【作者】 冯政夫；

【导师】 张志峰；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2010， 博士

【摘要】 对动物性别决定和性腺发育相关基因的研究是发育生物学重要的研究内容之一,也是水产养殖业中性别人工控制的理论基础。Dmrt基因最早是在果蝇和线虫中发现,随后在从无脊椎动物到哺乳动物的多种物种中均可获得dmrt基因,该家族蛋白享有共同的DM保守结构域,并在一定程度上显示了功能的保守性。在脊椎动物中它们参与了诸如性别决定、性腺分化和发育以及个体发育、组织功能维持等生物学过程。本文采用同源克隆技术,从栉孔扇贝精巢中克隆了dmrt基因家族中的Cf-dmrt4-like基因的全长cDNA及其2个拼接片段Cf-dmrt4a-like和Cf-dmrt4b-like,分析了它们的序列特征；利用半定量RT-PCR,相对定量qRT-PCR技术、整体和组织原位杂交方法,分析了该基因在栉孔扇贝胚胎发育、性腺发育和各组织中的时空表达特征和规律；进一步采用RNAi技术,通过体外注射Cf-dmrt4-like dsRNA到栉孔扇贝生长期性腺中,定量PCR检测了目的基因在体内的表达。栉孔扇贝Cf-dmrt4-like基因的cDNA全长为2312 bp, ORF 1110 bp,编码369个氨基酸；其氨基酸序列具有DMRT蛋白特有的DM保守结构域,且在C端拥有DMRT4和DMRT5蛋白共有的DMA保守结构域,以及DMRT4和DMRT5蛋白特有的短保守序列(short motif 1)。序列比对和系统进化分析显示,Cf-DMRT4-LIKE蛋白与其它物种的DMRT4蛋白的相似性最高,且首先与DMRT4蛋白聚类,这一结果也说明了Cf-dmrt4-like基因在进化上的保守性。本实验还克隆获得了Cf-dmrt4a-like基因cDNA片段,长1638bp,编码143个氨基酸以及Cf-dmrt4b-like基因cDNA片段,长1641bp,编码104个氨基酸,初步分析其为Cf-dmrt4-like基因的拼接产物。半定量RT-PCR, Cf-dmrt4-like mRNA在雌性成体组织的鳃、外套膜、肾、闭壳肌、性腺及雄性鳃和性腺中表达。荧光定量qRT-PCR分析结果显示,该基因在各发育期的雌雄两性性腺中均有表达,且成熟期精巢中的表达量与其余各期精巢中的表达量呈显著性差异,约是其它时期的20倍；然而各期卵巢以及增殖期与生长期精巢中的表达量未显示显著性差异,表明该基因参与了栉孔扇贝两性性腺的发育,尤其在成熟期精巢的发育中起重要的作用。原位杂交结果显示,Cf-dmrt4-like mRNA分布在各期卵巢的卵原细胞、卵母细胞的胞质和核中,其中成熟期卵巢各期生卵细胞中的阳性信号略强于增殖期和生长期；Cf-dmrt4-like mRNA也定位于各期精巢的精原、精母和精细胞中,成熟期各期生精细胞中的阳性信号更加明显；在性腺的体细胞中未见目的基因的表达。上述结果暗示Cf-dmrt4-like基因参与了生殖细胞的分化和功能维持。胚胎和幼虫的半定量RT-PCR和荧光定量qRT-PCR结果证实,Cf-dmrt4-like基因在未受精卵、受精卵、卵裂期胚胎、各期幼虫中均有表达。未受精卵Cf-dmrt4-like基因的表达量显著高于各期胚胎的表达量,D形幼虫的表达量显著高于壳顶幼虫和附着前幼虫的表达量(p<0.05)。原位杂交结果显示,Cf-dmrt4-like基因的蓝色阳性信号主要集中在未受精卵和早期胚胎的中间部位,随着发育的进行,信号相继集中在幼虫的外套膜和鳃处,暗示该基因参与栉孔扇贝鳃和外套膜组织的早期构建,进一步因其在成体的鳃和外套膜组织中也有表达,推测其在栉孔扇贝鳃和外套膜等组织的构建和功能维持中发挥一定的作用。Cf-dmrt4-like RNA干扰及qRT-PCR结果显示,体外注射dsRNA可引起栉孔扇贝精巢中Cf-dmrt4-like基因的瞬时沉默,注射后48h时,目的基因在体内的表达量较对照组个体无显著性的差异；然而,72h时实验组表达量显著性的低于注射生理盐水的对照组。本实验我们初步建立了栉孔扇贝RNA干扰技术。由于时间的关系,相关的功能分析(如两性性腺中各期生殖细胞发育状况以及Cf-vtg的表达)尚在检测中。更多还原

