IOCAS-IR  > 实验海洋生物学重点实验室
牙鲆dead end基因功能的初步探究
隋玉嫘
Subtype硕士
Thesis Advisor谭训刚
2016-05-27
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline生物工程
Keyword牙鲆 原始生殖细胞 亚细胞定位 剪切体
Abstract在鱼类的生命活动中,原始生殖细胞(primordial germ cells, PGCs)不仅能够生成配子将遗传信息传递给下一代,而且也调控着性别分化,因此,原始生殖细胞相关研究对鱼类资源保护、繁殖及性别调控至关重要。牙鲆是我国北方重要的海水养殖经济鱼类,PGCs发育调控机制的研究将为其资源的保护、遗传育种及性别分化的调控提供新的思路。获得牙鲆PGC发育相关基因并对其功能进行研究是PGC应用的前提条件。本研究针对牙鲆PGC标志基因dead end (dnd)进行了克隆、表达及功能的初步探究。
克隆得到了牙鲆PGCs相关基因dnd五个不同的剪切体:dndadndbdndcdndddnde。其中dnda所编码的蛋白具有完整的6个保守的功能结构域:1个NR、1个RRM以及4个CR;而其它四个分别缺失其中1个或者多个结构域。序列比对以及系统进化分析发现牙鲆Dnd与大菱鲆的Dnd聚于一小分支。
运用RT-PCR方法分析了dnd不同剪切体在牙鲆胚胎发育过程中的和性腺发育成熟过程中的表达。结果表明在胚胎发育过程中,不同剪切体的表达模式不同,有的持续表达,有的伴随着胚胎的发育出现逐渐降低的趋势,且原位杂交结果发现dnd基因特异性表达在胚胎期的PGCs中;而在性腺发育分化过程中,dndb的表达量最低,并且在在卵巢发育的I期、IV期及精巢发育的I期都没有表达;dndc在精巢中的I期中没有表达。
通过构建dnd-GFP重组表达载体,在HeLa细胞中研究了Dnd不同的剪切体的亚细胞定位,结果显示Dnda、Dndc和Dndd定位于细胞核质中,而Dndb和Dnde定位于细胞核中,因而Dnd结构域中RRM和NR、CR1可能分别是决定蛋白细胞核、质定位的功能结构域。
构建真核重组蛋白表达载体,在HEK 293T细胞表达蛋白,利用EMSA检测 Dnd与nanos3 3’UTR结合,发现Dnda、Dndc、Dnde可以和nanos3 3’UTR结合,而Dndb、Dndd不能和nanos3 3’UTR结合。
合成dndc-dsRNAi,通过注射到2.0cm左右的牙鲆幼鱼中,探究dndc的作用,结果显示敲降dndc可以促进幼鱼的生长,可能是dndc的敲降抑制了PGC的发育,从而PGC发育所需要的物质和能量被用于生长。
Other AbstractDuring the life cycle of fish, the primordial germ cells (primordial germ cells, PGCs) not only able to reproduce through generating sperm and egg cells, but also plays an important role in the sex differentiation process. Therefore, research on fish primordial germ cell is important for fish resource protection, reproduction and sex regulation. The olive flounder (Paralichthys olivaceus) is one of the important marine economic fish, research on the PGCs developmental regulation mechanisms will provide new ideas for the resources protection, genetic breeding and regulation of sex differentiation. It is a prerequisite to get PGC development related gene and study its function for the application of PGC in flounder. In this study, flounder PGC marker gene dead ene (dnd) was cloned, and its expression and function was analysis too.  
We cloned flounder PGC related gene dnd and found there was five dnd isoforms: dnda, dndb, dndc, dndd, dnde. The code sequecne of Dnda contained the complete Dnd conserved domains: one NR, one RRM and four CR domain, while the other four missing one or more domain.Sequence alignment and phylogenetic analysis found flounder Dnd grouped particularly with turbot Dnd in a branch.
The expression pattern of dnd during flounder embryonic development of gonadal development and differentiation was analysis through RT-PCR . The results show that flounder different isoforms of dnd show different expression pattern, some dnd isofroms was continuously expressed and the expression of some dnd isofroms decreased during embryonic development.And in situ hybridization indicated that  all dnd was expressed specifically in the PGCs during embryonic development. During gonad development and differentiation process, the expression of dndb was lower than other isoformsand dndb wasn’t expressed in the I phase and IV phase of ovaries development and I phase of testis development. The expression of dndc was not expressed in the I phase of testis. 
Dnd-GFP, recombinant expression vector, was constructed and expressed in HeLa cell to test the subcellular localization of different dnd isoforms. Dnda, Dndc and Dndd located in the nucleus and cytoplasms, whereas Dndb and Dnde were only expressed in the nucleus. Thus, RRM, and NR and CR1 domain of Dnd might be nuclear localization and cytoplasmic localization signal peptide, respectively.
Eukaryotic recombinant protein expression vectors were constructed and used to express Dnd different isoforms in HEK 293T cells. EMSA was used to detect Dnd binding to nanos3 3'UTR. Dnda,Dndc,Dnde can nanos3 3’UTR,while Dndb andDndd can not .
dndc-dsRNAi was synthesized and injected in juvenile flounder(about 2.0cm) and the function of dndc was explored. Results showed knockdown dndc could promote the growth of juvenile fish. It might be that knockdown dndc inhibited PGC development, and the material and energy for PGC were used for growth.
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/116983
Collection实验海洋生物学重点实验室
Affiliation中国科学院海洋研究所
Recommended Citation
GB/T 7714
隋玉嫘. 牙鲆dead end基因功能的初步探究[D]. 北京. 中国科学院大学,2016.
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