【作者】 吴萍；

【导师】 陈立侨；

【作者基本信息】 华东师范大学 ， 动物学， 2009， 博士

【摘要】 生殖细胞的发生是发育生物学研究的主要内容之一,卵子在卵巢中的形成更是个体发育中极其重要的时期,也是历来备受关注的生殖生物学课题。与其它模式生物相比,十足类中关于生殖的分子机制方面的研究相对薄弱,淡水虾类尤其突出,迄今对淡水虾类遗传资源的了解极为有限,NCBI数据库中收录的信息量与对虾类相比颇为匮乏。因此,大量挖掘、获取淡水虾类生殖相关的基因资源,无疑具有重要的意义和潜在的应用价值。同时,一些功能保守基因的发现,也为遗传背景薄弱的经济物种生殖细胞的研究带来了契机。本文以日本沼虾为研究对象,通过构建卵巢cDNA文库及表达序列标签分析,鉴别了29个与生殖、发育相关的基因;随后以日本沼虾卵巢发育过程为主线,以获得的表达序列标签为基础,结合同源克隆策略,分离获得了7个与卵母细胞形成、成熟有关的基因,并对其序列特征和时空表达进行了系统地分析。研究主要包括如下四个部分:1、日本沼虾卵巢cDNA文库的构建及生殖相关基因的鉴别为了研究日本沼虾卵巢发育过程中储存的遗传信息,以卵巢为材料构建了cDNA文库,文库重组率为82.93%,库容量为2.9×10⁶,插入片段大小为0.5-3kb。随机挑取文库中3294个克隆进行5’-3’端测序,获得了3256条高质量的表达序列标签（ESTs）,拼接后得到了1514条单基因簇,其中可推测功能的序列占47.5%。据序列相似性分析,发现了组织蛋白酶B和L、细胞周期蛋白B、周期蛋白依赖性激酶、DEAD-box家族蛋白等29个与生殖、发育相关的基因。此外,在构建的文库中还发现了与皮质棒形成密切相关的围食膜因子,预测在成熟卵缺少皮质棒结构的沼虾属种类中,这种基因同样存在。通过卵巢cDNA文库的构建及ESTs分析,初步明晰了与日本沼虾卵巢发育相关的基因,为进一步研究日本沼虾乃至十足类的功能基因组提供了重要的基础资料。2、日本沼虾DExD-box家族基因的分子克隆、特征分析及时空表达根据ESTs提供的信息,结合同源克隆策略,采用RACE技术成功克隆了以vasa为代表的DExD-box家族4个基因的全长cDNA序列。从日本沼虾卵巢中克隆得到的DExD-box基因Mn-vasa、Mn-p68和Putative Mn-DDX39 cDNA全长分别为2391、2174和1774bp,开放阅读框分别为1806、1623和1299bp,编码601、540和432个氨基酸的蛋白。其中PL10基因的RNA存在选择性剪接,得到Mn-PL10A、Mn-PL10B两种转录本,两者cDNA全长分别为2277和2649bp,开放阅读框分别编码485和709个氨基酸的蛋白,前者比后者在N端缺少224个氨基酸。所推测的5种蛋白均具有DExD-box家族特有的9个保守结构域和GG重复序列,并且在Q-motif上游17个氨基酸处除Mn-PL10A外都存在一高度保守的苯丙氨酸（F）。RT-PCR的研究结果表明,Mn-vasa基因仅在日本沼虾性腺中表达,并且在卵巢发育的增殖期表达量最高,此后随着发育进程逐渐下降,至消退期下调至最低。除Mn-vasa外,DExD-box家族的另外3个基因Mn-PL10、Mn-p68、Putative Mn-DDX39在日本沼虾组织中广泛分布,但都表现出在卵巢和肝胰腺中高表达的特征。它们在卵巢发育中的表达模式基本相似,都表现为消退期降至最低、次级卵黄发生期上调至最高。对日本沼虾DExD-box家族基因的研究结果表明,Mn-PL10基因的RNA存在选择性剪接,推测其属于通过选择启动子形成不同转录本的选择性剪接方式;Mn-vasa、Mn-PL10、Mn-p68和PutativeMn-DDX39基因在结构上高度保守,这些保守结构的存在保证了其功能的实施;Mn-vasa基因的主要作用可能体现在卵原细胞的形成上,而Mn-PL10、Mn-p68和Putative Mn-DDX39则可能在日本沼虾卵母细胞的成熟过程中具有调节作用。3、日本沼虾成熟促进因子（MPF）组成亚基基因的分子克隆、特征分析及时空表达成熟促进因子（MPF）由调节亚基周期蛋白B（cyclin B）和催化亚基细胞周期蛋白依赖性激酶(p34^cdc2)构成。从日本沼虾卵巢中克隆得到的Mn-cyclin B基因和Mn-Cdc2基因cDNA全长分别为2318和1639bp,开放阅读框分别编码398和299个氨基酸的蛋白。Mn-cyclin B具有989bp偏长的3’UTR,其中集中了包括2个多聚腺苷酸加尾信号、5个胞质聚腺苷化元件（CPEs）、1个翻译调控元件（TCE）和1个K-box在内的多种调节元件。Mn-cyclin B蛋白具有周期蛋白B特有的破坏框、周期蛋白识别框和磷酸化位点。进一步的多序列比对和同源性分析表明日本沼虾Mn-cyclin B和斑节对虾、日本囊对虾、锯缘青蟹、中华绒螯蟹cyclin B的同源性分别为69%、68%、60%和60%,系统进化分析证明Mn-cyclin B基因在进化上高度保守。日本沼虾周期蛋白依赖性激酶基因高度保守,和斑节对虾、锯缘青蟹、中华绒螯蟹Cdc2的同源性分别为92%、91%和91%;Mn-Cdc2蛋白具有蛋白激酶特有的ATP特异结合位点、PSTAIR结构域和丝氨酸/苏氨酸（Ser/Thr）蛋白激酶活化位点;此外,第14、15和161个氨基酸残基分别是Thr14、Tyr15和Thr161。空间表达的实时荧光定量PCR分析表明,Mn-cyclin B在卵巢中的表达量最高,此外在精巢、肌肉、肝胰腺、表皮和血液中都有较高的表达,而在肠中的表达极其微弱。Mn-Cdc2主要在精巢、卵巢、肝胰腺和表皮内表达,其中精巢中的表达量最高,其次是卵巢。时间表达的实时荧光定量PCR分析显示,Mn-cyclin B转录本在卵巢的发育过程中呈现周期性的波动:增殖期Mn-cyclin B基因的表达量处于一相对较高的水平,卵黄发生前期有所下降,至初级卵黄发生期开始上调,至成熟期上调至最高,此后急剧下降。Mn-Cdc2基因的表达量在除次级卵黄发生期之外的其它各期都基本一致,而在次级卵黄发生期则有所上调,但与其它各期相比没有显著差异。综合所得的结果,日本沼虾cyclin B的翻译或转录后调节过程是通过CPEs、TCE和K-box等复合结构的联合作用实施的;Mn-cyclin B和Mn-Cdc2蛋白的氨基酸序列高度保守,从结构上保证了其在细胞分裂中调节功能的实施,并且直接参与了卵母细胞的减数分裂,在日本沼虾卵母细胞的成熟中起着关键的作用。4、日本沼虾围食膜因子基因的分子克隆、特征分析及表达模式以日本沼虾卵巢cDNA文库中注释为围食膜因子（Peritrophin）同源蛋白的EST为基础,采用RACE技术成功地克隆了日本沼虾Peritrophin基因全长cDNA序列。