摘要
半滑舌鳎(Cynoglossus semilaevis)是一个优良的高效海水养殖品种。但半滑舌鳎雌、雄个体差异巨大,并且雄性个体生殖能力弱,因而导致繁殖能力差。因此开展性别调控,提高受精效率为主的繁殖生物学研究对于提高半滑舌鳎的养殖效率,增加养殖产量和经济效益,都有重要意义。近几年来,有关半滑舌鳎繁殖生物学的研究已全面展开,但仅限于组织学和胚胎学的研究,在基因水平上研究还未见报到,因此研究半滑舌鳎雌雄性腺的差异表达基因谱不仅对于研究造成其雌、雄发育差异的机制,研究鱼类性别决定和分化的过程有着重要的理论意义而且有利于探索半滑舌鳎性别调控以及提高受精效率的新方法。本文采用SSH的方法,建立了半滑舌鳎成体鱼卵巢与精巢的差减cDNA文库,通过筛选鉴定与测序获得了41个性腺差异表达的非重复克隆,进而通过RACE技术克隆了其中5个基因,并对其进行了序列特征和时空表达分析。此外还通过同源克隆的方法克隆了在精巢中特异表达的DMRT1基因,并对其进行了组织表达分析。
以成体半滑舌鳎卵巢作为检测子(tester),成体鱼的精巢作为驱赶子(driver),分别提取总RNA,逆转录合成双链cDNA。经两轮差减杂交,两轮抑制性PCR扩增后,取差减的PCR产物与T载体连接,构建抑制差减cDNA文库,随机挑取2000余个克隆,对其中1000多个克隆进行PCR扩增鉴定,发现近600个克隆中均含有插入片段,大小在250-2000bp之间,进一步对含有插入片段的近600个克隆进行了斑点杂交筛选,得到196个性腺差异表达的阳性克隆,经测序共获得166阳性克隆的结果,共41个非重复的克隆,其中已知基因20个未知基因21个。
利用RACE技术成功克隆到了半滑舌鳎的zp3a、Aquaporin1o、survivin、PABP和RacGAP1这5个与生殖相关的基因,这些基因在半滑舌鳎中都是首次被克隆鉴别。ZP3蛋白是卵膜的组成成分之一,其在卵子形成和受精中起到重要作用。在差减文库中筛选并克隆了一ZP3亚家族的成员,命名为zp3a,该亚家族中的另外一成员在本实验构建的全长cDNA文库中获得,命名为zp3b。两者的cDNA全长分别为1638bp和1135bp,开放阅读框分别编码518和312个氨基酸的蛋白质。序列比对及系统进化分析均表明两者属于ZP3亚家族。RT-PCR分析表明,二者在卵巢中大量表达,同时也在其它一些雌性的组织中有低量表达,有意思的是zp3b mRNA也在精巢和雄性的肾脏中被检测到。Aquaporin1o主要在卵巢中表达,与海水鱼类卵子成熟吸水排卵有关。在差减cDNA文库中克隆了半滑舌鳎的Aquaporin1o基因,同时通过同源克隆的方法克隆了非卵巢特异表达的Aquaporin1基因,二者的cDNA全长分别为993bp和1335bp,开放阅读框分别编码262和261个氨基酸的蛋白质。RT-PCR分析表明Aquaporin1o仅在卵巢中大量表达在雌性的肾脏中微量表达,Aquaporin1在雌性和雄性的多种组织中均有表达。Survivin是一种细胞凋亡抑制因子,其在卵子形成和胚胎发育中具有重要作用。克隆了半滑舌鳎的survivin基因,序列分析表明该基因cDNA全长704bp,开放阅读框编码147个氨基酸的蛋白质,该蛋白包含一个BIR结构域。RT-PCR分析表明该基因主要在卵巢中表达,随着胚胎发育表达逐渐减少,这可能表明该基因在半滑舌鳎卵子形成和早期胚胎发育中起到重要作用。ePABP是特异的在卵巢和早期胚胎表达的Poly(A)结合蛋白。从差减文库中克隆了一PABP基因,序列分析及RT-PCR分析组织及胚胎发育时期的表达,初步认为该基因可能为与其它物种的ePABP2基因同源。RacGAP1是小G蛋白Rac重要的调控因子,在调节细胞骨架,细胞移动,细胞周期调控以及细胞凋亡方面起重要作用。从差减文库中克隆了一主要在卵巢中表达的RacGAP1基因,这是第一次发现主要在卵巢中表达的RacGAP1基因,组织表达及胚胎发育的表达分析,可能暗示该基因在卵子形成和胚胎发育中起重要作用。
利用同源克隆的方法克隆了半滑舌鳎的DMRT1基因,该基因cDNA序列全长1232bp,编码258个氨基酸的蛋白质。RT-PCR分析表明,DMRT1基因在半滑舌鳎早期胚胎不同发育时期和孵化后5天、13天、18天、22天和35天的仔鱼均有表达,在孵化后22天表达量最高。在成体鱼中只在雄性精巢中有特异表达,其他组织均无表达,表明该基因可能参与半滑舌鳎雄性性腺的发育或精子的形成。
Half-smooth tongue sole (Cynoglossus semilaevis) is a rare marine flatfish and one of the most valuable fishes in Chinese coastal. In nature, the size difference between females and males of half-smooth tongue sole is great. It is very important to search method for sex control and improving the efficiency of fertilization because of weak reproduction of male. Recent years, the research of reproduction in half-smooth tongue sole has carried out in histology and embryology, but no report about gene profile. Therefore, identification of differentially expressed genes in gonad of half-smooth tongue is not only help to understand the different development mechanism but also help to look for new method for sex control and improving the efficiency of fertilization. In this paper, a subtracted cDNA library was constructed between ovary and testis by SSH. A total of 41 nonredundant clones were identified, from which 5 genes were cloned by RACE method, followed by analysis of characterization and spatio-temporal expression. In addition, the DMRT1 gene was cloned using a homologous cloning strategy and its tissues expression was analysed.
