摘要
本文利用RACE方法克隆得到了两种泥鳅SoxE亚族全部成员基因和大鳞副泥鳅DMRT1基因,并利用半定量RT-PCR和荧光定量RT-PCR对各个基因的表达模式进行了研究,为了更进一步确定目的基因的表达部位,我们利用RNA原位杂交法分别对两种泥鳅的性腺和不同发育时期的胚胎进行了杂交,主要研究结果包括下面三个方面：
1.泥鳅SoxE亚族基因的克隆和表达模式分析
本研究根据其它物种Sox8和Sox10基因HMG保守区序列,设计两对简并引物,并在此基础上采用RACE方法从泥鳅脑中分离得到了SoxE亚族各成员cDNA全序列：MaSox8a基因cDNA全长为2555bp,包括53bp5'非翻译区,1212bp3'非翻译区(含polyA)和编码429个氨基酸的开放阅读框(基因注册号为GU166139)；MaSox8b基因cDNA全长为1725bp,包括85bp5'非翻译区,395bp3'非翻译区(含polyA)和编码414个氨基酸的开放阅读框(基因注册号为GU166140)；MaSox9a基因cDNA全长为3192bp,包括326bp5'非翻译区,1408bp3'非翻译区(含polyA)和编码485个氨基酸的开放阅读框；MaSox9b基因的cDNA全长为1782bp,包括217bp5'非翻译区,179bp3'非翻译区(含polyA)和编码462个氨基酸的开放阅读框；MaSox10基因的cDNA全长为2096bp,包括311bp5'非翻译区,312bp3'非翻译区(含polyA)和编码490个氨基酸的开放阅读框。
半定量RT-PCR和荧光定量RT-PCR结果显示：在泥鳅胚胎发育的各个时期,MaSox8a和MaSox8b的表达趋势相反但是都从原肠胚开始一直持续到卵黄吸尽期,MaSox9a和MaSox9b的表达量都逐渐升高,在孵出期期表达量最高,MaSox10的表达量逐渐增加,孵出期达到最高；在泥鳅成体的不同组织中,SoxE亚族各个基因都广泛存在,MaSox8a和MaSox8b在脑中的表达量较高,MaSox9a和MaSox9b在性腺中表达量最高,Sox10在心脏中表达量最高。
原位杂交试验结果显示：SoxE亚族的五个成员都在泥鳅未成熟的生殖细胞(卵巢的卵原细胞、初级卵母细胞,精巢中的精原细胞和精母细胞)中表达,因此推测SoxE亚族的五个成员可能调控泥鳅性腺的发育和分化；整体胚胎杂交发现,SoxE亚族的五个成员均在体节形成期的体节中大量表达,之后一直到尾芽期的胚胎脊柱中都有很强的阳性杂交信号,孵出期以后SoxE亚族的五个成员的表达主要集中在泥鳅的脊柱、体节和头部。
2.大鳞副泥鳅SoxE亚族基因的克隆和表达模式分析
本研究根据其它物种Sox8和Sox10基因HMG保守区序列,设计两对简并引物,并在此基础上采用RACE方法从泥鳅脑中分离得到了SoxE亚族各成员cDNA全序列：PdSox8a基因cDNA全长为2467bp,包括53bp5'非翻译区,1127bp3'非翻译区(含polyA)和编码428个氨基酸的开放阅读框；PdSox8b基因cDNA全长为2166bp,包括201bp5'非翻译区,720bp3'非翻译区(含polyA)和编码414个氨基酸的开放阅读框；PdSox9a基因cDNA全长为3232bp,包括329bp5'非翻译区,1439bp3'非翻译区(含polyA)和编码487个氨基酸的开放阅读框；PdSox9b基因的cDNA全长为1946bp,包括219bp5'非翻译区,341bp3'非翻译区(含polyA)和编码461个氨基酸的开放阅读框；PdSox10基因的cDNA全长为2053bp,包括312bp5'非翻译区,262bp3'非翻译区(含polyA)和编码492个氨基酸的开放阅读框。
半定量RT-PCR和荧光定量RT-PCR结果显示：在大鳞副泥鳅胚胎发育的各个时期,PdSox8a、PdSox8b、PdSox9b和PdSox10的表达量都逐渐下降,PdSox9a的表达量则相反呈现上升趋势；在大鳞副泥鳅成体的不同组织中,SoxE亚族各个基因中广泛存在,并且都在性腺中大量表达,除此之外PdSox8a在脑中的表达量也很高,PdSox8b、PdSox9a和PdSox9b在肝中的表达量均较高,PdSox10在心脏中表达量高。
SoxE亚族的五个成员在大鳞副泥鳅性腺和胚胎中的原位杂交试验结果与泥鳅的一致。
3.大鳞副泥鳅DMRT1基因的选择性剪接和时空表达模式分析
本研究根据其它物种DMRT1基因DM保守区序列,设计一对简并引物,并在此基础上采用RACE方法获得了大鳞副泥鳅DMRT1基因的cDNA全长。结果表明：在大鳞副泥鳅中DMRT1基因存在选择性剪接。大鳞副泥鳅中得到五条DMRT1基因,分别命名为PdDMRT1al(1903bp)、PdDMRT1a2(1714bp), PdDMRT1a3(S81bp)、PdDMRT1b(724bp)和PdDMRT1c(946bp),它们也都含有DM基序和polyA尾。其中PdDMRT1a1-3具有相同的开放阅读框,包含5个完整的外显子,编码267个氨基酸,其差异主要在于3'非翻译区长度和序列不同；PdDMRT1b缺失了第四个外显子3'末端的9bp和第五个外显子,编码220个氨基酸；PdDMRT1c则同时缺失了第四和第五个外显子,并利用一段长63bp的额外序列在其编码框末端充当第五个外显子,编码196个氨基酸。
半定量RT-PCR/荧光定量RT-PCR和原位杂交技术用于检测大鳞副泥鳅DMRTl基因在成体不同组织和胚胎发育不同时期的表达谱。结果表明：大鳞副泥鳅DMRT1基因只在精巢中强烈表达,而在卵巢中不表达或只有极微弱表达,这与其它物种中的研究结果相一致；在胚胎发育的各个时期大鳞副泥鳅DMRT1基因均有表达,但表达强度又各不相同,呈现先上升后下降的表达趋势。由此,推断大鳞副泥鳅的DMRT1与雄性性腺分化和发育具有明显的相关性。这些结果为阐述DMRT1在脊椎动物体内对性别决定和性别分化的作用奠定了基础。
综上所述本研究结果证明：1.两种泥鳅SoxE亚族至少由五个成员组成：Sox8a、Sox8b、Sox9a、Sox9b和Sox10；2.本实验首次发现在两种泥鳅中Sox8基因都具有两个亚型Sox8a和Sox8b,目前仅发现河豚Sox8基因具有以上两种亚型,其它物种中未见到相关报道；3.两种泥鳅中Sox9基因也都具有两个拷贝Sox9a和Sox9b,而Sox10基因只存在一个拷贝,这与部分已报道的物种一致；4.我们发现大鳞副泥鳅的DMRT1基因存在选择性剪接；5.本实验首次利用多种检测方法对各个目的基因的表达模式进行了系统研究。SoxE亚族各成员之间存在功能冗余,它们对两种泥鳅神经系统和生殖系统的分化具有重要作用；DMRT1基因可能是一个与精巢的形成和精巢的功能维持有关的性别分化基因。