该序列全长654bp,包含一个192bp的5’UTR和一个314bp的3’UTR,开放阅读框长291bp,编码96个氨基酸。生物信息学分析发现此预测蛋白具有1个跨膜螺旋,一个CBM 14功能域,含有19个信号肽的裂解位点,具有蛋白激酶Ⅱ磷酸化位点、N-豆蔻酰化位点和蛋白激酶C磷酸化位点等修饰。RT-PCR的研究结果表明,Mn-Peritrophin基因在卵巢和肝胰腺中表达量最高,而在雄性生殖腺中的表达则极其微弱;其表达量在日本沼虾的卵巢发育中表现出初级卵黄发生期平稳、成熟期高表达的特征。研究结果提示Mn-Peritrophin基因可能参与了卵母细胞后期成熟过程中卵黄之外的物质的积累,和卵母细胞的最终成熟有关;并且Mn-Peritrophin蛋白可能是日本沼虾皮质颗粒的组成成分之一,从而推测日本沼虾成熟卵母细胞中虽然不含皮质棒的结构,却依然存在皮质颗粒,并且肝胰腺可能是Mn-Peritrophin蛋白的主要合成位点之一。通过以上研究,鉴别、挖掘了29个与日本沼虾生殖、发育相关的基因,对于日本沼虾这种遗传背景相对薄弱而经济价值又较高的淡水虾类,不仅为其繁殖生物学在分子水平的研究积累了宝贵的资料,而且为今后进一步开展与生殖相关的功能基因组的研究奠定了基础。通过对Mn-vasa基因的克隆和表达特征研究,为其成为日本沼虾潜在的原始生殖细胞形成的分子标记提供了依据。此外,对日本沼虾这种卵母细胞中缺乏皮质棒结构的真虾类却有组成皮质棒成分的Peritrophin转录本存在的发现及Mn-p68和Mn-DDX39等基因的结构和表达特征的研究,为进一步完善十足类的卵子发生、繁殖发育等理论问题提供了更为全面的参考资料。尤其对于DDX39的功能迄今尚未阐明,本研究对Mn-DDX39功能的推测更是为DDX39功能的研究开启了一扇窗户,建议今后对DDX39的功能研究可以侧重探讨其与配子发生之间的关系。综上所述,本研究为揭示日本沼虾卵子成熟调控的分子机制提供了线索,为解决一直困扰着日本沼虾养殖业的卵巢提前成熟现象提供了基础资料,也为日本沼虾的遗传改良提供了理论依据。另一方面,以基因库的形式进行了日本沼虾种质资源的保存,对于丰富淡水虾类的遗传学资源也具有一定的意义。更多还原

【Abstract】 Ontogenesis of germ cells is an important subject in developmental biology.The animal ovary is functionally important in reproduction and secretion of hormones for growth and development regulation.Unfortunately,the regulative mechanism for ovarian maturation of decapods at the molecular level is quite limited compared with some model organism,as well as in freshwater prawn.For most prawns,however,the number of expressed sequence tags（ESTs）available is inadequate.There were only 11 ESTs of Macrobrachium in GenBank,and no any EST of oriental river prawn was found.Acquiring plenty of reproduction-related genes may prompt prawn genomics and developmental biology.Currently,some functionally conserved genes through evolution have been discovered,which might be useful for an extensive investigation on germ cells of non-model animals.In this thesis,an ovary cDNA library of oriental river prawn Macrobrachium nipponense was constructed and ESTs were analyzed.29 genes related to reproduction and development were discovered.cDNAs of reproduction-related genes including DExD-box family and Mn-cyclin B,Mn-cdc2, Mn-Peritrophin were gained using a homologous cloning strategy or basis on expressed sequence tags,followed by analysis of characterization and spatio-temporal expression.There were four major parts,listed as follow:1、Gene discovery from an ovary cDNA library of Macrobrachium nipponense by ESTs annotationA high-quality cDNA library of M.nipponense was constructed from the ovary tissue.A total of 3,294 successful sequencing reactions yielded 3,256 expressed sequence tags（ESTs）longer than 100bp.The cluster and assembly analyses yielded 1,514 unique sequences including 414 contigs and 1,168 singletons.About 719 （47.5%）unique sequences were identified as orthologs of genes from other organisms. By sequence comparability analysis,29 important genes including cathepsin B, chromobox protein,Cdc2,cyclin B,DEAD box protein and ADF/Cofilin protein