【作者】 张凤英；

【导师】 陈立侨；

【作者基本信息】 华东师范大学 ， 动物学， 2011， 博士

【摘要】 精子卵子成熟后,经过受精进入胚胎发育阶段。在这一过程中,卵子不仅将母体的遗传物质传递给子代,而且其细胞质中储存的母体效应因子是动物胚胎发育所需营养的主要源泉。卵巢是卵子发生的场所,因而卵巢和胚胎的发育成为发育生物学研究的重要内容。大型十足目虾蟹类由于具有较高的经济价值和营养价值,其卵巢和胚胎发育研究自然成为众多学者关注的一个热点。目前,虾蟹类的卵巢和胚胎发育研究主要集中在形态学和生理学方面,而有关发育的进程及其相关机理的研究甚少。本研究首先克隆了日本沼虾卵子发生和胚胎发育的5个相关基因,分析了这些基因的分子特征。通过研究它们在卵子发生和胚胎发育过程中的时空表达模式,探讨了这些基因在日本沼虾卵子发生和胚胎发育中的作用,以期为日本沼虾乃至甲壳类发育的分子调控机理提供有益的借鉴。本研究主要包括以下四个部分：1、日本沼虾Mago nashi和Tsunagi基因在卵子发生和胚胎发育中的表达模式采用RACE技术克隆得到日本沼虾Mago nahi和Tsunagi cDNA全长序列,分别命名为MnMago和MnTsu。试验获得了2个MnMago基因的转录本,cDNA全长分别是746bp和683bp,两个序列只是在5’-非翻译区(5’-UTR)长度不同,两者之间相差63bp,开放阅读框(ORF)和3’-非翻译区(3’-UTR)则完全相同,该ORF编码147个氨基酸。MnMago两个转录本的5’-UTR长度不同,表明MnMago基因在日本沼虾体内存在不同的剪切方式。MnTsu cDNA全长为847bp,ORF长度为486bp,编码161个氨基酸。通过对这两个蛋白的序列分析发现它们分别含有Mago和Tsunagi蛋白相应的功能域：Mago superfamily和RNA recognition motif (RRM)。利用蛋白重叠软件对这两个蛋白进行相互作用预测,结果表明它们能嵌合成为一个完整的蛋白复合体,提示这两个蛋白可能与其它模式生物一样也以复合体的形式参与日本沼虾发育过程。荧光定量PCR(RT-QPCR)结果表明,这两个基因在卵巢和胚胎发育过程中的表达模式完全一致。MnMago和MnTsu基因在卵巢的增殖期表达量很低。随着卵巢的发育,其表达量逐步升高,至次级卵黄发生期达到最高,而到了成熟期显著下降,至恢复期又有所升高,说明MnMago和MnTsu基因对日本沼虾卵子的发生、成熟具有调节作用。胚胎发育过程的RT-QPCR分析表明,在胚胎发育早期MnMago和MnTsu基因的表达量处于比较平稳的状态,至原蚤状幼体期急剧升高,提示这两个基因与日本沼虾胚胎后期的形态发生紧密相关。MnMago和MnTsu基因在胚胎和卵巢发育过程中表达模式的高度一致性,提示它们在胚胎和卵巢发育过程中协同发挥作用。成体组织的RT-QPCR结果显示,MnMago和MnTsu基因在肌肉中表达量最高,而在精巢中的表达量很低。在肌肉中表达量高的原因可能是肌细胞处于增殖状态,与MnMago和MnTsu参与活跃mRNA转录、翻译和核质定位有关。以上结果说明MnMago和MnTsu基因具有多样的生物学功能,同时提示它们与日本沼虾卵子形成紧密相关。2、日本沼虾Gustavus基因在卵子发生和胚胎发育中的时空表达分析Gustavus是与Vasa蛋白相互作用的一种蛋白,在生殖质的定位和生殖细胞的形成中具有重要作用。Gustavus蛋白具有SPRY和SOCS功能域,研究认为SPRY功能域与肌质网中Ca2+释放调节有关,而SOCS功能域与蛋白质降解有关。本试验从日本沼虾卵巢中克隆得到Gustavus cDNA序列(MnGus),序列全长为1099bp,其ORF长度为786bp,编码261个氨基酸。通过对其氨基酸序列功能域分析结果显示,日本沼虾MnGus具有Gustavus蛋白特有的SPRY和SOCS功能域。卵巢发育的RT-QPCR分析表明,MnGus基因在卵巢发育过程中表现为先升高后降低的表达模式,至初级卵黄发生期表达量达到最高值,这提示MnGus基因参与了卵子发生过程,而与卵子的最后成熟关系不大。胚胎发育的RT-QPCR分析表明,在卵裂期有一定表达量,至囊胚期表达量下降,到原肠期又逐渐升高,一直到蚤状幼体期达到最高值,这种逐渐上调的表达模式提示MnGus基因在日本沼虾胚胎发育后期的腹部形成中具有重要的调节作用。成体组织的RT-QPCR表明该基因在肌肉中的表达量非常高,这可能是因为MnGus蛋白中SPRY功能域参与了肌质网中Ca2+活动,并在其中起着主要的作用。3、日本沼虾泛素化结合酶Ubc9基因的时空表达及其作用泛素(ubiquitin, Ub)介导的蛋白质降解通路是蛋白质功能的主要调节者和终结者,控制着几乎所有动植物的生命活动,包括细胞增殖、分化、凋亡、DNA复制和修复、转录和蛋白质质量控制等。而泛素结合酶(ubiquitin conjugating enzyme, E2)是泛素化过程中介导泛素活化酶(activating enzyme, E1)和泛素连接酶(ligase, E3)之间的关键酶。Ubc9是其中一种结合酶,它参与类泛素化修饰(small ubiquitin-like modifier, SUMO)途径,这个途径参与转录调控和细胞周期进程。本研究克隆得到日本沼虾Ubc9 cDNA全长序列(MnUbc9),其ORF长度为483bp,编码160个氨基酸。经生物信息学分析发现,MnUbc9蛋白结构中具有泛素结合酶的UBC功能域和泛素连接酶的结合位点。RT-QPCR结果表明,除在血液和精巢中表达量较低外,MnUbc9基因广泛分布于其它组织。随着卵巢的发育,MnUbc9基因表达量逐渐增强,至次级卵黄发生期达到最高值,而后显著下降,这与日本沼虾卵母细胞增殖、成熟过程是高度一致的。MnUbc9基因在胚胎的卵裂期有一定的表达量,至囊胚期下降,而后又逐渐升高,到胚胎发育后期达到最高。这可能是因为随着胚胎体积的增大,细胞有丝分裂活动逐渐增强,因而MnUbc9基因表达量逐渐升高。综上所述,结合酶MnUbc9基因通过类泛素化修饰途径调节着日本沼虾的卵子发生和胚胎发育过程。4、日本沼虾MnvWD-Kazal基因在卵子发生过程中的表达分析从日本沼虾卵巢中克隆到一个具有von Willebrand factor D domain (vWD)和Kazal型两种功能域的新基因,将其命名为MnvWD-Kazal。vWD功能域存在于多种蛋白中,如血浆糖蛋白、载脂蛋白、黏液素、整联蛋白和透明带粘附素,一般具有粘附作用。Kazal功能域是Kazal型蛋白酶抑制剂的主要结构域。本研究获得的日本沼虾MnvWD-Kazal cDNA全长2713bp,其ORF长度为2574bp,编码857个氨基酸。生物信息学分析发现MnvWD-Kazal蛋白的氨基端有一个长173个氨基酸的vWD功能域,在羧基端有3个Kazal功能域。RT-QPCR结果表明,MnvWD-Kazal基因在整个日本沼虾胚胎期表达量甚微,在成体组织如肠、卵巢、心脏、胸神经节等都有一定表达量,从卵巢的增殖期开始MnvWD-Kazal基因的表达量稳步上升,至次级卵黄发生期达到最高值,提示该基因参与了日本沼虾卵细胞的成熟过程。