【作者】 李诗豪；

【导师】 相建海； 李富花；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2012， 博士

【摘要】 单性化养殖在提高水产养殖动物的生产效率和养殖产量方面具有重要应用前景，其成功开展需要成熟的性别控制技术作为基础。然而由于对虾类性别决定和性别分化分子机制的了解仍处于初步阶段，因此开发有效的性控技术实现大规模养殖尚存在很大的难度。本论文通过构建对虾促雄性腺消减杂交文库和全长cDNA文库，对其性别决定和性别分化的分子机理进行了初步的探索，并尝试开发了对虾早期发育阶段性别探针，为阐述对虾性别决定和性别分化分子机制、开发有效的对虾性控技术提供了理论基础和指导。本论文的主要进展如下：1、构建的促雄性腺相关组织全长cDNA文库中，共测序得到745个unigene，对序列进行全长分析，在745条序列中，有455条序列有相关同源信息（E<1×e-5），其中全长336条，完整性比率为334/455=73.4%。利用抑制性消减杂交（SSH）技术，构建了对虾促雄性腺相关组织的消减文库，对测序成功的402个有效EST片段进行生物信息学分析，最终得到48个contig和104个singlet；选取11个差异表达基因，利用半定量RT-PCR技术对其在促雄性腺相关组织和对虾其它各组织的表达模式进行了检测，结果显示除1个基因在促雄性腺相关组织和精巢中都表达外，其余基因均特异地在促雄性腺相关组织表达，证明了我们构建的消减文库效率很高；对上述选取的11个基因进行两轮的RT-PCR检测后，筛选了1个在对虾早期发育阶段可用于鉴定性别的分子探针。为了进一步研究感兴趣基因在对虾中的生物学功能，选取文库中获得的transformer2（FcTra-2）、sex lethal（FcSxl）、doublesex（FcDsx）、insulin-likeandrogenic gland hormone（FcIAG）和crustacean hyperglycemic hormone（FcCHH）等基因进行相关研究分析，为深入探索对虾性别决定和性别分化的分子机制奠定了基础。2、成功获得中国明对虾FcTra-2、FcSxl和FcDsx基因。通过选择性剪切，FcTra-2基因产生三种形式的成熟mRNA，它们在性腺中的表达量均显著高于其它组织；其中一种形式的FcTra-2（Fctra-2c）在卵巢中的表达量显著高于其在精巢中的表达量，并且它在对虾性腺分化之前的糠虾时期表达量急剧上升，暗示着它可能参与了对虾的性别决定过程；对FcSxl和FcDsx进行了初步的表达分析，发现它们在卵巢和精巢中的表达水平都存在明显的差异。3、克隆得到Fc-IAG基因的全长cDNA和DNA序列，并发现Fc-IAG的前体mRNA通过选择性剪切产生两种不同形式的成熟mRNA Fc-IAG1和Fc-IAG2，分析发现都具有其它甲壳动物物种中IAG的保守结构特征。进一步的研究结果显示，Fc-IAG1和Fc-IAG2可能具有不同生物学功能；研究还通过分析基因的5’侧翼序列转录因子结合位点，初步揭示了它们可能的转录表达调控机制，为后续的功能研究奠定了基础。4、分离得到中国明对虾两种不同形式的FcCHH基因序列（FcCHH1和FcCHH2），序列分析发现它们的推导氨基酸序列具有78%的相似性，但来源于基因组中两个不同的基因拷贝；与其他研究结果不同的是，FcCHH特异地在雄性对虾精荚囊内壁的上皮细胞中表达；发育过程表达分析显示FcCHH随精荚囊出现开始表达；利用原核表达技术进行了FcCHH的体外重组表达，并制备了兔源多克隆抗体，为蛋白水平揭示其功能提供了条件。更多还原

【Abstract】 Monosex culture has a great priority on increasing production efficiency andyield in shrimp aquiculture. Mature technologies on sex control are the basis forsuccessful development of monosex culture. However, it is still difficult to developsuch effective technologies to implement large-scale cultivation due to lack ofunderstanding on the molecular mechanism of sex determination and differentiation inshrimp. In the present dissertation, suppression subtractive hybridization (SSH)library and full-length cDNA library of shrimp androgenic gland related tissues(including androgenic gland and part of the spermatophore sac) were constructed andprimary studies were performed on the molecular mechanism of sex determinationand differentiation in shrimp. Molecular markers were also tentatively developed toidentify shrimp gender at early developmental stages. These data provide theoreticalbasis and guidance for illustrating the molecular mechanism of sex determination anddifferentiation and developing effective technologies on sex control in shrimp. Themain progresses are as follows:Firstly, in the full-length cDNA library,745unigenes were obtained and455ofthem had homologous annotations (E<1×e-5). Full-length analysis showed that334unigenes were completed cDNA sequences, which took part of73.4%(334/455) ofthe annotated unigenes. the SSH library and full-length cDNA library of shrimpandrogenic gland related tissues were constructed, respectively.402expressedsequence tags (ESTs) were obtained from the SSH library.48contigs and104singletswere assembled based on the ESTs.11differentially expressed transcripts wereselected to detect their expression profiles in androgenic gland and other tissues inshrimp via semi-quantitative RT-PCR. Results revealed that all selected transcriptswere exclusively expressed in the androgenic gland related tissues except for one candidate, which was also detected in testis. These results indicated an high efficiencyof the SSH library. Two round RT-PCR were carried out on the11selected candidatesand one of them was proved to be an molecular marker distinguishing shrimp genderat the early developmental stages. In order to investigate their biological function inshrimp, a serial interesting genes including transformer2(FcTra-2), sex lethal(FcSxl), doublesex (FcDsx), insulin-like androgenic gland hormone (FcIAG) andcrustacean hyperglycemic hormone (FcCHH) were further analyzed, which laid afoundation for exploring the molecular mechanism of sex determination anddifferentiation in shrimp.Secondly, Fctra-2, FcSxl and FcDsx gene were successfully obtained. Threemature variants of FcTra-2generated by alternatively spliced were isolated, all ofwhich exhibited higher expression level in gonad than in other tissues. One of theFctra-2variants (Fctra-2c) showed significantly higher expression level in ovary thanin testis, and its expression level sharply increased at mysis stage before gonaddifferentiation, which indicated that Fctra-2c might be involved in the sexdetermination process in shrimp. Transcription ananlysis revealed that FcSxl andFcDsx also displayed sexual dimorphism on the expression level.Thirdly, full length cDNA sequence and genomic DNA sequence of Fc-IAG wereobtained. Two alternatively spliced variants (Fc-IAG1and Fc-IAG2) of Fc-IAG wereisolated and they all possessed the conserved sequence structure with IAGs from othercrustacean species. Further study revealed that Fc-IAG1and Fc-IAG2should performdifferent biological function. Transcription factor binding sites were predicted on the5’-flanking sequence of FcIAG gene, which primarily revealed the possiblytranscriptional regulation mechanism and laid a foundation for gene function study.Fourthly, two isoforms of FcCHH gene (FcCHH1and FcCHH2), whosededuced amino acid sequences had a similarity of78%, were isolated fromFenneropenaeus chinensis. Sequence analysis revealed they were two different gene copies from the genomic DNA. In contrast with previous studies, FcCHH transcriptswere exclusively expressed in the endo-epithelium cells of spermatophore sac.Expression analysis during shrimp developmental stages showed that FcCHHtranscripts started to turn up along with the appearance spermatophore sac.Prokaryotic recombinant protein of FcCHH (His-CHH) was expressed in vitro and itsrabbit polyclonal antibody was prepared for investigating the gene function on theprotein level.更多还原