were expressed.These genes may be involved in reproductive and developmental functions in prawn.Peritrophin consisting of cortical rods was also found in this species.2、Cloning and expression profile of reproduction-related DExD-box family genes from M.nipponenseFour genes of DExD-box family,including Mn-vasa、Mn-PL10、Mn-p68 and Putative Mn-DDX39 were cloned using rapid amplification of cDNA ends（RACE）. Three of them such as Mn-vasa、Mn-p68 and Putative Mn-DDX39 cDNAs isolated from M.nipponense ovary with the size of 2391 bp,2174 bp,1774 bp respectively. The open reading frames（ORFs）of them are 1806 bp,1623 bp and 1299 bp,which encode 601aa,540 aa and 432 aa respectively.We first discovered that there were two alternative spliced isoforms of Mn-PL10（Mn-PL10A and Mn-PL10B）.Mn-PL10A and Mn-PL10B with the size of 2247 bp and 2649 bp,encode 485 aa and 709 aa respectively.There is 224 aa residues absent in the N-terminal in Mn-PL10A isoform. Mn-vasa,Mn-PL10A,Mn-PL10B,Mn-p68 and Putative Mn-DDX39 proteins share nine conserved motifs and a GG doublets with other DExD-box members.The conserved phe is in 17 amino acid residues upper the Q-motif except Mn-PL10A.Mn-vasa contains a zinc-finger CCHC motif conserved among vasa-related proteins.There is a RGG repeat in Mn-vasa and three RGGs in Mn-PL10B,which only found in vasa-related and PL10-related proteins.All the conserved structures ensure the RNA helicase function.RT-PCR analysis of spatial expression revealed a specific expression of Mn-vasa in gonads,suggesting that Mn-vasa might be involved in M. nipponense gametogenesis.To compare with the relative amounts of mRNA for Mn-vasa between stages during oogenesis,ovary samples from development stages were subjected to quantitative Real-time RT-PCR analysis using beta-actin as an internal reference.The level of Mn-vasa transcript is the highest at perinucleolus stage, but drops at followed development stages and reaches the lowest point at paracmasis stage.The results indicated that Mn-vasa might play a role during the oogonium formations.In addition,RT-PCR results also indicated that mainly expression in ovary and hepatopancreas of Mn-PL10,Mn-p68 and Putative Mn-DDX39,little expression in other tissues.The expression profiles of the transcripts during oogenesis are similar.The levels of Mn-PL10,Mn-p68,Putative Mn-DDX39 are the highest at yolk granule stage and reach the lowest point at paracmasis stage,which suggested that Mn-PL10,Mn-p68,Putative Mn-DDX39 might be involved in prawn oocyte maturation.This is the first report on p68 and DDX39 in crustacean and the discovery of expression profile of Mn-DDX39 during oogenesis might prompt DDX39 function research.3、Cloning and expression profile of cyclin B and Cdc2 genes from M.nipponenseThe meiotic maturation of oocyte in animals is regulated by maturation promation factor（MPF）,a complex of Cdc2 and cyclin