall topology of the tree showed the tongue sole Sox10 clusters with all Sox10. Alignment of amino acid residues of the tongue sole Sox10 gene with those from other vertebrate indicated high level conservation of amino acid sequence. The RT-PCR analysis demonstrated that the tongue sole Sox10 was highly expressed in brain, gills, skin and eyes, intermediately in spleen, heart, head–kidney and muscles, weakly expressed in kidneys and intestine and no expression in liver and gonad. The Sox10 was also expressed weakly in germ cell and zygote. We cannot detect the expression of the Sox10 in 8-cells stage. However it resumed expression weakly from blastula stage to middle of gastrula. And it expressed highly from neurula stage to 25 dah (day after hatching). It suggested that the Sox10 was involved in the development of embryos and larvae in tongue sole.     

cDNA cloning and expression analysis of two distinct <Emphasis Type="Italic">Sox8</Emphasis> genes in <Emphasis Type="Italic">Paramisgurnus dabryanus</Emphasis> (Cypriniformes)

The Sox9 gene attracts a lot of attention because of its connection with gonadal development and differentiation. However, Sox8, belonging to the same subgroup SoxE, has rarely been studied. To investigate the function as well as the evolutionary origin of SOXE subgroup, we amplified the genomic DNA of Paramisgurnus dabryanu using a pair of degenerate primers. Using rapid amplification of the cDNA ends (RACE), it was discovered that P. dabryanu has two duplicates: Sox8a and Sox8b. Each has an intron of different length in the conserved HMG-box region. The overall sequence similarity of the deduced amino acid of PdSox8a and PdSox8b was 46.26%, and only two amino acids changed in the HMG-box. This is the first evidence showing that there are two distinct duplications of Sox8 genes in Cypriniformes. Southern blot analysis showed only one hybrid band, with lengths 7.4 or 9.2 kb. Both semi-quantitative RT-PCR and real-time quantitative PCR assay displayed that both PdSox8a and PdSox8b are downregulated during early embryonic development. In adult tissues, the two Sox8 genes expressed ubiquitously, and expression levels are particularly high in the gonads and brain. In gonads, both PdSox8a and PdSox8b are expressed at a higher level in the tesis than in the ovary. PdSox8a and PdSox8b may have functional overlaps and are essential for the neuronal development and differentiation of gonads.     

马氏珠母贝Sox11基因的克隆及时序表达模式分析

为探究Sox(SRY-related HMG-box genes)基因家族在马氏珠母贝个体发育及性别分化中的作用, 研究首先利用兼并引物从马氏珠母贝基因组中克隆到一个HMG框(high mobility group box), 利用RACE-PCR技术从SMART cDNA文库中克隆到一个Sox基因的cDNA全长, 通过Clustal X和MEGA 4软件对该序列进行比对分析并构建系统进化树; 通过荧光定量PCR技术对该基因在不同组织及发育不同时期性腺中的表达情况进行分析。结果显示, 马氏珠母贝该Sox基因的cDNA全长为1579 bp, 其中开放阅读框(ORF)为1008 bp, 编码336个氨基酸, 5'非编码区为126 bp, 3'非编码区为445 bp。同源性分析表明, 马氏珠母贝Sox基因与太平洋牡蛎Sox11基因的同源性(Identity)最高, 为80%, 故命名为pmSox11; 系统进化树分析也显示pmSox11与太平洋牡蛎Sox11基因的亲缘关系最近。荧光定量PCR分析组织表达特异性显示, pmSox11在马氏珠母贝神经节分布较多的组织如外套膜、鳃、足、消化盲囊等大量表达, 在神经节相对较少的闭壳肌和卵巢中表达量较少; 时序表达图谱显示, pmSox11在3月龄幼贝性腺和1年龄发育早期精巢中表达量最高, 在2年龄成熟精巢和2年龄性转换性腺中表达量降低, 而在2年龄卵巢中表达量最低。研究表明, pmSox11基因可能在马氏珠母贝早期神经系统发育和性别发育的调控方面起到重要作用。     

