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博碩士論文 etd-0807107-160322 詳細資訊
Title page for etd-0807107-160322
論文名稱
Title
雞胚胎早期性別決定相關轉錄體之鑑別
Identification of transcripts related to sex determination in early chicken embryogenesis
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
180
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2007-07-27
繳交日期
Date of Submission
2007-08-07
關鍵字
Keywords
胚胎發育、性別決定、扣減cDNA基因庫、雞胚
subtractive cDNA libraries, chicken embryos, embryogenesis, sex-determination
統計
Statistics
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中文摘要
在大多數哺乳動物中,性別的結果是由位在Y染色體上存在的SRY基因所轉錄出睪丸決定因子來決定。同樣地,家禽也是經由基因體來決定性別。雞胚孵化發育的第3.5天 (stage 22; HH),性腺原基會開始形成。因此,為了鑑別早期雞胚發育的性別決定基因,本研究建立了孵化發育3天的雄性減雌性與雌性減雄性的cDNA基因庫。首先經由使用Dynabeads收集並純化過的雄性雞與雌性雞的全部mRNAs,在使用平頭端限制性酵素Rsa I切過cDNA後,接著進行tester cDNA的引子接合。當完成第一次與第二次的cDNA雜交後,那些在tester與driver之間沒有重複出現的cDNA接著會被兩次的PCR放大擴增。隨後即進行TA-cloning並使用M13 primers進行PCR放大擴增cDNA片段。菌落的PCR產物會先經由差異性雜交方法減少負陽性產物。然後,基因分析則由線上資料庫GeneBank (NCBI, USA)來進行資料開採。最後,在雄性減雌性基因庫有40個已知與71個未知轉錄體被鑑別出,而在雌性減雄性基因庫則有88個已知與128個未知轉錄體被鑑別出。在雄性減雌性基因庫中,有3個被鑑別出的已知基因是位在Z性染色體上,分別為WD repeat domain 36 (WDR36), PC4 and SFRS1 interacting protein 1 (PSIP1), serum response factor binding protein 1 (SRFBP1) and glycine dehydrogenase (decarboxylating) (GLDC).另外有2個鑑別出的已知基因分別為laminin alpha 1 (LAMA1) and leukocyte cell derived chemotaxin 1 (LECT1)則是曾被報導與細胞的分化發育有關。在雌性減雄性的基因庫中,只有鑑別出的Wpkci-8是位在在W性染色體上。其他被鑑別出的已知基因如slowmo homolog 2 (Drosophila) (SLMO2), collagen, type IV, alpha 1 (COL4A1), anterior gradient 2 homolog (Xenopus laevis), transcript variant 2 (AGR2), solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6 (SLC25A6) and prolyl endopeptidase (PREP)也被發現在人類的卵巢表現量比睪丸高。PREP被認為或許扮演一個經由調控thyrotropin釋放賀爾蒙類似物程度及調節與necrozoospermia有關精子死亡的角色。這些位在W或Z性染色體上所鑑別出的轉錄體可能在性別決定機制上扮演重要的角色。
Abstract
In most mammals, sexual fate is determining genetically by the presence of the SRY gene which encoded the testis-determining factors on the Y chromosome. Likewise, avian sex is determined genetically. At day 3.5 (stage 22; HH) in chicken embryogenesis, the gonadal primordium begins forming. Thus, to identify the novel sex-determinating genes in early chicken embryos, subtractive cDNA libraries from male-minus-female (M-F) and female-minus-male (F-M) of 3 Dpc. embryos were established. Both collected male and female chicken total mRNAs were purified using Dynabeads. After a blund-end restriction endonuclease Rsa I digestion of cDNA, adaptor ligation for tester cDNA was performed. When first and second cDNA hybridization was finished, those nonredundant cDNA between tester and driver will be amplified by two rounds of PCR. Subsequently, TA-cloning was performed and the cDNA fragments were PCR-amplified using M13 primers. PCR products of Clones were first screened by differential screening hybridization to decrease false positive inserts. Then, gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA). Finally, 40 known and 71 novel transcripts of M-F cDNA library, 88 known and 128 novel transcripts of F-M cDNA library were identified. In M-F subtracted library, 4 identified known genes were located on Z sex chromosome such as WD repeat domain 36 (WDR36), PC4 and SFRS1 interacting protein 1 (PSIP1), serum response factor binding protein 1 (SRFBP1) and glycine dehydrogenase (decarboxylating) (GLDC). Another two identified known genes, laminin alpha 1 (LAMA1) and leukocyte cell derived chemotaxin 1 (LECT1) were reported be relate to cell differentiation and development. In F-M subtracted library, only Wpkic-8 was located on W sex chromosome. Other identified genes like slowmo homolog 2 (Drosophila) (SLMO2), collagen, type IV, alpha 1 (COL4A1), anterior gradient 2 homolog (Xenopus laevis), transcript variant 2 (AGR2), solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6 (SLC25A6) and prolyl endopeptidase (PREP) were also found expressed higer in human ovary then testis. PREP was proposed that it may play a role in mediating sperm death by regulating the levels of thyrotropin-releasing hormone analogs and in mediating sperm death associated with necrozoospermia. These transcripts located on W or Z sex chromosome identified from subtracted libraries may play an important role in sex determination mechanism.