B.In this study,full-length cDNAs of cyclin B and Cdc2 was cloned using RACE technique.The prawn cyclin B cDNA was 2318 bp containing a long 3’ untranslation region（UTR）of 989 bp and an open reading frame encoding for a protein of 398 amino acids.There are two cytoplasmic polyadenylation signals,five cytoplasmic polyadenylation elements（CPEs）,a translation control element（TCE）and a K-box motif in the 3’UTR of prawn cyclin B transcription,which suggested that the translation or post-transcription of Mn-cyclin B might be regulated cooperatively via all the present elements.The deduced amino acid sequence of Mn-cyclin B has high identity with the reported cyclin B sequences of other species in the GenBank,sharing 69%,68%,60%and 60%identity with Penaeus monodon,Marsupenaeus japonicus,Scylla serrata,Eriocheir Sinensis respectively. Several obvious sequence motifs or domains are present in the Mn-cyclin B:a cyclin family signature,the consensus sequence known as the destruction box and the amino acid residues of pkA site.Poylogenetic analysis showed that Mn-cyclin B was highly conserved through evolution.A Blast searching GenBank database revealed that the deduced amino acid sequence of the prawn Cdc2 kinase shared 92%identity with P. monodon,91%with S.serrata and 91%with E.sinensis.Mn-Cdc2 kinase include several signature domains,such as elements involved in ATP binding,catalytic domain PSTAIR,Serine/Threonine protein kinase active-site,conserved phosphorylation site Thr161,dephosphorylation sites Thr 14 and Thrl5.RT-PCR results indicated mainly expression in the ovary of Mn-cyclin B transcript,also expression in the testis,muscle,hepatopancreas,skin and hemolymph,little expression in the intestine.The expression of the prawn Cdc2 mRNA was detected mainly in the ovary,testis,hepatopancreas and skin.Quantitative Real-time RT-PCR analysis revealed that the levels of Mn-cyclin B transcript were cyclincal fluctuation during oogenesis.It has a relative high quantity at perinucleolus stage,and drops at fusion nucleolus stage,but a little increase at oil globule stage and reaches the highest point at yolk granule stage,then drops remarkably at paracmasis stage.The fluctuation characteristic is consistent with the role of the cyclin B in cell cycle regulation.The high level of Mn-cyclin B at maturation stage indicated that it is closely related to oocytes meiotic maturation in the prawn ovary.Quantitative Real-time RT-PCR analysis also revealed that the levels of Mn-Cdc2 mRNA showed no statistically significant difference during oogenesis,which is consistent with the mechanisms of Cdc2 kinase regulating cell cycle.4、Cloning and expression profile of Peritrophin genes from M.nipponensePeritrophin is one of the major components of cortical rods in the jelly layer of shrimp and is proposed to play a role in the protection of spawned eggs.At the end of