CLONING AND EXPRESSION ANALYSIS OF THE SEALI GENE IN MESOCENTROTUS NUDUS
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摘要: seali基因隶属于PIWI超家族, 其编码的RNA结合蛋白在生殖细胞发育过程中发挥重要作用。研究经同源比对从光棘球海胆(Mesocentrotus nudus)性腺转录组数据库中筛选得到seali基因片段, 随后通过cDNA末端快速扩增技术(Rapid amplification of cDNA ends, RACE), 获得其全长cDNA序列。光棘球海胆seali基因cDNA全长3462 bp, 其中3′UTR长度为416 bp, 5′UTR长度为180 bp, 其中3′UTR的加尾信号并非经典的AAUAAA或AUUAAA, 而是较少见的AAUACA。开放阅读框(Open Reading Frame, ORF)2862 bp, 编码954个氨基酸, 具有保守的PIWI和PAZ结构域, 多重序列比对和系统进化分析结果表明其属于Argonaute家族的PIWI亚家族成员。荧光定量PCR技术检测结果表明, Mnseali基因在光棘球海胆性腺、肠、管足和体腔细胞中均有表达, 在性腺中表达量最高。此外, Mnseali基因为母源因子, 在整个胚胎发育时期均有表达。在卵巢中, 随着卵母细胞的成熟, Mnseali的表达量逐渐升高, 而仅在成熟期的精巢中表达量显著上调。RNA原位杂交结果表明, Mnseali在光棘球海胆性腺的生殖细胞中特异表达, 是光棘球海胆生殖细胞标记基因。该研究为海胆生殖细胞发育相关的研究提供了支撑。Abstract: The seali gene belongs to the PIWI superfamily and plays an important role in germ cell development. In this study, a unigene annotated as seali was screened from the gonad transcriptome database of Mesocentrotus nudus and named Mnseali. Subsequently, the full-length Mnseali cDNA sequence was obtained by 5′ and 3′RACE (rapid amplification of cDNA ends). The full-length cDNA of Mnseali was 3462 bp, containing an 84 bp 5′UTR, a 447 bp 3′UTR and a 2862 bp open reading frame (ORF), which encodes a protein of 954 amino acids. The polyadenylation signal of the 3′UTR was not the classic “AAUAAA” or “AUUAAA” sequence but “AAUACA”. Similar to that of other species, Mnseali also had highly conserved PIWI and PAZ domains and belonged to the PIWI subfamily of the Argonaute family based on the results of domain and phylogenetic analyses. Real-time quantitative PCR (RT-qPCR) analysis revealed that Mnseali was expressed in the gonad, intestine, tubular foot and coelomic fluid, with the highest expression level in gonads. In addition, Mnseali is a maternal factor and can be detected throughout embryogenesis. The Mnseali level significantly increased along with oogenesis. However, there was no significant difference in expression in the testis before maturation, and expression peaked at Stage IV. Moreover, in situ hybridization (SISH) of tissue sections showed that Mnseali was specifically expressed in the germ cells. The current study identified a germ cell marker to further study the mechanism of germ cell development in sea urchin.
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Keywords:
- Mesocentrotus nudus /
- Seali /
- Piwi /
- Germ cells
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图 1 MnSeali蛋白结构域预测及其蛋白质三级结构预测
黑色框内为沙蚕PIWI蛋白的疏水核心区域及由此推测的MnSeali蛋白疏水核心区域
Figure 1. Prediction of the MnSeali protein domain and protein tertiary structure
The black box indicates the hydrophobic core region of the PIWI protein and the predicted MnSeali protein hydrophobic core region
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图 3 Mnseali组织表达特异性分析(以精巢为参照)
Figure 3. Mnseali expression in adult tissues (Relative fold to testis)
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图 4 Mnseali在不同性腺发育时期的动态表达分析(以恢复期为参照)
Figure 4. Dynamic expression analysis of Mnseali in different stages of gonadal (Relative fold to stage Ⅰ)
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图 5 切片原位杂交分析Mnseali在不同发育时期性腺中的表达情况
NP 营养细胞; Sperm 精细胞; oocyte 卵细胞; germ cell 生殖细胞, 对照组均为成熟期
Figure 5. SISH analyses of Mnseali transcripts at different stages of gonadal
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图 6 Mnseali在胚胎发育时期的表达分析(以受精卵为参照)
Figure 6. Expression analysis of Mnseali in embryonic samples (Relative fold to egg)
表 1 PCR所用到的引物序列
Table 1 Sequences of the primers used for PCR
引物Primer 引物序列Primer sequence (5′－3′) 用途Application seali-H-F TACAGCCTTGAGATCAGTGACCCAG 同源性核心片段克隆 seali-H-R AACTCAGCTGCTTTGGATGCGTCTC seali-RE-31F TACAGCCTTGAGATCAGTGACCCAG RACE seali-RE-32F GGATCTAGCCATGCACACAAGAGT seali-RE-33F TCAGGGAGACAGCTGCCTCTGGAGA seali-RE-51R GCGTGGTAGGGTTGTAGAAATGACG seali-RE-52R GGTCCTATGTAACGAGTCTGTGCTA seali-RE-53R GCGTGGTAGGGTTGTAGAAATGACG seali-Q-F TCTTCCTACCTCACAAACTTCCTC 荧光定量 seali-Q-R TGGTAGGGTTGTAGAAATGACGT seali-P-F GGAGCAGTCCTCTTCCTACCTCAC 原位杂交 seali-P-R TAATACGACTCACTATAGGATAGCGAGTCAGAACCACTGTCCC seali-CP-F TAATACGACTCACTATAGGGGAGCAGTCCTCTTCCTACCTCAC seali-CP-R ATAGCGAGTCAGAACCACTGTCCC 
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