We obtained the full length cDNA of SoxE from two loaches and DMRT1 of Paramisgurnus dabryanus by RACE. Semi-quantitative RT-PCR and fluorescent real-time RT-PCR were conducted to determine the expression profiles. We also examined the expression patterns of the corresponding genes in gonad and early embryonic gonads of two loaches by in situ hybridization. The major research results of this study are as follow:
1. Isolation and Expression of SoxE Genes in Misgurnus anguillicaudatus
In this article, to amplify genomic DNA of Misgurnus anguillicaudatus, degenerate primers were designed according to the conservative sequences in HMG-box of Sox% and Sox10 genes. Based on these results, the full-length cDNA of above five fragments were isolated from the brain of Misgurnus anguillicaudatus by using 5'-and 3'-rapid amplification of cDNA ends (RACE). The full-length cDNA of MaSox8a was 2555bp (GenBank accession no. GU166139), containing the 53bp 5'-untranslated region, 1212bp 3'-untranslated region [including poly(A)] and encoding a putative protein of 429 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:86-164);The full-length cDNA of MaSox8b was 1725 bp (GenBank acc. no. GU166140), containing the 85bp 5'-untranslated region,395bp 3'-untranslated region [including poly(A)] and encoding a putative protein of 414 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:89-167); The full-length cDNA of MaSox9a was 3192bp, containing the 326bp 5'-untranslated region,1408bp 3'-untranslated region [including poly(A)] and encoding a putative protein of 485 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:105-183);The full-length cDNA of MaSox9b was 1782bp, containing the 217bp 5'-untranslated region,179bp 3'-untranslated region [including poly(A)] and encoding a putative protein of 462 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:97-175); The full-length cDNA of MaSox10 was 2096bp, containing the 311bp 5'-untranslated region,312bp 3'-untranslated region [including poly(A)] and encoding a putative protein of 490 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:105-183).
Semi-quantitative RT-PCR and fluorescent real-time RT-PCR reactions were performed to analyze expression profiles using the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene as the reference. During early embryonic development, the expression of MaSox8a and MaSox8b were opposite, and both begain from the gastrulae to the yolk-sac absorption phase. MaSox9a、MaSox9b and MaSox10 all showed a notable up-regulation and highest expression in hatched stage; In adult tissues, the expression level of SoxE varied among tissues, MaSox8a and MaSox8b with stronger expression detected in brain, MaSox9a and MaSox9b with stronger expression detected in testis, MaSox10 with stronger expression detected in heart.
