【作者】 邵明瑜；

【导师】 包振民； 张志峰；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2007， 博士

【摘要】 生殖细胞的发生是发育生物学研究的重要领域。模式动物中的深入研究正在逐步揭示生殖细胞起源和发育的分子机制。同时,功能保守基因的发现也为大量非模式动物的生殖细胞研究提供了契机。本文采用同源克隆策略,在栉孔扇贝中分离获得了生殖相关的DEAD-box家族基因中的Cf-vasa、Cf-PL10和Cf-p68以及DAZ家族基因中的Cf-boule的cDNA,对其进行了序列特征和时空表达分析,并以Cf-vasa mRNA为标记,鉴定了栉孔扇贝原生殖细胞起源的方式和时间,及其在早期幼虫发育中的迁移。DEAD-box家族基因编码一类ATP依赖的RNA解旋酶,该家族蛋白被分为VASA, PL10和p68三个亚家族。其中,vasa在许多生物中为原生殖细胞形成和配子发生所需,一些PL10相关基因(人的DBY和果蝇的bel)则参与了配子发生。从栉孔扇贝精巢中克隆得到的DEAD-box基因Cf-vasa、Cf-PL10和Cf-p68的cDNA全长分别为5578 bp、2494 bp和2950 bp,开放阅读框分别编码801、760和703个氨基酸的蛋白。Cf-VASA、Cf-PL10和Cf-p68蛋白具有DEAD-box家族共有的9个保守结构域和GG重复序列,Cf-VASA、Cf-PL10具有特有的ARKF框,Cf-VASA的N端区域存在3个特有的锌指结构(CCHC框)。系统进化分析显示3个蛋白首先与各自的亚家族成员聚类,证明栉孔扇贝Cf-vasa、Cf-PL10和Cf-p68基因在进化上高度保守。空间表达的RT-PCR分析和性腺发育周期的原位杂交结果显示,Cf-vasa在除成熟精子外的所有生殖细胞中特异表达,表明该基因可能参与栉孔扇贝两性配子的发生。胚胎和幼虫RT-PCR分析检测到受精卵至原肠胚的各期胚胎都含有Cf-vasa mRNA。整体原位杂交结果显示Cf-vasa mRNA在受精卵植物极呈高密度集中分布,并随卵裂传递至早期胚胎的特定分裂球,原肠胚中含Cf-vasa mRNA的细胞增至2个,且随之后的幼虫发育逐渐增多。根据以上结果推测,Cf-vasa mRNA可能作为母源生殖质成分参与栉孔扇贝原生殖细胞的决定形成。表达Cf-vasa的原生殖细胞形成于原肠胚时期,在担轮幼虫中对称分布于口凹两侧,并随幼虫发育迁移至D型幼虫末端后,继续沿边缘向腹面迁移。另外,RT-PCR分析检测到Cf-PL10和Cf-p68在性腺和其它成体组织中都有表达。增殖期和成熟期性腺的Cf-PL10表达水平明显强于外套膜、闭壳肌和鳃,Cf-p68在卵巢中随性腺发育成熟表达渐强,暗示这两个基因可能参与了栉孔扇贝的配子发生。boule是DAZ基因家族的一员,编码一类RNA结合蛋白,该蛋白调控减数分裂起始的功能在进化上具有保守性。栉孔扇贝Cf-boule cDNA从精巢中克隆得到,全长1879 bp,编码278个氨基酸的蛋白。Cf-BOULE蛋白具有DAZ家族特有的RNP-1和RNP-2结构域,以及1个DAZ重复序列。RT-PCR结果显示,Cf-boule在性腺、闭壳肌和外套膜中表达,性腺中的表达量明显高于后者。生长期性腺的表达水平高于增殖期,卵巢的表达强于相同发育阶段的精巢。原位杂交结果进一步表明,Cf-boule在除成熟精子外的所有生殖细胞中都有表达,推测栉孔扇贝Cf-boule可能参与调控精子发生中的减数分裂。胚胎和幼虫的RT-PCR分析检测到Cf-boule mRNA含量在受精卵至囊胚的各期胚胎中相似,原肠胚和担轮幼虫中明显增加,表明栉孔扇贝早期胚胎中的Cf-boule mRNA是母源性的,原肠胚和担轮幼虫中则有明显的合子表达。整体原位杂交结果显示,Cf-boule mRNA在受精卵至原肠胚的各期胚胎中弥散分布,与Cf-vasa mRNA的分布差异表明Cf-boule mRNA不作为生殖质成分参与栉孔扇贝原生殖细胞的形成。担轮幼虫的原生殖细胞和面盘上方少数细胞中可见Cf-boule的高水平表达,但在随后的发育中消失。Cf-boule在栉孔扇贝胚胎发育包括生殖细胞发生和分化中的作用尚待验证。更多还原