综上所述,本研究首次克隆得到5个与日本沼虾卵子发生和胚胎发育相关基因的cDNA全长,并采用RT-QPCR技术研究了它们在日本沼虾卵巢和胚胎发育过程的时空表达模式,分析了它们在发育过程中的生物学功能,为日本沼虾的发育生物学提供了有价值的资料。由MnMago和MnTsu等蛋白组成的外显子拼接复合体可以识别转录时mRNA中提前出现的无义终止密码子,在转录的水平上调节细胞增殖活动,而MnUbc9在蛋白质水平上调节细胞的有丝分裂和减数分裂活动。虽然迄今尚没有足够的证据表明MnvWD-Kazal与日本沼虾胚胎发育直接相关,但与其它4个基因相同的是在卵细胞形成和增殖过程中具有重要作用。通过对这几个基因分子特征和RT-QPCR表达的研究,为日本沼虾等十足类的卵子发生、胚胎发育等生殖生理过程提供更为全面的参考资料。尤其是新基因MnvWD-Kazal的发现为甲壳类卵细胞成熟的调节机理研究提供了新的契机。综上所述,本研究为日本沼虾胚胎发育和卵子发生分子调控机制提供了线索,结果可为日本沼虾苗种种质改良提供必备的基础。同时,本研究为虾蟹类的发育机理研究提供有益的借鉴,并进一步丰富了虾蟹类的发育生物学资料。更多还原

【Abstract】 Sexual gametes combine to form fertilized eggs, which develop to adult through embryonic development. Oocytes play an important role in the process because they carry the genetic information and nutritious material. Oocytes are formed in the ovary. So ovary and embryonic development is the core of developmental biology research. Because large-scale shrimps and crabs have high economic and nutritional value, their developmental research has naturally been concerned by many scientists. However, researchers tend to focus on the morphology and physiology in recent years, and the molecular mechanisms on development has been little studied. In this study, five genes related to the development of oogenesis and embryos were cloned from M. nipponense. At the same time their molecular characterization and expression patterning in the process of ovary and embryo development were analyzed. We further discussed their role on oogenesis and embryonic development of M. nipponense. This study provides a useful reference for molecular mechanism of development of M. nipponense as well as crustaceans. This study includes the four parts as follows:1. Molecular cloning, characterization and expression of Mago nashi and Tsunagi in M. nipponenseMago nashi and Tsunagi genes, named as MnMago and MnTsu, were cloned from M. nipponense according to homogenous cloning and the established EST information using rapid amplification of cDNA ends (RACE). Two transcripts of Mago nashi were isolated from M. nipponense with the size of 746bp and 683bp respectively. It was found that the same open reading frame (ORF) and 3’-untranslated region (3’-UTR) but different 5’-UTR in the two cDNA sequences encoding 147 amino acids. The ORF of MnTsu cDNA was 486bp, encoding a protein of 161 amino acids. The Mago domain and RNA recognition motif (RRM) were detected in MnMago and MnTsu proteins respectively based on the motif scan at http://hits.isb-sib.ch/cgi-bin/motifscan and Blast p at http://blast.ncbi.nlm.nih.gov/Blast.cgi. Three-dimensional structure prediction of interaction between MnMago and MnTsu proteins showed that they apparently constituted a Mago-Tsunagi complex, which indicated that they may play a biological role in M. nipponense as in other model organisms. Real-time quantitative PCR (RT-QPCR) analyses revealed that the expression patterns of MnMago and MnTsu gene in the process of embryo and ovary development were consistent completely. In the early phase of embryo, their expression level kept steady, and