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研究生:謝佳吟
研究生(外文):Chia-Yin Hsieh
論文名稱:荷蘭牛產乳性能相關之差異表現轉錄基因
論文名稱(外文):Differential Expressed Transcripts Associated with Milk Production Performance in of Holstein
指導教授:吳建平吳建平引用關係洪炎明洪炎明引用關係黃木秋黃木秋引用關係
指導教授(外文):Chean-Ping WuYan-Ming HorngMu-Chiou Huang
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:動物科學系研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
畢業學年度:97
語文別:中文
中文關鍵詞:荷蘭牛cDNA微陣列乳量
外文關鍵詞:Holstein cowcDNA MicroarrayMilk yield
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自1995 年開始,全面性分析基因表現的微陣列技術日漸普及,本試驗即利用cDNA 微陣列技術進行與荷蘭牛產乳相關差異表現轉錄基因之探討。荷蘭牛為重要經濟動物之ㄧ,其泌乳性能為選拔的重要指標。試驗依照DHI 乳量記錄收集不同乳量之荷蘭母牛血液,萃取純化出mRNA 以建立荷蘭牛血液之cDNA 基因庫,從中逢機挑選出2880 個菌落,抽取質體DNA 並進行聚合&;#37238;連鎖反應(PCR),增殖接入之cDNA 片段,供cDNA 微陣列晶片之點製;另外以管家基因(18S rRNA、GADPH、β-Actin)及DMSO 溶液作為對照組。晶片採單光設計,將牛隻依DHI 校正乳量高低分組後,自高產組(2 頭)與低產組
(3 頭)個別萃取血液之總RNA,經Fluorescein-dCTP 反轉錄標定及
純化後,與自製cDNA 微陣列晶片(8736 點)雜交,以TSA 訊號放大
技術放大螢光訊號,並以雷射波長532 mm 之螢光掃描擷取影像並量
化,最後將原始數值匯入分析軟體進行數據正規化、對數轉換與統計
分析。結果發現有49 個cDNA 的表現達顯著差異(P < 0.05),且差異表現1.5 倍以上。經序列分析與GenBank 資料庫比對後去除重複,發現差異表現轉錄基因(P < 0.05)有7 個,分別為核醣體蛋白L13a( ribosomal protein L13a, RPL13A)、L31 ( ribosomal protein L31, RPL31)、L37a(ribosomal protein L37a, RPL37A)、S12(ribosomal protein S12, RPS12)、thymosin β10、聚泛素(polyubiquitin)和酪胺酸激&;#37238;鍵結蛋白(tyrosine kinase binding protein, TYROBP),另有23 個差異表現轉錄體比對結果與粒線體DNA 序列相符。此外,利用微序列法(minisequencing)分析差異表現胸腺素β10與酪胺酸激&;#37238;鍵結蛋白
基因之部分序列的A/G 變異,發現各有3 種基因型,分別為TT、TC
及CC 型。胸腺素β10基因各基因型之基因頻率為0.687、0.265 及
0.048,基因型與性狀之間無顯著差異。酪胺酸激&;#37238;鍵結蛋白基因各基因型之基因頻率為0.11、0.52 及0.37,其TT 型之乳產量顯著高於TC型與CC 型(P < 0.05),且TC 型之蛋白質含量(Protein content, PC)亦有高於CC 型之趨勢(P < 0.1)。酪胺酸激&;#37238;鍵結蛋白基因TT型之乳產量雖有顯著增加,但因樣本總數只有82 隻,仍需進一步擴大樣本數以追蹤評估。荷蘭母牛酪胺酸激&;#37238;鍵結蛋白基因之部分序列的A/G 變異,將可作為荷蘭母牛育種與選拔之新候選標記,而其餘本試驗所篩出之差異表現轉錄基因是否與荷蘭牛之產乳性能相關,則有待更進一步之探討。

The purpose of this study was to investigate the differentially expressed transcripts in milk yield of Holstein cow using cDNA microarray. We were isolated and purified the mRNA from Holstein cow blood. The mRNA was
pooled for cDNA library construction with Creator SMART cDNA Library Construction Kit. Plasmid DNA was extracted from random selected 2,880 colonies in cultured plates and the inserts was amplified by PCR. The amplified housekeeping genes (18S rRNA, GADPH, β-Actin) and blank
solution (DMSO) was used as control for array fabrication. The pooled target RNA was collected from 5 Holstein cow, two of high milk yield cows and three of low milk yield cows and labeled with fluorescein-dCTP for hybridization with probes in-house cDNA chips. After washing, the hybridization signals were amplified by TSA technology and scanned by fluorescent scanner at 532 nm for image acquisition and quantification. Data normalization, log transformation and statistics were carried out by software. The results showed that 49 transcripts were differentially
expressed significance (P < 0.05) and those transcripts were observed in fold change > 1.5 at low milk yield cows relative to high milk yield cows. Several sequences of differentially expressed transcripts were found after
sequencing and alignment by BLAST (GenBank). Seven of these were ribosomal protein L13a (RPL13A), ribosomal protein L31 (RPL31), ribosomal protein L37a (RPL37A), ribosomal protein S12 (RPS12), thymosin β10, polyubiquitin, and tyrosine kinase binding protein (TYROBP). The others were mitochondrion DNA sequences. We were analysis A/G point mutation in partial nucleotide sequence of thymosin β10
