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黑鲪核受体基因FTZ-F，TRα and TRβ的克隆及表达分析

作　者: Muhammad Shafi
导　师: 张全启
学　校: 中国海洋大学
专　业: 海洋生物学
关键词: Cloning Expression Sebastes schlegelii
分类号: S917.4
类　型: 博士论文
年　份: 2012年
下　载: 20次
引　用: 0次
阅　读: 论文下载

内容摘要

The Sebastes schlegelii is distributed along the coast of Japan, Korea and China. It is wellestablished that rock fish Sebastes schegelii is dominant for coastal fisheries and withcomparison to other fishes it is likely because of its fast growth and large size hence thecage culture of black rockfish （Sebastes schlegeli） have been set out and gotten intofruitful aquaculture industry since1990. Now cage farming has become major part ofmarine fish farming culture. The black rock fish is a viviparous which belongs to familyscorpaenidae. The sex determination in mammals is relatively stable and the sexdetermination and differentiation in fishes vary greatly among species. The mechanismsresponsible for the variability are still not clear. Thus sex determination research in fishwill provide important insight into the plasticity of the sex determination process invertebrates besides it is the important role of thyroid hormone receptors in fishes needednot to be ignored but meager information is known about thyroid hormone receptors forgrowth and developmental impact. In the present work we focused the FTZ-F1and thecloning of TRα and TRβ in the mature black rockfish from testis and ovary respectively.We also characterize the investigation of their expression patterns in the various tissues ofimmature and mature fish as well as in fry and juveniles in order to understand thefunction of these genes. The detailed output of classical genes in black rock fish are beingdescribed below. FTZ-F1is a transcriptional factor regulating the expression of the fushi tarazugene that belongs to a member of the orphan nuclear receptors. The sex related gene ofFTZ-F1was isolated from testis tissue of black rockfish Sebastes schlegeli by homologycloning. The full-length sequence of S. schlegeli FTZ-F1（ssFTZ-F1） contains a232bp5′UTR,1449bp ORF encoding482amino acids with an estimated molecular weight of5.4kD and a105bp3′UTR. Sequence, tissue distribution and phylogenic analysis of theFTZ-F1showed that the ssFTZ-F1belonged to FTZ group, with highly conserved regionsof I, II, III FTZ-F1box and AF-2hexamer. Relatively high expressions were observed atdifferent larva stages detected. In105days old juveniles, ssFTZ-F1expressed in alltissues with high levels in testis, ovary, spleen and brain, while lower levels in othertissues. In mature fish, high expression was observed in testis, ovary, spleen and brain.Trace expression was seen in liver and absolutely no expression was detected all othertissues. The highest expression was always observed in gonads in both the juvenile andthe mature fish. Male tissues showed higher expression than their female counterpart （p<0.05）.Thyroid hormones triiodothyronine （T₃） and thyroxine （T₄） play a vital role fordevelopmental and physiological functions in vertebrates. They are mediated via thyroidhormone receptors （TR）. In the present paper we describe the cloning and characterizationof two TR genes, thyroid hormone receptor alpha （TRα） and thyroid hormone receptorbeta （TRβ） from ovary mRNA of a ovoviviparous fish Sebastes schlegelii. Resultsrevealed that main domains and motifs of the two genes were highly conserved.Quantitative real time PCR showed that the mRNA expression levels of both TRα and TRβ were high at5dps and slightly lower and constant during10,20and33dps. In105days old juveniles and three years mature fish, the expression level was higher in gonads,brain, liver and kidney than in other tissues. The highest expression was observed in thegonads. The developmental and tissue expression patterns indicated their involvement inthe growth and gonadal development. TRα exhibited higher expression than TRβ at alldevelopmental stages and in all detected tissues except female liver, which might indicatethe functional differences of these two genes.

全文目录

Chapter 1  12-42
  1. General Introduction  12-19
    1.1. Employment in Fisheries sector  14
    1.2. Aquaculture production and value for major groups  14-15
    1.3. Culture of Major groups in China  15-16
    1.4. Cage culture  16-17
    1.5. Species of genes Sebestaes and their distribution  17-18
    1.6. Biology of Sebastes species  18-19
  2. Literature review  19-32
    2.1. History of FTZ-F1  19-21
    2.2. Nuclear receptor 5A family  21-23
    2.3. Synthesis of steroids in mammals  23-25
    2.4. SF-1 in steroid bioynthesis  25-28
    2.5. Synthesis of Cholesterol  28-29
    2.6. Role of FTZ-F1 for sex determination inTeleost  29-32
  3. Thyroid hormone  32-36
    3.1. Thyroid hormone role for Development  34
    3.2. Thyroid hormone role for Growth  34-35
    3.3. Thyroid hormone role for Metabolism  35-36
    3.4. Nuclear Receptor Superfamily  36
  4.  36-42
    4.1. Materials and Methods in detail  36-37
    4.2. RNA extraction  37-38
    4.3. cDNA Synthesis  38
    4.4. Gel Extraction Protocol  38-40
    4.5. Ligation  40
    4.6. Transformation protocol  40-41
    4.7 Aims and objectives of dissertation  41-42
Chapter 2 Molecular cloning and expression analysis of FTZ-F1 in black rock fish Sebastes schlegelii  42-57
  Introduction  42-44
  2. Materials and Methods  44-47
    2.1 RNA extraction and multi-tissue RT-PCR  44
    2.2 The FTZ-F1 gene cloning and Sequencing  44-46
    2.3 Phylogenetic analysis  46-47
    2.4 Quantitative real-time PCR  47
  3. Results  47-49
    3.1 Identification of ssFTZ-F1 gene  47-48
    3.2 Phylogenetic analysis  48-49
    3.3 Expression of the black rockfish FTZ-F1 gene  49
  4. Discussion  49-57
Chapter 3 Isolation and characterization of Thyroid Hormone Receptor alpha (TRα) and beta TRβ) in black rock fish Sebastes schlegelii  57-101
  1. Introduction  57-101
    1.1. Materials and methods  60
    1.2. Experimental fish  60
    1.3. RNA Isolation and Synthesis of the first strand cDNA  60-61
    1.4. Gene cloning and sequencing  61
    1.5. 5’ and 3’ RACE-PCR  61-62
    1.6. Phylogenetic analysis TRα and TRβ with others known species of vertebrate  62-63
    1.7. Quantitative real-time PCR  63-64
    1.8. Results  64-66
    1.9. Phylogenetic analysis  66
    1.10. Expression of ssTRα and ssTRβ  66-67
    1.11. DISCUSSION  67-74
    1.12 References  74-98
    1.13. Self Introduction  98-99
    1.14. List of Publications  99-100
    1.15. Acknowledgement  100-101

黑鲪核受体基因FTZ-F，TRα and TRβ的克隆及表达分析

内容摘要

全文目录

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