【Abstract】 Study on animal sex determination-and gonad development-related genes, an important part in developmental biology, is a theoretical basis for artificial sex control in aquaculture. Dmrt gene, originally found in drosophila and nematode, was isolated in succession in many vertebrates and invertebrates. These dmrt genes in different animals share a DM conserved domain and show a functional conservation to a certain extent. They are involved in some biological processes such as sex determination, gonadal differentiation and development, ontogeny and maintaining tissue function in vertebrate.In the present study, full-length cDNA of Cf-dmrt4-like gene and two alternative splice isoforms (Cf-dmrt4a-like and Cf-dmrt4b-like) were isolated from Chlamys farreri testis using a homologous cloning strategy, followed by analysis of sequence characteristic and spatio-temporal expression with methods of RT-PCR, qRT-PCR, in situ hybridization (ISH). To detect the function of this gene, RNA interference (RNAi) was conducted by injecting dsRNA of Cf-dmrt4-like gene into testes at the growing stage, and then mRNA levels were determined by qRT-PCR.cDNA of Cf-dmrt4-like gene is 2312 bp long containing an open reading frame (ORF) of 1110 bp that encodes the protein of 369 amino acids. The amino acid sequence shares the DM domain conserved in DMRT proteins, and contains both the DMA conserved domain and the characteristic short conserved domain (short motif 1), sharing with DMRT4 and DMRT5 proteins at C end. Sequence comparation and phylogenetic analysis showed that Cf-DMRT4-LIKE protein was most similar to other DMRT4 and appeared more closely related to DMRT4 protein, indicating that Cf-dmrt4-like gene is highly conserved through evolution. The Cf-dmrt4a-like isoform includes 1638bp encoding 143 amino acids, and Cf-dmrt4b-like isoform includes 1641 bp encoding 104 amino acids, which were preliminary speculated as the alternative splice isoforms.Cf-dmrt4-like gene was expressed in the gill, mantle, kidney, adductor muscle, and gonad of adult female, and the gill and gonad of adult male detected by RT-PCR. The real time qRT-PCR also showed a widely expression of Cf-dmrt4-like gene during the gonadal development cycle both in male and female. The expression level in mature testis was 20-time higher significantly than those in other stages, but no significant difference was observed between testes at other development stages, which showed similar express level with all ovary stages. These results reveal that this gene is involved in the gonadal development both of male and female and especially play an important role in the mature of testis. The ISH assay showed that Cf-dmrt4-like mRNA was distributed in the cytoplasm and nucleus of oogonia and oocytes during ovary development, and the positive signal appeared stronger in mature ovary than in proliferative and growing stages. During testis development, Cf-dmrt4-like mRNA was located in spermatogonia, spermatocytes and spermatids, and the signal was stronger in germ cells at the mature stage. No signal was found in somatic cells of the gonad. Above results imply that Cf-dmrt4-like gene is involved in differentiation and functional maintenance of germ cells.The semi-quantitative RT-PCR assay showed that Cf-dmrt4-like mRNA was distributed in unfertilized eggs, fertilized eggs, cleavage embryos and all larva stages. The expression level in unfertilized eggs was higher than those in other embryo stages, and the level in D-shaped larva higher than those in umbo larva and creeping larva (p <0.05). Results of ISH showed that the blue positive signal of Cf-dmrt4-like mRNA mainly appeared in the center of unfertilized eggs and early embryos, and migrated to the gill and mantle of larvae with the embryogenesis, which suggests this gene is involved in early establishment of gill and mantle. It is supposed that this gene also play a role in the functional maintenance of some tissues including gill and mantle due to the expression in adult tissues.The RNAi and qRT-PCR showed that injection of dsRNA can instantly silence the expression of Cf-dmrt4-like gene. The expression level of target gene in dsRNA treatment was same as the control at 48 h post injection, but was significantly lower than the control at 72 h post injection. In this study, a RNAi method for scallop was preliminary established. Further functional analysis including morphological observation of germ cell development in gonads and the expression variety of Cf-vtg are being performed.更多还原