目次 Table of Contents
中文摘要...............................................................................................I
Absreact.................................................................................III
Table of contents..................................................................V
List of tables and figures..................................................VII
List of appendix..................................................................VIII
Chapter 1. Literature review...............................................1
1.1. Sex Determination in Vertebrates.............................1
1.2. Gonadal development and differentiation in chicken...................................................................................2
1.3. Sex Determination in Birds........................................3
1.4. Genes involved in sex determination in Birds........4
1.4.1. Protein kinase C inhibitor (PKCI).........................5
1.4.2. Doublesex and mab-3-related transcription factor (DMRT1)......................................................................8
Chapter 2. Identification of Candidate Sex Differentiation Genes Using Subtractive cDNA Libraries in the Early Chicken Embryogenesis............11
2.1. Introduction.................................................................11
2.2. Materials and methods............................................13
2.2.1. Chicken embryos collection and preparations........................................................................13
2.2.2. Identification of the genetic sex..........................14
2.2.3. Construction of M-F and F-M subtracted embryo cDNA libraries ....................................................................14
2.2.4. Identification of the cDNA insert and sequence analysis................................................................................16
2.2.5. Differential screening hybridization...................17
2.2.6. Identification of the cDNA insert and sequence analysis................................................................................17
2.2.7. Protein expression and antibody preparation..........................................................................18
2.3. Results........................................................................20
2.3.1. Sex identification in chicken embryos...............20
2.3.2. Sex identification in chicken embryos...............21
2.3.3. Differential expression of genes identified by SSH.......................................................................................21
2.3.4. Identification of differentially expressed transcripts from subtracted cDNA libraries...................22
2.3.5. Analysis of mRNA expression profile................23
2.4. Disscussion...............................................................25
Tables..................................................................................28
Figures..............................................................................154
References.......................................................................159
Appendix............................................................................163
參考文獻 References
Achermann JC, Ito M, Ito M, Hindmarsh PC, Jameson JL (1999) A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans. Nat Genet, 125-126.
Berta P, Hawkins JR, Sinclair AH, Taylor A, Griffiths BL, Goodfellow PN, Fellous M (1991) Genetic evidence equating SRY and the testis-determining factor. Nature 348, 448 – 450.
Browder DM, Lalli JS (1991) Review of research on sight word instruction. Res Dev Disabil. 12(3):203-28.
Brzoska PM, Chen H, Zhu Y, Levin NA, Disatnik MH, Mochly-Rosen D, Murnane JP, and Christman MF (1995) The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor. Proc. Natl. Acad. Sci. USA 92, 7824–7828.
Burtis KC and Baker BS (1989) Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell 56, 997-1010.
Butlin R (2002) Evolution of sex: The costs and benefits of sex: new insights from old asexual lineages. Nature Reviews Genetics 3, 311-317.
Calvari V, Bertini V, De Grandi A, Peverali G, Zuffardi O, Ferguson-Smith M, Knudtzon J, Camerino G, Borsani G, Guioli S (2000) A new submicroscopic deletion that refines the 9p region for sex reversal. Genomics 65, 203-212.
Carmichael AN, Fridolfsson AK, Halverson J, Ellegren H (2000) Male-biased mutation rates revealed from Z and W chromosome-linked ATP synthase
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