Using suppression subtractive hybridization (SSH), a subtractive cDNA library was constructed with the ovary as tester and testis as driver. The total RNA was isolated from tester and driver respectively and double-strand cDNA molecules were synthesized. After two times of subtractive hybridization and then two times of PCR amplification, the forward subtracted PCR products were ligated with T vector (pMD18T) and the subtractive cDNA library construction was finished. 1000 clones of the total 2000 clones were detected by nest PCR, and about 600 clones contained the inserted fragments with the length between 250bp and 2000bp. Dot blot was performed on the nest PCR products of the 600 clones hybridized with the Dig-High Prime labeled probes and 196 clones were obtained as positive, which were analyzed by sequencing, and 166 generated adequate sequencing results. Comparison of the obtained cDNA sequences against GenBank databases resulted in the identification of 41 nonredundant cDNAs, of which 20 corresponded to known genes and 21 to unkown genes.
Five reproduction-related genes including zp3a, Aquaporin1o, survivin, PABP and RacGAP1 were colned using RACE method. All these genes were first identified in half-smooth tongue sole. ZP3 protein is one of Zona pellucida (ZP) proteins and it play important role in oogenesis and fertilization. Two zp3 gene were cloned from a subtractive cDNA library and a full-length cDNA library, named zp3a and zp3b. zp3a and zp3b cDNAs were 1638bp and 1135bp. The open reading frames(ORFs) encode proteins of 518 and 312 amino acids. The two genes belong to ZP3 sub-family by characterization analysis and phylogenetic analysis. They were all highly expressed in ovary and low expressed in some other tissues of female by RT-PCR analysis. Interestingly the mRNA of zp3b also was identified in testis and kidney of male. Aquaporin1o was mainly expressed in ovary and related to egg hydration. From subtractive cDNA library, aquaporin1o was cloned and aquaporin1 was cloned by homologous cloning strategy. Aquaporin1o and aquaporin1 cDNAs were 993bp and 1335bp. The open reading frames(ORFs) encode proteins of 262 and 261 amino acids. Aquaporin1o was highly expressed in ovary and slightly in kidney of female.Survivin of one kind IAPs (inhibitor of apoptosis proteins) gene was cloned in Cynoglossus semilaevis. Sequence analysis revealed that the full-length cDNA was 704bp containing 444bp open reading frame (ORF). This sequence encoded 147 amino acids. RT-PCR analysis showed that the gene was highly expressed in ovary and slightly in kidney of female. In different embryo developmental stages, its expression was decline gradually . This result indicated that the survivin may play important role in oogenesis and early embryogenesis. One PABP gene was cloned from the subtractive cDNA library. It was considered that the PABP gene might be homologous to ePABP2 gene of other species by sequence analysis and expression analysis. RacGAP1 was one of the main regulator of Rac, one small G protein. One RacGAP1 was cloned and was mainly expressed in ovary. Until now, it is the first time to find one RacGAP1 that is mainly expressed in ovary. The expression pattern of different tissue and embryo development suggests that the RacGAP1 might play important role in oogenesis and embryogenesis.
The DMRT1 gene was obtained by homologous cloning in Cynoglossus semilaevis. Sequence analysis revealed that the full-length cDNA was 1232bp containing 774bp open reading frame (ORF). This sequence encoded 258 amino acids. RT-PCR analysis showed that the gene expressed in all the analyzed developmental stages, from early embryo to 35-day juveniles, but the expression reached the highest level in juveniles at 22-days after hatching. In adult fish, however, its expression was detected only in male testis. This result indicated that the DMRT1 may play important role in the gonad differentiation and maturation of male C. semilaevis.
引文
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