Aj-vasa全长cDNA的克隆及其在仿刺参（Apostichopus japonicus）生殖系中的发育表达图式
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摘要
生殖细胞的发生是发育生物学研究的重要领域。vasa基因在许多动物生殖系细胞中具有表达特异性,被广泛用作生殖细胞的分子标记物,在动物生殖细胞发生和生殖调控等研究中起到了重要的作用。仿刺参(Apostichopus japonicus)俗称刺参,是我国主要的棘皮动物经济种类,然而迄今尚未见对其生殖系起源和性腺发生的研究报道。本文采用同源克隆策略和cDNA末端快速扩增(RACE)技术,克隆了刺参Aj-vasa全长cDNA序列,对其进行了序列特征和时空表达分析,并以Aj-vasa mRNA为标记,研究了刺参原生殖细胞的起源;进一步采用组织学技术,观察了刺参性腺的早期发生。
克隆得到的Aj-vasa基因cDNA序列全长2167 bp,含有开放阅读框1593 bp,编码530个氨基酸。5′非翻译区(UTR)为449 bp,3′UTR为125 bp。推导的氨基酸序列具有DEAD-box家族蛋白的全部9个保守结构域以及GG重复序列。同时在该基因的蛋白序列中还发现了ARKF框,该框只在DEAD-box家族VASA和PL10蛋白中保守存在,而在其他亚家族中并未发现。同源性比对和进化分析显示该基因编码蛋白与其他物种的VASA相关蛋白具有较高的相似性,证明VASA蛋白在进化上高度保守。蛋白三维结构预测蛋白功能显示该蛋白Aj-VASA具备了DEAD-box家族蛋白RNA连接,ATP酶和解旋酶的功能。
半定量RT-PCR分析显示,Aj-vasa基因在成体性腺组织中特异表达;原位杂交结果表明Aj-vasa mRNA主要在精原、精母细胞和卵母细胞的胞质中表达显著,推测该基因可能参与刺参两性配子的发生。胚胎和幼虫半定量RT-PCR分析检测到从未受精卵到耳状幼虫都含有Aj-vasa mRNA。表达量自未受精卵至囊胚期逐渐升高,原肠胚至耳状幼虫表达量逐渐降低。整体原位杂交结果显示:从未受精卵到囊胚期,Aj-vasa mRNA在各细胞胞质中均有分布,原肠胚阶段Aj-vasa阳性细胞集中在内、中胚层处,小耳状幼虫阶段阳性细胞分布在消化道及水体腔囊处,随后出现在水体腔及左右体腔处,并在樽形幼虫及五触手幼虫期分布在身体左右两侧的球状体处,到稚参期,强阳性信号前移至身体前端,到1mm幼参期,表达Aj-vasa的细胞已集中于背系膜处,这些细胞即为原始生殖细胞。
对1mm至3cm的幼参进行石蜡切片,结果显示:1～3mm幼参中,原始生殖细胞数个集群被包在背系膜处,形成性腺原基;5mm幼参性腺原基中开始出现空腔,具有了性腺雏形;至3cm幼参,性腺开始出现分支,其形态接近成体性腺形态。
Ontogenesis of germ cells is an important field in developmental biology. Up to now, vasa expression is exclusively restricted to the germ line cells in most animals, so vasa gene provides the most reliable molecular marker for studies on the germ cell development and reproductive regulation. Sea cucumber, Apostichopus japonicus is one of the most commercially important Echinodermata in China. While, the germline origin and the gonad genesis of A. japonicus haven't been reported till now. In this thesis, full-length cDNA of Aj-vasa from A. japonicus was first cloned using a homologous cloning strategy and rapid amplification of cDNA end (RACE) technology, and then the spatio-temporal expression of Aj-vasa transcripts was analysed by RT-PCR and in situ hybrization. Furthermore, Aj-vasa mRNA was used as a molecular marker to establish the origin model of sea cucumber primordial germ cells (PGCs). Finally, early genesis of A. japonicus gonad was observed by histological technology.
