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博碩士論文 etd-0707105-154531 詳細資訊
Title page for etd-0707105-154531
論文名稱
Title
雞隻腦部高產相關cDNA之選殖、註解及其表現分析
Cloning, annotation and mRNA expression analysis of brain cDNA related to high-egg yield in chickens
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
163
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
2005-06-20
繳交日期
Date of Submission
2005-07-07
關鍵字
Keywords
臺灣土雞、產蛋率、定量反轉錄聚合、腦下垂體、cDNA序列資料庫、下視丘
Quantitative RT-PCR, hypothalamus, egg yield, Taiwan Country Chicken (TCC), chicken, pituitary gland, subtractive hybridization cDNA library
統計
Statistics
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中文摘要
本研究為了搜尋及探討在下視丘及腦下垂體中與雞隻繁殖能力相關的基因,藉由抑制性減除雜合聚合
Abstract
To identify known genes or expressed sequence tags (ESTs) which are expressed specifically or preferentially in the chicken hypothalamus and pituitary gland related to highly reproductive performance, two reciprocal cDNA libraries were constructed using a subtractive hybridization strategy. Two different strains, L2 (dam line; n=12) and B (sire line; n=12) of Taiwan Country Chickens (TCCs), which were originated from one single strain and further subjected to 40-wk egg production and comb size, body weight, respectively since 1982, were used in our study. A total of 324 and 370 clones were identified from L2-subtract-B and B-subtract-L2 hypothalamus/pituitary cDNA libraries. 311 and 360 single inserted sequences from each cDNA library, 53 and 23 non-redundant candidate genes were identified. Quantitative reverse-transcription (RT)-PCR were used to validate the association of mRNA expression profiles of the identified candidate genes and high-egg yield trait in another 118 hypothalamuses and pituitary glands that were dissected from seven different chicken stocks, including B-, L2-, Black-, Red-feather TCCs, commercial Single-Comb White Leghorn (WL) layer at National Chung-Hsing University (NCHU) and Red-feather TCCs grouped into high eggs (Red-high) & low eggs (Red-low) to 40 wks of age at National Chiayi University (NCYU). Among identified genes including known genes and novel genes, involving 33 screened genes, Inhibitor-1 of protein phosphatase type 2A (ANP32A), 3-hydroxybutyrate dehydrogenase (BDH), Contactin (CNTN1), Deiodinase iodothyronine type II (DIO2), Inhibitor of growth family, member 3 (ING3), Lysosomal-associated transmembrane protein 4 beta (LAPTM4B), Neural cell adhesion molecule 1 (NCAM1), DJ-1 protein (PARK7), Prostaglandin D2 synthase (PGDS), Prolactin (PRL), Protocadherin 1 (PCDH1), Pleiomorphic adenoma gene 1 (PLAG1), GTP-binding protein SAR1a (SAR1A), Secretogranin II (SCG2), Stathmin 2 (STMN2), T-box protein 2 (TBX2) were up-regulated in B-subtract-L2 cDNA library. Among above-mentioned 16 identified genes, there were 9 genes related to high-egg yield in chickens., including BDH, NCAM1, PCDH1, PGDS, PLAG1, PRL, SAR1A, SCG2, STMN2.
目次 Table of Contents
Contents
Introduction-----------------------------------------------------------------------------1
Materials--------------------------------------------------------------------------------6
Methods----------------------------------------------------------------------------------8
Construction of the L2-subtract-B and the B-subtract-L2 cDNA libraries-----------8
Extraction of total RNA from chicken hypothalamuses and pituitary glands---------8
Isolation of poly (A)+ mRNA from total RNA---------------------------------------8
Reverse transcription polymerase chain reaction (RT-PCR)-------------------------9
Preparation of tester and driver cDNA-------------------------------------------12
Suppression subtractive hybridization (SSH)-------------------------------------15
Cloning of differentially expressed sequences-----------------------------------17
Identification of cDNA insert and sequence analysis-----------------------------17
Quantitative reverse-transcription (RT) polymerase chain reaction (PCR)---------18
Results---------------------------------------------------------------------------------23
Extraction of total RNA and purification of mRNA--------------------------------23
Analysis of Rsa I digestion-----------------------------------------------------25
Analysis of adaptor ligation----------------------------------------------------26
PCR analysis of subtraction efficiency------------------------------------------27
Construction of reciprocal subtract cDNA libraries------------------------------28
Identification of cDNA inserts and sequence analysis----------------------------29
Quantitative RT-PCR analysis of identified candidate
genes related to highly reproductive performance--------------------------------29
Discussion------------------------------------------------------------------------------39
References------------------------------------------------------------------------------53
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