摘要

雄性甲壳动物特有的内分泌器官—促雄性腺（androgenic gland，AG）—是调控雄性性别分化和性腺发育的重要器官，其分泌的胰岛素样促雄性腺素（insulin-like androgeic gland hormone，IAG）在这些过程中发挥主导作用。研究表明，位于甲壳动物眼柄中的X-器官/窦腺复合体（X-organ/sinus gland complex，XO/SG）所分泌的神经肽类激素对IAG基因的表达具有负向调控作用，而促雄性腺分泌的IAG基因则与位于精巢的受体互作，它们之间通过“眼柄-促雄性腺-精巢”的内分泌轴共同调控着甲壳动物雄性性别发育。然而，XO/SG复合体分泌的神经肽类激素对IAG基因表达的调控机制以及IAG调节精巢发育的作用途径尚不明确。在本论文中，我们以凡纳滨对虾作为研究对象，通过眼柄摘除、比较转录组、RNA干扰、DNA pull-down等实验手段，对影响IAG基因表达调控及其所影响的精巢中下游靶基因进行了筛选鉴定，以期为IAG基因的表达调控及其作用机制提供分子证据。主要研究进展如下：

（1）通过对单侧眼柄摘除前后以及LvIAG基因干扰前后的雄性凡纳滨对虾促雄性腺组织进行转录组Illumina测序和分析，分别鉴定获得了67个（其中上调26个，下调41个）和168个（其中上调78个，下调90个）差异表达基因（differentially expressed genes，DEGs）。LvIAG和部分参与性别发育过程的基因在眼柄摘除后均发生明显上调，而在RNAi后则发生明显下调。同时，蜕皮激素信号通路中的核激素受体4基因（LvHR4）在眼柄摘除后显著下调。进一步通过定量PCR检测发现LvIAG与LvHR4等蜕皮通路基因在不同蜕皮阶段均存在周期性表达变化并且表达模式存在明显的相关性。注射外源蜕皮激素20-羟基蜕皮酮（20E）后，LvIAG的表达水平在不同蜕皮时期均受到剂量依赖的下调表达。结果表明，蜕皮激素调控通路可能在“眼柄-促雄性腺”调控途径中发挥着桥梁作用。

（2）制备了LvIAG启动子区DNA序列，利用DNA Pull-down和蛋白质谱技术，获得了与LvIAG启动子区DNA具有潜在结合能力的核蛋白信息，共鉴定获得20个蛋白，其中两个具有潜在的DNA结合能力的TDP-43和PCBP3蛋白，它们在眼柄摘除后的对虾促雄性腺中发生差异表达，暗示着它们可能是LvIAG基因转录表达的调控因子。

（3）开展了单侧眼柄摘除前后以及LvIAG基因干扰前后对虾的精巢比较转录组测序分析，共分别鉴定获得267个（其中上调238个，下调29个）和97个（其中上调68个，下调29个）DEGs。功能注释及分析发现，细胞色素P450酶系和泛素化系统等相关基因在眼柄摘除后发生明显的上调表达，而在LvIAG基因干扰后部分基因表达下调，表明这些过程可能参与了IAG调控的精巢发育的过程。

通过本文的研究，初步发现了部分参与IAG基因表达调控及其对精巢发育作用相关的基因信息，为理解IAG基因的功能调控机制提供了新的认识，为后续深入开展雄性性别发育机制研究提供了重要数据基础。

关键词：凡纳滨对虾，雄性性别发育，“眼柄—促雄性腺—精巢”内分泌轴，胰岛素样促雄性腺素，蜕皮激素

Male sexual differentiation and gonad development in crustaceans is regulated by the male-specific androgenic gland (AG). A hormone secreted by the AG tissue of crustaceans named insulin-like androgenic gland hormone (IAG) plays a dominant role in regulating male differentiation and maintaining male sexuality. Studies have shown that several neurohormones secreted by X-organ/sinus gland complex (XO/SG), which locates in the eyestalk of crustacean, negatively regulate the expression of IAG while IAG secreted by AG interacts with its receptor in testis. Their effects on controlling male sex development is coordinated by the "eyestalk-androgenic gland (AG)-testis" endocrine axis. However, the mechanisms about how the neurohormones regulate the expression of IAG and how IAG promotes testis development are still unclear. In this study, eyestalk-ablation, RNA interference and DNA Pull-down techniques were preformed to identify genes related to IAG expression regulation. The data will provide new evidence in the IAG expression regulation and functioning mechanism. The major research progresses are as follows:

       (1) Through Illumina sequencing and transcriptome analysis of androgenic gland in L. vannamei after eyestalk ablation and LvIAG dsRNA injection, 67 and 168 differentially expressed genes (DEGs) were identified in AG after eyestalk ablation or  LvIAG silencing. LvIAG and some of the DEGs involved in sexual development showed up-regulated expression after eyestalk ablation and down-regulated expression after LvIAG dsRNA injection. Meanwhile, the hormone receptor 4 (LvHR4), which is the element of ecdysone signaling pathway, was significantly down-regulated after eyestalk ablation treatment. RT-qPCR further revealed the periodic variations in expression of LvIAG and LvHR4 throughout the molting cycle and the expression profiles of these two genes were negatively correlated with each other. After 20-hydroxyecdysone (20E) injection, the expression level of LvIAG was down-regulated in a dose-dependent manner at all molting stages. The results showed that ecdysone signaling pathway might function as a bridge in the "eyestalk-AG" endocrine axis.

