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牙鲆dead end基因功能的初步探究
Thesis Advisor谭训刚
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。
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。
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.
Document Type学位论文
First Author AffilicationInstitute of Oceanology, Chinese Academy of Sciences
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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