中华鳖Dmrt1基因的克隆、表达及在性逆转中的变化分析

在大部分脊椎动物中,Dmrt1基因在雄性性别决定和性腺分化中起重要的调控作用.本文从m RNA和蛋白水平分析Dmrt1基因的组织差异性表达、在不同发育阶段性腺中的细胞定位及在性逆转中的表达变化,研究Dmrt1基因在中华鳖性别分化中的调控作用.Rapid-amplification of c DNA ends(RACE)结果显示,Dmrt1基因c DNA序列全长2409 bp,其中5′非编码区为230 bp,3′非编码区为1072 bp,开放阅读框为1107 bp,编码368个氨基酸,具有一个高度保守的DM结构域.荧光定量PCR和免疫组化结果显示,Dmrt1在性腺分化之前的第16期雄性性腺中开始表达,先于Amh和Sox9基因表达.随着性腺的发育,Dmrt1蛋白主要定位于性腺Sertoli细胞的细胞核上,在雌性性腺发育过程中并未见其表达.此外,在雌二醇诱导的雄性转雌性性逆转胚胎性腺中,Dmrt1表达显著下调;在芳香化酶抑制剂诱导的雌性转雄性性腺中,Dmrt1表达则显著上升.上述研究表明,Dmrt1基因是中华鳖雄性特异性基因,参与雄性性腺的发育过程,可能在中华鳖早期性别决定中起重要的调控作用.     

翘嘴鲌Sox9基因的克隆及CpG岛甲基化与基因表达的关系

为了揭示翘嘴鲌(Culter alburnus)性别决定与分化的作用机制, 进而更好地发展性别控制育种技术, 研究重点分析了Sox9基因在翘嘴鲌性腺分化过程中的作用。通过RT-PCR和RACE方法获得了翘嘴鲌2个旁系同源基因Sox9a和Sox9b的cDNA序列: Sox9a全长1642 bp, 编码458个氨基酸; Sox9b全长1673 bp, 编码456个氨基酸。序列分析表明两者相似度达到73.95%, 编码HMG盒区域极其保守。蛋白质次级结构预测显示Sox9a和Sox9b除了保守的HMG盒结构域外, 还存在2个核定位信号; 两者的三维结构都存在多个螺旋结构。系统进化树分析发现翘嘴鲌Sox9a与罗非鱼关系最近, 但Sox9b形成单独的一支。利用实时荧光定量PCR技术分析了翘嘴鲌Sox9a和Sox9b基因在各成体组织中的表达水平, 结果显示Sox9a在脑和精巢中表达量最高, 其次是肌肉、鳍条、眼睛和卵巢, 在肾脏、脾脏、肝脏中相对较低; Sox9b只在脑、鳍条、眼睛和精巢中检测到一定水平的表达。通过重亚硫酸氢盐DNA测序方法分析了翘嘴鲌性腺组织Sox9a启动子CpG岛甲基化修饰模式, 结果显示在精巢中CG位点几乎不发生甲基化, 然而卵巢中的甲基化程度非常高。这些结果表明启动子CpG甲基化可以调控Sox9a的性别异形表达, 表观遗传修饰在翘嘴鲌性腺发育过程中可能具有重要的生物学功能。     

Molecular characterization of a 14-3-3 zeta gene from Plodia interpunctella: A potential marker for phylogenetic inference

The 14-3-3 proteins are a large family of approximately 30 kDa acidic proteins and acting in the regulation of many biological processes. In this study, a 14-3-3 zeta (Pi14-3-3z) gene from the Indian meal moth, Plodia interpunctella (Lepidoptera, Pyralidae) was isolated and characterized. The full-length cDNA of Pi14-3-3z is 1382 bp, including a 5'-untranslated region (UTR) of 141 bp, 3′-UTR of 497 bp and an open reading frame (ORF) of 744 bp encoding a polypeptide of 247 amino acids which contains a 14-3-3 homologues domain (PF00244). The deduced Pi14-3-3z protein sequence has 81%–100% identity with the homologues in comparison to with other individuals. qPCR analysis revealed that Pi14-3-3z was expressed at the four developmental stages and in all tissues tested. Based on the amino acid of 14-3-3z, phylogenetic analysis demonstrated a similar topology with the traditional classification, suggesting 14-3-3z protein has the potential value in phylogenetic inference.