increased substantially in the later, which suggested that MnMago and MnTsu were related to morphogenesis of later embryonic development. In the development of ovary, the expression level of MnMago and MnTsu gradually increased from the perinucleolus (PN) stage to the yolk granule (YG), and then decreased suddenly in the maturation stage (MA). However, the expression patterns of two genes in mature tissues were a little uniform. The maximum level of MnMago and MnTsu occurred in muscle tissue which indicated that the muscle cell may be in active proliferation. Interestingly, their expression levels in testis were both very low. These suggested that MnMago and MnTsu not only played critical roles in oocyte maturation but also performed multiple biological functions in oriental river prawn.2. Cloning, characterization and expression of gustavus in the development of M. nipponenseThe protein of the gustavus (GUS) gene contains a SPRY domain and a SOCS box. The SPRY domain is thought to mediate Ca2+release from the sarcoplasmic reticulum, and the SOCS box belongs to the suppressor of cytokines signaling family (SOCS) and functions on proteasomal degradation. In this study, the GUS gene was firstly indentified and termed as MnGus in oriental river prawn M. nipponense. Bioinformatics analyses showed that this gene encodes a protein of 261 amino acids with a predicted molecular mass of 29.70 kDa. Phylogenetic analysis showed that MnGus protein was clustered into the clade of SSB-1 in other species. Real-time quantitative PCR (RT-QPCR) analyses revealed that the expression level varied significantly in the process of embryo and ovary development and changed substantially in other tissues. In embryos, the expression level of MnGus was slightly higher in the cleavage stage (CS) than in the blastula stage (BS). Following an increase in the blastula stage (BS), the MnGus reached a maximum in the zoea stage (ZS). In the ovary, the minimum expression of MnGus occurred in the perinucleolus (PN) stage, while the maximum was in the oil globule (OG) stage. The MnGus gradually decreased from the yolk granule (YG) stage to the paracmasis (PM) stage. The expression level of MnGus in muscles was much higher than that in other tissues in a mature prawn. The differential expressions of MnGus in the embryo, ovary and other tissues suggest that the gustavus gene performs multiple biological functions in oriental river prawn.3. cDNA cloning and expression of Ubc9 in the developing embryo and ovary of M. nipponenseUbiquitin-mediated protein degradation pathway is the main regulator of protein function and the terminator. In the same way the small ubiquitin-like modifier (SUMO) pathway in eukaryotes is an essential biological process involving cellular processes, development and organelle biogenesis. In a sequential enzymatic action, Ubc9 is an important conjunction enzyme in the SUMO pathway. Although the Ubc9 has been found in vertebrates, its expression in crustaceans is little known. In this study, the Ubc9 was identified