In site hybridization results demonstrated that SoxE genes express in premature germ cells (ogania, primary oocytes in ovaries, spermatogonias and spermatocytes in testes) of gonads during gonadal development, suggesting roles of SoxE in Misgurnus anguillicaudatu gonads development and differentiation; The zygotic expression of SoxE all occure in the middle of segmentation, and persists throughout tail-bud formed, strong positive hybrid signals could be detected in the spine. Till hatched larva, the major tissues expressing SoxE include notochord, somites and eyes.
2. Isolation and Expression of SoxE Genes in Paramisgurnus dabryanus
In this article, to amplify genomic DNA of Paramisgurnus dabryanus, degenerate primers were designed according to the conservative sequences in HMG-box of Sox8 and Sox10 genes. Based on these results, the full-length cDNA of SoxE were isolated from the brain of Paramisgurnus dabryanus by RACE. The full-length cDNA of PdSox8a was 2467bp, containing the 53bp 5'-untranslated region,1127bp 3'-untranslated region [including poly(A)] and encoding a putative protein of 428 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:86-164); The full-length cDNA of PdSox8b was 2166bp, containing the 201bp 5'-untranslated region,720bp 3'-untranslated region [including poly(A)] and encoding a putative protein of 414 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:89-167); The full-length cDNA of PdSox9a was 3232bp, containing the 329bp 5'-untranslated region,1439bp 3'-untranslated region [including poly(A)] and encoding a putative protein of 487 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa: 105-183); The full-length cDNA of PdSox9b was 1946bp, containing the 219bp 5'-untranslated region, 341bp 3'-untranslated region [including poly(A)] and encoding a putative protein of 461 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:97-175); The full-length cDNA of PdSox10 was 2053bp, containing the 312bp 5'-untranslated region,262bp 3'-untranslated region [including poly(A)] and encoding a putative protein of 492 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:106-184).
Semi-quantitative RT-PCR and fluorescent real-time RT-PCR reactions were performed to analyze expression profiles using the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene as the reference. During early embryonic development, the expression of PdSox8a、PdSox8b、PdSox9b and PdSox10 showed a downward trend from the gastrulae to the yolk-sac absorption phase, PdSox9a was opposite, with a notable up-regulation; In adult tissues, the expression level of SoxE varied among tissues, and all with strong expression detected in gonad. Moreover, PdSox8a with stronger expression detected in brain, PdSox8b、PdSox9a and PdSox9b with stronger expression detected in liver, PdSox10 with stronger expression detected in heart.
The results of in site hybridization in Paramisgurnus dabryanus were same as Misgurnus anguillicaudatus.
3. mRNA Spatio-temporal expression analysis and multiple alternative splicing of DMRT1 in Paramisgurnus dabryanus
To isolate DMRT1 gene, we amplified the DM-domain by PCR, using degenerate primers designed on the basis of the conserved region amino acid sequence of the DMRT1 genes registered in Genbank. Based on these results, the full-length cDNA of DMRT1 were cloned from Paramisgurnus dabryanus using 5'-and 3'-RACE. Five isoforms generated by alternative splicing were cloned from Paramisgurnus dabryanus, which all have the DM domain and 3'polyA tail yet.They were named PdDMRT1a1 (1903bp, encode 267aa), PdDMRT1a2(1714bp, encode 267aa), PdDMRT1a3(887bp, encode 267aa), PdDMRT1b(724bp, encode 220aa) and PdDMRT1c(946bp, encode 196aa) respectively. PdDMRT1a1,2 and 3 all have five exons, by the difference between them was only in 3'UTR. PdDMRT1b lacks 9bp of exon4 in its 3'end and the whole exon5. PdDMRT1c lacks both exon4 and exon5 but uses a 63bp region of intron sequence as its exon 4 at the 3'ends.
Several technologies, such as Semi-quantitive PCR, fluorescent quantitative RT-PCR and in situ hybridization, were used to analyse the expression pattern of DMRT1. The result revealed that the expression of PdDMRT1 was testis-specific in adults, with no or very week expression in ovary. This pattern is in line with that of other species's DMRT1. Contrary to expectations, all the DMRT1 transcripts were detectable in all embryos tested. However, take the result by and large, each isoform's expression trend was risie at first and then decline. Taken together, these results provided the basic data for elucidating DMRT1 role(s) for sex-determination and gonadal differentiation in vertebrates. The differential expression of these transcripts provides new insight into roles of alternative splicing of DMRT1 in governing sex differentiation and development of Paramisgurnus dabryanus. These results provided the basic data for elucidating DMRT1 role(s) for sex-determination and gonadal differentiation in vertebrates.
In conclusion, my study established that 1) Subgroup E, at least including five members in two loaches; 2) In this study, two distinct Sox8 genes were identified in two loaches, so far only one case reported that two duplicates of Sox8 were identified in Takifugu rubripes; 3)We also obtained two distinct Sox9 genes and one copy of Sox10 from two loaches; 4) We identified that alternative splicing have been occured in Paramisgurnus dabryanus; 5) This experiment first used a variety of methods to detect the expression patterns of each objective gene. Results indicated that SoxE have a possible functional redundancy in some domains and they are essential for the differentiation of nervous and reproductive system. DMRT1gene was relevant to the formation and function maintainance of testis.
引文
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