       (2) The DNA sequence of IAG promoter region was prepared to screen for the nuclear proteins with the potential ability to bind to LvIAG promoter region by using DNA pull-down and protein mass spectrometry. Twenty proteins were identified in total and two of these proteins with potential DNA-binding ability, TDP-43 and PCBP3, expressed differentially in AG tissue after eyestalk ablation, which showed that TDP-43 and PCBP3 could work as transcription factors to regulate IAG expression.

       (3) Through Illumina sequencing and transcriptome analysis of testis in L. vannamei with eyestalk ablation and LvIAG dsRNA injection, 267 and 97 DEGs were identified in testis after eyestalk ablation and or LvIAG silencing. Based on the functional annotation and analysis, some genes in cytochrome P450 enzyme system and ubiquitylation system showed significant up-regulation after eyestalk ablation treatment while some of these genes were down-regulated after LvIAG silencing, which indicated that these genes might play important roles in testis development under the regulation of IAG.

       This study found some preliminary information about genes involved in IAG expression regulation and functional mechanism in testis development, which will provide new insights into understanding the functional and regulatory mechansims of IAG gene and lay a solid data basis for further studies in sexual development of male crustacean.

Key words：Litopenaeus vannamei; male sexual differentiation; "eyestalk-androgenic gland (AG)-testis" endocrine axis; insulin-like androgenic gland hormone; ecdysteroids

目录 摘要.................................................................................................................................................. I Abstract....................................................................................................................................... III 目录................................................................................................................................................ V 第一章 绪论............................................................................................................................... 1 1.1 国内外研究进展......................................................................................................1 1.1.1 甲壳动物性别决定研究进展........................................................................1 1.1.2 甲壳动物促雄性腺及 IAG 基因研究进展.................................................3 1.1.3 IAG 表达调控机制研究..................................................................................6 1.1.4 精巢分化发育机制.......................................................................................... 7 2.1 本研究的目的与意义........................................................................................... 8 第二章 材料与方法............................................................................................................. 10 2.2 材料与方法...............................................................................................................10 2.2.1 主要仪器与试剂.............................................................................................10 2.2.2 实验材料准备................................................................................................. 10 2.2.3 凡纳滨对虾眼柄摘除实验方法.................................................................10 2.2.4 凡纳滨对虾 LvIAG-dsRNA 设计与注射................................................... 10 2.2.5 不同蜕皮周期样品选取...............................................................................16 2.2.6 蜕皮激素 20E 注射实验方法......................................................................16 2.2.7 总 RNA 提取与 cDNA 合成.............................................................................17 2.2.8 差异基因定量验证方法与统计分析........................................................18 2.2.9 凡纳滨对虾眼柄摘除与LvIAG-dsRNA注射前后AG及精巢转录组测 序分析.............................................................................................................................19 2.2.10 LvIAG 基因启动子区 DNA Pull-down 实验方法................................24 第三章 研究结果...................................................................................................................27 3.1 转录组测序数据的质控....................................................................................27 3.2 IAG 表达的调控基因鉴定和功能分析.....................................................34凡纳滨对虾 IAG 基因的表达调控与作用机制研究 VI 3.2.1 单侧眼柄摘除实验 AG 差异基因鉴定和功能分析.............................. 34 3.2.2 LvIAG 基因敲降后 AG 差异基因鉴定和功能分析................................38 3.2.3 LvIAG 基因表达与蜕皮过程的关系.........................................................45 3.2.4 LvIAG 基因启动子区 DNA 结合蛋白的筛选和分析............................. 47 3.3 LvIAG 基因表达水平对精巢中基因表达的影响错误！未定义书签。 3.3.1 眼柄摘除实验精巢差异基因鉴定与功能分析..................................... 51 3.3.2 LvIAG 基因敲降后精巢差异基因鉴定与功能分析............................. 59 第四章 讨论............................................................................................................................. 69 4.1 凡纳滨对虾 IAG 基因的表达调控.............................................................69 4.1.1 蜕皮激素信号通路........................................................................................ 69 4.1.2 甲基法尼酯代谢通路................................................................................... 70 4.1.3 调控 IAG 表达的潜在转录因子.................................................................72 4.2 AG 组织中性别发育相关基因与 IAG 协同表达................................73 4.2.1 热休克蛋白家族.............................................................................................73 4.2.2 微小染色体维持蛋白................................................................................... 74 4.3 IAG 对精巢发育的调控作用.......................................................................... 75 4.3.1 细胞色素 P450 酶系......................................................................................75 4.3.2 泛素化系统......................................................................................................76 4.3.3 DNA 修复与表观修饰.....................................................................................78 第五章 结论与展望............................................................................................................. 80 5.1 本文结论....................................................................................................................80 5.2 研究展望....................................................................................................................80 参考文献..................................................................................................................................... 82 附录 I 主要试剂目录.........................................................................................................91 附录 II 主要仪器设备.......................................................................................................92 致谢................................................................................................................................................ 93 作者简历及攻读学位期间发表的学术论文与研究成果...............................