池蝶蚌Sox2 基因在不同组织及不同月龄精巢中的表达
EXPRESSION OF THE SOX2 GENE IN DIFFERENT TISSUES AND DIFFERENT MONTHS ON TESTIS OF HYRIOPSIS SCHLEGELII
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摘要: Sox 基因家族在胚胎发育过程和性别分化中起重要作用, 为研究池蝶蚌中Sox 基因的功能, 以人SRY基因HMG-box 保守区的序列设计简并引物, 以雌、雄池蝶蚌基因组DNA 和精巢cDNA 为模板进行扩增, 获得了2 个不完全相同的序列, 分别为DNA-HMG1、DNA-HMG2 和cDNA-HMG, 长度均为220 bp, 编码73个氨基酸。与人等物种Sox1、Sox2、Sox3 及Sox14 有很高的同源性, 雌雄个体之间没有序列差异性。采用RACE-PCR 扩增获得了池蝶蚌性腺Sox2 部分cDNA 片段, 长度为1774 bp, 该序列核苷酸与欧洲帽贝的SoxB和人类的Sox2 的同源性最高; 在部分开放阅读框249 个氨基酸残基中, 具有Sox 家族典型的HMG-box 结构域, 与人类、小鼠、原鸡和斑马鱼等Sox2 的HMG-box 同源性为98%。为了解该基因在各组织中的表达情况,采用实时荧光定量PCR 方法分析了外套膜、闭壳肌、鳃、肠、肝、肾、精巢和卵巢在内的8 种组织hs-Sox2的表达情况, 结果显示, hs-Sox2 基因在8 种组织中均有表达, 其中在肾脏中的表达量最高, 其次是肠与闭壳肌, 在雄性性腺中的表达量明显高于雌性性腺, 在肝脏中的表达量最低; 为了解hs-Sox2 在不同性腺发育时期的表达情况, 采用实时荧光定量PCR 方法分析了5 个不同月龄的精巢组织中hs-Sox2 的表达情况, 结果显示在39 月龄性腺的表达量最高, 其次是16 月龄性腺, 63 月龄蚌中的表达量最少。以上结果表明, hs-Sox2 基因可能参与了池蝶蚌精巢的发育及功能的维持。Abstract: The Sox genes family comprises several transcription factors that share a highly conserved HMG (High-Mobility-Group) box and has been studied in many species, including a variety of vertebrates and several of invertebrates such as fruit fly, nematode and Portunustrituberculatus. But there are only few reports on Sox gene family of freshwater bivalve so far, which have important value in evolution and production. H. schlegelii, which originated from Lake Biwa in Japan and was introduced into China in 1998, is one of the representative freshwater pearl mussels. It has been widely applied in the Chinese freshwater pearl industry for its high quality pearl bearing ability. In order to know the function of Sox genes in this mussel, a degenerate PCR, referred to the HMG box of human SRY gene, was used to amplify the conserved sequence of HMG domains of Sox genes. Two different HMG sequences were got from DNA and the testis cDNA, named DNA-HMG1, DNA-HMG2 and cDNA-HMG. Amino acids sequences analysis showed that those HMG sequences had high homology with the Sox1, Sox2, Sox3 and Sox14 genes from other animals including human being. But there showed no difference between the male and female. A partial cDNA sequence of Sox2 gene (refereed as hs-Sox2) with 1774 bp including partial ORF and complete 3' untranslated region (UTR) was cloned from the testis cDNA by RACE-PCR methods. DNA sequences analysis showed that it had high homology with the SoxB gene in Patella vulgata and the Sox2 gene in human being. The putative 249 amino acid sequence contained one conserved HMG box like the human SYR gene, and exhibited 98% homology with human, mouse, chicken and zebrafish. For further know the expression level of Sox2 gene, real-time PCR method was used to examine its mRNA level in different tissues and different months on testis of H. schlegelii. Results showed that the hs-Sox2 mRNA was ubiquitously expressed in all the tissues, with the highest in kidney, followed by intestine, adductor muscle, and the expression level in the testis was higher than that in the ovary. The result from the different months on testis revealed that hs-Sox2 was transcribed mainly in the 39 months, followed by 16 months and lowly in the 63 months. These results suggested that hs-Sox2 may be involved in the development of on testis in the H. schlegelii and may constitute clues for future work in order to better understand Sox protein.
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Keywords:
- Hyriopsis schlegelii /
- Sox2 gene /
- Gonad /
- Real time PCR /
- Expression analysis
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