Aj-vasa cDNA was 2167 bp in length, comprising an open reading frame (ORF) of 1593 bp encoding 530 amino acids, a 5' untranslated region (UTR) of 449 bp and a 3' UTR of 125 bp. The putative amino acid sequence of Aj-VASA shared all the nine conserved motifs characteristic and GG repeats of the DEAD-box protein family. An ARKF motif, appeared in VASA and PL 10 proteins, but not in other DEAD-box helicases, was also found in the Aj-VASA protein. Homology and phylogenetic analysis showed that Aj-VASA appeared more closely related to the other members of VASA sub-familie, which indicates that this gene is highly conserved through evolution. The three-dimensional structure of Aj-VASA suggested that this protein should possess essential functions of DEAD-box proteins such as RNA binding, ATPase and helicase activities.
Semi-quantitative RT-PCR showed that Aj-vasa was specifically expressed in the gonads of adult sea cucumbers and in situ hybridization analysis revealed that Aj-vasa mRNA located abundantly in the cytoplasm of spermatogonia, spermatocytes and oocytes, which suggested that Aj-vasa could be involved in gametogenesis of A. japonicus. Aj-vasa mRNA was detected by RT-PCR from fertilized eggs to auriculariaes. The expression level increased rapidly after fertilization, and reached the peak at blastulas, then gradually decreased from gastrulaes to auricularias. Whole-mount in situ hybridization showed that Aj-vasa mRNAs were detected in each cell in early developmental stage from unfertilized egg to blastula. Cells containing Aj-vasa mRNAs were observed in endoblast and mesoblast during gastrulation. At early auricularia, Aj-vasa mRNAs were detected in the cells of alimentary tract and hydroentorocoel. These positive cells subsequently appeared at hydroeoele and somatocoel and located symmetrically at hyaline spheres at both sides of the body during doliolaria and pentactula stage. Then the signals appeared at the forside of the body and finally centralized at the dorsal mesentery at juveniles. These cells were the primodial germ cells(PGCs).
The gonad genesis was invesgated by observing serial sections of 1mm to 3 cm juveniles. The results suggested that several PGCs were embedded in the dorsal mesentery in 1-3mm juveniles, these PGCs and the dorsal mesentery composed the gonad rudiment. When A. japonicus developed to 5mm juvenile, a cavum occurred at the gonad rudiment and an embranchment appeared at 3cm juvenile. At this time, its configuration is very near to the adult's.
克隆得到的Aj-vasa基因cDNA序列全长2167 bp,含有开放阅读框1593 bp,编码530个氨基酸。5′非翻译区(UTR)为449 bp,3′UTR为125 bp。推导的氨基酸序列具有DEAD-box家族蛋白的全部9个保守结构域以及GG重复序列。同时在该基因的蛋白序列中还发现了ARKF框,该框只在DEAD-box家族VASA和PL10蛋白中保守存在,而在其他亚家族中并未发现。同源性比对和进化分析显示该基因编码蛋白与其他物种的VASA相关蛋白具有较高的相似性,证明VASA蛋白在进化上高度保守。蛋白三维结构预测蛋白功能显示该蛋白Aj-VASA具备了DEAD-box家族蛋白RNA连接,ATP酶和解旋酶的功能。
半定量RT-PCR分析显示,Aj-vasa基因在成体性腺组织中特异表达;原位杂交结果表明Aj-vasa mRNA主要在精原、精母细胞和卵母细胞的胞质中表达显著,推测该基因可能参与刺参两性配子的发生。胚胎和幼虫半定量RT-PCR分析检测到从未受精卵到耳状幼虫都含有Aj-vasa mRNA。表达量自未受精卵至囊胚期逐渐升高,原肠胚至耳状幼虫表达量逐渐降低。整体原位杂交结果显示:从未受精卵到囊胚期,Aj-vasa mRNA在各细胞胞质中均有分布,原肠胚阶段Aj-vasa阳性细胞集中在内、中胚层处,小耳状幼虫阶段阳性细胞分布在消化道及水体腔囊处,随后出现在水体腔及左右体腔处,并在樽形幼虫及五触手幼虫期分布在身体左右两侧的球状体处,到稚参期,强阳性信号前移至身体前端,到1mm幼参期,表达Aj-vasa的细胞已集中于背系膜处,这些细胞即为原始生殖细胞。
对1mm至3cm的幼参进行石蜡切片,结果显示:1～3mm幼参中,原始生殖细胞数个集群被包在背系膜处,形成性腺原基;5mm幼参性腺原基中开始出现空腔,具有了性腺雏形;至3cm幼参,性腺开始出现分支,其形态接近成体性腺形态。