vitellogenesis in penaeid shrimp,oocytes maturation was characterized by the appearance of rod-like bodies arranged radially around the periphery of the oocyte plasma membranes.When the oocytes are released into seawater,the contents of cortical rods are released,forming a jelly layer,a corona,around the egg. Nevertheless,in oriental river prawn,cortical rods were not found even in the fully matured oocytes.It is generally believed that this prawn species lacks cortical rod protein in the oocytes.Peritrophin was found only one EST homology in constructed ovary cDNA library.The full-length cDNA of Mn-Peritrophin was cloned using RACE method.It was 654 bp,including a 5’UTR of 192 bp,a 3’UTR of 314 bp and an open reading frame of 291 bp excoding a polypeptide of 96 amino acids.The protein has a transmembrane helice,a CBM₁4 domain and a cleavage site containing 19 amino acid signal peptide.In addition,it has a casein kinaseⅡphosphorylation site,a myristyl N-myristoylation site and a protein kinase C phosphorylation site too. Quantitative Real-time RT-PCR analysis revealed that mainly expression in the ovary and hepatopancreas of the Mn-Peritrophin transcript and little expression in the testis, which indicated that Mn-peritrophin has important role in oogenesis not in spermatogenesis.Further quantitative RT-PCR analysis during the oogenesis showed that the level of Mn-Peritrophin transcript was no difference at oil globule stage but was the highest at the maturation stage,which suggested Mn-Peritrophin might contribution to nutrient accumulation of the oocyte except for yolk proteins.Moreover, it occurs only at the later stage during the oogenesis.The result indicated that the ovaries might contain cortical rod protein or a homologous protein which was a chemical component of cortical rods in penaeid prawn even without the formation of cortical rods in these species.The expression profile in the tissues of the Mn-Peritrophin mRNA also indicated that hepatopancreas might be the main Peritrophin protein synthesis site.The high level of Mn-Peritrophin at maturation stage also suggested that Mn-Peritrophin played a critical role prompting oocyte maturation finally.This dissertation provides sufficient basic data for research on decapods developmental biology.The identification of EST sequences in M.nipponense would improve our understanding on the genes that regulate reproduction and development in prawn species.This study also lays the groundwork for development of molecular markers related to ovary development in other prawn species.Further study on these genes might prompt prawn genomics and developmental biology.It not only provided oriental river prawn gene sequences information for researchers who use the data from NCBI to perform character string searches of the annotations but also facilitated research in crustacean molecular genetics.更多还原