in the embryo and ovary of a freshwater prawn M. nipponense for the first time and it was denoted as MnUbc9. Bioinformatics analyses showed that this gene encodes a protein of 160 amino acids with predicted molecular mass of 18.32 kDa. Real-time quantitative PCR analyses demonstrated that the expression levels varied significantly in the developing embryo and ovary. In the embryo, the expression level of MnUbc9 was higher at the cleavage stage (CS) than at the blastula stage (BS), and reached even higher levels at the protozoea stage (PS) and the zoea stage (ZS). In the ovary, the MuUbc9 expression was low at the early stage, but reached the highest at the yolk granule stage (YG), and then abruptly declined at the maturation stage (MA). The differential expressions of MnUbc9 in the embryo and ovary suggest that MnUbc9 may play an important role in embryogenesis and oogenesis of M. nipponense.4. Molecular cloning, characterization and expression of MnvWD-Kazal gene in M. nipponenseA new gene, named as MnvWD-Kazal, containing the von Willebrand factor D (vWD) and Kazal-type domain was cloned from the ovary of M. nipponense. The full length of MnvWD-Kazal cDNA was 2713bp with the ORF of 2574bp encoding a protein of 857 amino acids. Bioinformatics analyses showed that there were a vWD domain at amino terminal and three Kazal domains at carboxyl terminal of MnvWD-Kazal protein. Real-time quantitative PCR (RT-QPCR) analysis revealed that the expression level of MnvWD-Kazal was very low in the process of embryo, but a regular expression in ovary development. The level of MnvWD-Kazal gene increased gradually from perinucleolus stage to yolk granule stage, and decreased suddenly at maturation. This suggested that MnvWD-Kazal may participate in the process of oocyte maturation of M. nipponense. RT-QPCR analysis of mature tissues showed that the high level MnvWD-Kazal transcript occurred in these tissues, such as intestines, thoracicganglia, yolk granule and heart, especially in intestines. The high expression level in intestines may be related to Kazal domain because the proteinase inhibitor with Kazal domain can restrain prematurity of insulin-activated enzyme zymogen.This is the first report that five genes related to oogenesis and embryo development were cloned from M. nipponense, and their spatio-temporal expression patterns were studied in the development of embryo and ovary by RT-QPCR technique. The two genes of MnGus and MnUbc9 participated in the whole process of embryonic development, while the other two genes of MnMago and MnTsu were extremely relevant to late morphogenesis of embryo. The five genes were all involved in formation and proliferation of oocytes. However the new gene, MnvWD-Kazal, was little to do with the embryo development of M. nipponense. In conclusion, this research not only provided clues to molecular mechanism of oogenesis and a theoretical basis for seedling quality improvement from M. nipponense, but also enriched the developmental biological resources of crustacean.更多还原