Ontogenesis of germ cells is an important field in developmental biology. Up to now, vasa expression is exclusively restricted to the germ line cells in most animals, so vasa gene provides the most reliable molecular marker for studies on the germ cell development and reproductive regulation. Sea cucumber, Apostichopus japonicus is one of the most commercially important Echinodermata in China. While, the germline origin and the gonad genesis of A. japonicus haven't been reported till now. In this thesis, full-length cDNA of Aj-vasa from A. japonicus was first cloned using a homologous cloning strategy and rapid amplification of cDNA end (RACE) technology, and then the spatio-temporal expression of Aj-vasa transcripts was analysed by RT-PCR and in situ hybrization. Furthermore, Aj-vasa mRNA was used as a molecular marker to establish the origin model of sea cucumber primordial germ cells (PGCs). Finally, early genesis of A. japonicus gonad was observed by histological technology.
Aj-vasa cDNA was 2167 bp in length, comprising an open reading frame (ORF) of 1593 bp encoding 530 amino acids, a 5' untranslated region (UTR) of 449 bp and a 3' UTR of 125 bp. The putative amino acid sequence of Aj-VASA shared all the nine conserved motifs characteristic and GG repeats of the DEAD-box protein family. An ARKF motif, appeared in VASA and PL 10 proteins, but not in other DEAD-box helicases, was also found in the Aj-VASA protein. Homology and phylogenetic analysis showed that Aj-VASA appeared more closely related to the other members of VASA sub-familie, which indicates that this gene is highly conserved through evolution. The three-dimensional structure of Aj-VASA suggested that this protein should possess essential functions of DEAD-box proteins such as RNA binding, ATPase and helicase activities.
Semi-quantitative RT-PCR showed that Aj-vasa was specifically expressed in the gonads of adult sea cucumbers and in situ hybridization analysis revealed that Aj-vasa mRNA located abundantly in the cytoplasm of spermatogonia, spermatocytes and oocytes, which suggested that Aj-vasa could be involved in gametogenesis of A. japonicus. Aj-vasa mRNA was detected by RT-PCR from fertilized eggs to auriculariaes. The expression level increased rapidly after fertilization, and reached the peak at blastulas, then gradually decreased from gastrulaes to auricularias. Whole-mount in situ hybridization showed that Aj-vasa mRNAs were detected in each cell in early developmental stage from unfertilized egg to blastula. Cells containing Aj-vasa mRNAs were observed in endoblast and mesoblast during gastrulation. At early auricularia, Aj-vasa mRNAs were detected in the cells of alimentary tract and hydroentorocoel. These positive cells subsequently appeared at hydroeoele and somatocoel and located symmetrically at hyaline spheres at both sides of the body during doliolaria and pentactula stage. Then the signals appeared at the forside of the body and finally centralized at the dorsal mesentery at juveniles. These cells were the primodial germ cells(PGCs).
The gonad genesis was invesgated by observing serial sections of 1mm to 3 cm juveniles. The results suggested that several PGCs were embedded in the dorsal mesentery in 1-3mm juveniles, these PGCs and the dorsal mesentery composed the gonad rudiment. When A. japonicus developed to 5mm juvenile, a cavum occurred at the gonad rudiment and an embranchment appeared at 3cm juvenile. At this time, its configuration is very near to the adult's.
引文
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