gene and tyrosine kinase binding protein gene using minisequencing. The results showed that the frequencies of TT, TC, and CC were 0.687, 0.265, 0.048 in thymosin β10 geneand 0.11, 0.52, 0.37 in tyrosine kinase binding protein gene, respectively. The relationship between genotypes and
reproductive traits was also investigated. The results showed that milk yield in TT genotypes were higher than TC and CC genotype (P < 0.05) in tyrosine kinase binding protein gene. The A/G point mutation in partial nucleotide sequence in tyrosine kinase binding protein gene of Holstein cow may be a novel marker applicable to the breeding selection programs of Holstein cow. Other differentially expressed transcripts may involve in high
milk production performance of Holstein cow, further study will be performed in the further.

目錄------------------------------------------------------------------------- I
圖次-------------------------------------------------------------------------- IV
表次-------------------------------------------------------------------------- VI
中文摘要------------------------------------------------------------------- VII
英文摘要------------------------------------------------------------------ VIII
前言------------------------------------------------------------------------- 1
第一章文獻探討--------------------------------------------------------- 3
一、乳牛乳腺之發育與功能------------------------------------------ 3
(一)乳腺之發育------------------------------------------------- 3
(二)乳腺之功能------------------------------------------------- 7
二、乳牛乳量相關基因之研究--------------------------------------- 8
(一)受體素------------------------------------------------------- 8
(二)二醯基甘油基醯基轉移酶------------------------------- 8
(三)生長激素---------------------------------------------------- 10
(四)其他---------------------------------------------------------- 10
三、生物晶片技術原理與應用--------------------------------------- 14
(一)背景---------------------------------------------------------- 14
(二)原理與種類------------------------------------------------- 14
(三) 微陣列基因晶片之製備---------------------------------- 19
(四)螢光標定與雜交反應-------------------------------------- 23
(五)影像偵測、處理與資料分析----------------------------- 32
(六)應用----------------------------------------------------------- 32
第二章材料與方法------------------------------------------------------ 34
一、試驗樣本之採集-------------------------------------------------- 34
二、總RNA 之製備--------------------------------------------------- 34
三、cDNA 基因庫之建立-------------------------------------------- 36
(一)mRNA 之純化----------------------------------------------- 36
(二)cDNA 基因庫之構築-------------------------------------- 36
四、cDNA 微陣列晶片之產製-------------------------------------- 39
(一)製備質體DNA ---------------------------------------------- 39
(二)點製cDNA 晶片-------------------------------------------- 41
五、螢光標定總RNA、cDNA 晶片之雜交反應與螢光訊號偵測41
(一)cDNA 之合成與標定-------------------------------------- 41
(二)cDNA 之純化----------------------------------------------- 44
(三)cDNA 微陣列晶片之雜交反應與清洗----------------- 44
(四)TSA 之偵測------------------------------------------------- 44
六、晶片影像擷取與資料分析-------------------------------------- 46
