Inad-like基因在黄颡鱼性别决定和精巢发育过程中的功能探究

Identification of sex chromosome-linked DNA markers is very useful for studying the sex-determination mechanism and sex-control breeding in fish species. In our previous work, we have identified a Y-linked marker Pf62-Y in yellow catfish and its linked gene Inad-like that is specially expressed in the testis. The Inad-like mutated yellow catfish has been successfully created by CRISPR/Cas9 technology. However, the expression regulation and physiological function of Inad-like are unknown. In this project, firstly, we will investigate whether Inad-like gene mutation results in sex reversal of XY yellow catfish and defects of testis development, and determine whether Inad-like transgene leads to sex reversal of XX yellow catfish. The physiological functions of Inad-like during sex-determination and testis development will be demonstrated. Further, we will investigate the temporal and spatial expression patterns of Inad-like and the regulation of its flanking sequences, thus providing a clue how Inad-like was regulated during the male development. In order to study the molecular mechanism that Inad-like is involved, comparative transcriptome and immunoprecipitation in combination with mass spectrometry will be performed to reveal downstream genes and signaling pathways of Inad-like in fish sex determination and testis development. If this project is supported, the data will be made to clarify the genetic basis of sex determination in yellow ctafish.

性染色体连锁DNA 标记的鉴定对于研究鱼类的性别决定机制和性控育种都有重要的意义。我们前期鉴定出黄颡鱼Y 染色体标记Pf62-Y 的连锁基因Inad-like，发现该基因特异在精巢组织中表达，并运用CRISPR/Cas9技术构建了黄颡鱼Inad-like基因的突变体。然而，该基因的表达调控和生理功能还不清楚。本项目将首先研究Inad-like基因突变是否造成XY雄性黄颡鱼逆转为雌性或影响精巢发育，以及Inad-like转基因能否诱导XX黄颡鱼向雄性逆转，揭示Inad-like在黄颡鱼性别决定和精巢发育中的生理功能。进而研究Inad-like的时空表达模式和侧翼序列的调控方式，揭示Inad-like的表达调控机制。最后运用比较转录组和免疫沉淀结合质谱等分析，解析Inad-like调控的下游分子信号并揭示其发挥的作用机制。本项目的实施将有助于阐明黄颡鱼性别决定的遗传基础。

黄颡鱼是我国重要的经济鱼类之一，雄性个体比雌性个体生长快，性别连锁分子标记Pf62-Y和Pf62-X已经运用于全雄黄颡鱼的可持续生产，但黄颡鱼的性别决定分子机制还不清楚。本研究构建了一个高质量的YY黄颡鱼BAC DNA文库，从中筛选出含有性别标记Pf62-Y的BAC阳性克隆。结合黄颡鱼性腺转录组数据库，我们从获得的BAC序列中分析预测得到一个含有PDZ结构域的转录本，通过基因组共线性分析和蛋白结构分析显示其与其它含有PDZ结构的蛋白区别较大，所以将其命名为pfpdz1基因（本项目中名称为inad-like，最终命名pfpdz1）。利用组织半定量PCR、制备anti-Pfpdz1多克隆抗体及western blot分析来研究其表达模式，结果显示pfpdz1特异表达于精巢组织。免疫组化结果显示Pfpdz1蛋白在早期表达于精原细胞和体细胞，而在成熟精巢组织中主要分布于精原细胞、初级和次级精母细胞。为进一步研究其功能，我们构建了Tol2-pfpdz1转基因载体进行过表达分析，结果显示转基因XX鱼的性腺呈现雌性向雄性逆转现象；通过Crispr/Cas9系统对pfpdz1基因进行了敲除实验，结果显示pfpdz1 XY-mutated个体的性腺呈现由雄性向雌性逆转的趋势。综上，我们认为pfpdz1基因对于黄颡鱼雄性性别的分化和维持起重要作用。. 同时，我们利用 CRISPR/Cas9 基因编辑技术分别构建了amh和dmrt1基因的斑马鱼突变体，以探究它们在鱼类生殖细胞发育和性别分化中的作用。amh和dmrt1突变体中均出现了雌鱼比例显著升高的现象，说明amh和dmrt1对于斑马鱼正常的性别分化是至关重要的。研究结果表明：在斑马鱼中，amh抑制精原细胞的过度增殖，促进生殖细胞的分化，以保证增殖和分化间的平衡；而dmrt1则负责雄性生殖细胞的自我更新、增殖及分化；且amh和dmrt1在表达上存在相互反馈调节。因此，amh和dmrt1协同调控斑马鱼雄性生殖细胞的自我更新和分化，保证正常的精巢发育及精子形成。. 本项目的开展为鱼类的性别控制育种及生殖调控提供了理论基础。