【Abstract】 Ontogenesis of germ cells is an important problem in developmental biology. The outstanding studies in model animals are revealing the molecular mechanisms of germ cell origin and development, in which some functionally conserved genes through evolution have been discovered. These genes might be useful as markers for an extensive investigation on germ cells of non-model animals. In this thesis, cDNAs of reproduction-related genes including Cf-vasa, Cf-PL10 and Cf-p68 of DEAD-box family and Cf-boule of DAZ family were isolated from Zhikong scallop Chlamys farreri using a homologous cloning strategy, followed by analysis of characterization and spatio-temporal expression. Cf-vasa mRNA was used as a molecular marker to establish the early developmental pattern of scallop primordial germ cells (PGCs) including the model and timing of PGCs origin and the migration in early larvae.DEAD-box family genes encode ATP-depended RNA helicase proteins, which are divided into VASA, PL10 and p68 sub-family by phylogenetic analysis. The vasa gene is essential both for germ cell formation and gametogenesis. Some PL10-related genes, such as human DBY and Drosophila bel, are involved in the gametogenesis. Cf-vasa, Cf-PL10 and Cf-p68 cDNAs isolated from C. farreri testis are 5578 bp, 2494 bp and 2950 bp long. The open reading frames (ORFs) encode proteins of 801, 760 and 703 amino acids. Cf-VASA, Cf-PL10 and Cf-p68 proteins share nine conserved motifs and a GG repeat with other DEAD-box members. ARKF motifs only found in VASA- and PL10-related proteins are present in Cf-PL10 and Cf-VASA, which also contains three zinc-finger CCHC motifs conserved among VASA-related proteins. Phylogenetic analysis showed that Cf-VASA, Cf-PL10 and Cf-p68 appeared more closely related to other members of their sub-families, respectively, which indicates that these genes are highly conserved through evolution. RT-PCR analysis of spatial expression and in situ hybridization experiments on developmental cycles of gonads revealed a specific expression of Cf-vasa in all germ cells except mature spermatozoids, suggesting that Cf-vasa might be involved in C. farreri gametogenesis. Cf-vasa mRNAs were detected by RT-PCR in early developmental stages from fertilized oocyte to gastrula. Whole-mount in situ hybridization showed that Cf-vasa mRNAs were localized to the vegetal pole of fertilized oocyte and transmitted to a specific blastomere during cleavages. Two cells containing Cf-vasa mRNAs were observed in gastrula, and the number of these cells increased in following stages. Based on above results, it is postulated that the C. farreri PGCs are determined at early development by maternal germ plasm containing Cf-vasa mRNAs. The putative PGCs with Cf-vasa expression form in gastrula stage, and are located symmetrically at both sides of the stomodeum in trochophore. These cells subsequently migrate to the posterior pole of D-shaped larva and then migrate towards abdomen. Moreover, expression of Cf-PL10 and Cf-p68 in gonads and other adult tissues were detected by RT-PCR. Expression level of Cf-PL10 in proliferative and mature gonads was higher than that in mantle, aductor muscle and gill. Cf-p68 expression in ovary increased along with gonad development. These suggest that Cf-PL10 and Cf-p68 might be involved in C. farreri gametogenesis.boule, a member of DAZ gene family, encodes an RNA-binding protein, which play a functionally conserved role in regulating the meiosis entry. A full length Cf-boule cDNA of 1879 bp was isolated from C. farreri testis and encodes a protein of 278 amino acids. Cf-BOULE protein shares the RNP-1, RNP-2 motifs and a DAZ repeat with other DAZ members. Cf-boule expression was detected by RT-PCR in gonads, aductor muscle and mantle, with the most abundance in gonads, where expression level in growing stage was higher than that in proliferative stage. Experiments of in situ hybridization revealed an extensive expression of Cf-boule in all germ cells except mature spermatozoids, suggesting that Cf-boule might be involved in regulating meiosis during C. farreri spermatogenesis. Similar amount of Cf-boule mRNAs in early embryos from fertilized oocyte to blastula was detected by RT-PCR, and that in gastrula and trochophore increased obviously. Above results indicate that Cf-boule mRNAs in early embryos are maternally inherited, and zygotic expression occurs in gastrula and trochophore. Whole-mount in situ hybridization showed a non-specific localization of Cf-boule mRNAs in early development from fertilized oocyte to gastrula. The distributional difference between Cf-boule and Cf-vasa mRNAs suggests that Cf-boule mRNAs are not involved in assembling germ plasm and determining PGCs fate in C. farreri. A high level expression of Cf-boule was observed in the putative PGCs and several cells above velum in trochophore, and disappeared in following stages. The role of Cf-boule in embryonic development including the origin and differentiation of C. farreri germ cells remains unknown.更多还原