七、序列分析與身份確認-------------------------------------------- 46
八、基因之鹼基變異位置與形式----------------------------------- 46
(一)萃取荷蘭母牛血液基因組DNA ------------------------- 46
(二)引子設計與PCR 增殖------------------------------------- 47
(三)PCR 產物酵素消化---------------------------------------- 47
(四)雙去氧單一鹼基延伸反應-------------------------------- 51
(五)微序列產物之純化----------------------------------------- 51
(六)自動化毛細管電泳與軟體分析基因型----------------- 51
九、統計分析----------------------------------------------------------- 51
第三章結果與討論------------------------------------------------------ 52
一、荷蘭牛血液cDNA 基因庫------------------------------------- 52
二、cDNA 微陣列分析----------------------------------------------- 52
三、差異表現株系之基因身份確認-------------------------------- 60
四、差異表現基因鹼基變異位置之微序列分析與基因型分析65
結論-------------------------------------------------------------------------- 72
參考文獻-------------------------------------------------------------------- 73
附錄-------------------------------------------------------------------------- 88
圖次
圖1 乳牛乳房於懷孕期、泌乳期及衰退期之荷爾蒙調控圖--- 4
圖2 泌乳物種之乳腺概略描述圖------------------------------------ 6
圖3 牛DGAT1 基因之基因多態性、位置與單一組合型--------- 9
圖4 牛隻GH 片段之長度與於GH 基因上之位置圖-------------- 12
圖5 cDNA 微陣列晶片概略圖-------------------------------------- 15
圖6 光學刻蝕法於玻片表面合成寡核苷酸序列----------------- 17
圖7 三種不同的蛋白質晶片:平板式玻片、立體膠墊式晶片以
及微孔型晶片----------------------------------------------------- 18
圖8 打點針-------------------------------------------------------------- 20
圖9 玻璃片表面塗覆之特異性功能基----------------------------- 22
圖10 直接標定法之標定流程--------------------------------------- 25
圖11 氨基化間接標定之流程圖------------------------------------ 26
圖12 TSA 之標定流程圖--------------------------------------------- 28
圖13 Dendrimer 結構圖----------------------------------------------- 29
圖14 3DNA dendrimer 偵測過程簡略圖--------------------------- 30
圖15 化學標定之簡圖------------------------------------------------ 31
圖16 試驗流程圖------------------------------------------------------ 35
圖17 構築cDNA 基因庫之操作流程圖--------------------------- 37
圖18 TSA 試驗流程圖------------------------------------------------ 43
圖19 TSA 偵測之流程圖--------------------------------------------- 45
圖20 荷蘭母牛胸腺素β10基因其部份序列上之A/G 點突變
位置--------------------------------------------------------------- 49
圖21 荷蘭母牛酪胺酸激酶鍵結蛋白基因其部份序列上之A/G
點突變位置------------------------------------------------------ 50
圖22 荷蘭母牛血液cDNA 基因庫之構築------------------------ 53
圖23 荷蘭母牛血液cDNA 基因庫之PCR 產物電泳圖-------- 54
圖24 荷蘭母牛血液cDNA 基因庫之PCR 產物電泳圖-------- 55
圖25 管家基因之電泳分析圖------------------------------------ 56
圖26 荷蘭母牛血液總RNA 之電泳分析圖---------------------- 57
圖27 荷蘭母牛血液cDNA 微陣列晶片影像圖------------------ 58
圖28 荷蘭母牛血液cDNA 微陣列晶片資料經“Avadis”套裝軟
體分析所得之對數座標圖與叢集分析圖----------------- 59
圖29 荷蘭母牛血液cDNA 基因庫之序列分析------------------ 61
圖30 荷蘭母牛TMS-β10基因之為序列分析結果-------------- 66
圖31 荷蘭母牛TYROBP 基因之為序列分析結果-------------- 69
表次
表1 生長激素之寡核苷酸引子序列及位置----------------------- 11
表2 SNPs 微陣列之16 個候選基因------------------------------ 13
表3 常用於DNA 與蛋白質微陣列技術之螢光物質的結構與光
學特性-------------------------------------------------------------- 24
表4 添加於荷蘭母牛血液cDNA 晶片之管家基因引子----- 42
表5 用於微序列分析之荷蘭母牛血液差異表現基因之特異引子48
表6 荷蘭母牛血液之差異表現基因--------------------------------- 62
表7 荷蘭母牛血液之差異表現轉錄體(粒線體DNA) -------- 63
表8 荷蘭母牛TMS-β10 基因之基因型與交替基因頻率------ 67
表9 荷蘭母牛TMS-β10 基因之基因型與產乳性狀相關性--- 68
表10 荷蘭母牛TYROBP 基因之基因型與交替基因頻率------ 70
表11 荷蘭母牛TYROBP 基因之基因型與產乳性狀相關性--- 71

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