臺灣博碩士論文加值系統

本研究目的主要探討雞心臟脂肪酸結合蛋白（Heart fatty acid binding protein, H-FABP）基因之篩選，以不同分子層面分析並比較台灣土雞、肉雞及蛋雞其品種-品系間之差異。利用逢機複製多態性DNA（Random amplified polymorphic, RAPD）技術分別對各品種及品系進行分析以區分品系內及品系間之相關性。並藉由DNA序列分析作同源性比對，利用限制片段長度多態性（Restriction fragment length polymorphism, RFLP）進行H-FABP基因多態性之檢測，以作為篩選高肉質基因之遺傳標記。於RAPD試驗中，使用40個逢機引子擴增14種不同的土雞品系、肉雞及蛋雞之基因組DNA，結果發現，僅有19個逢機引子可以擴增出多態性片段。根據此19個逢機引子所擴增出之RAPD態樣顯示，不同品系土雞及不同品種之間有不同之RAPD指印，且發現部分土雞品系之遺傳純度較低，可能是因為土雞之品系育種制度未完全整合之故。在DNA片段分析中，自行設計雞H-FABP基因之四種引子（F26, F132, R462及R500），做為聚合酶連鎖反應用。在雞之H-FABP基因中，以F132及R500兩種引子所擴增出448 bp片段，經由DNA定序後顯示具有一個79 bp之intron，在不同品種-品系間具有3個變異點，以HpaII及ScrFI兩種限制酶進行RFLP分析，獲致以HpaII切割F132及R500所擴增之片段，在所有雞隻品種-品系之間皆可區分出三種基因型，分別為AA、Aa及aa；以ScrF I亦可區分出三種不同的基因型，分別為BB、Bb及bb。雜合子型（A/a與B/b）有較高的態樣頻率。本研究初步探討H-FABP基因，尚需針對特異性之態樣做進一步分析。

The purpose of this study was to investigate for the screening of heart fatty acid binding protein （H-FABP）gene by molecular analysis to compare the variation of breed and line in native chicken, broiler and layer. The polymorphism of random amplified polymorphic DNA （RAPD） was used to set apart the relationship between breed and line. The homology of DNA sequence was compared. The restriction fragment length polymorphism （RFLP） was used to identify the polymorphism of H-FABP gene that provide genetic marker to screen the meat quality. In RAPD experiment, forty arbitrary primers was used to amplify genomic DNA of fourteen different lines in native chicken, broiler and layer. The results showed that only nineteen arbitrary primers produced polymorphic fragments. According to the patterns of RAPD from nineteen arbitrary primers showed that present the different patterns between native chicken lines and breed. There were lower homology in partial native chicken lines. The DNA fragments of the chicken H-FABP gene were amplified by polymerase chain reaction（PCR）, using the four primers (F26, F132, R462, R500). The amplification fragments were 448 bp. After DNA sequencing, the amplification fragments contained a 79 bp intron and observed three point mutation. The amplification fragments with primers of F132, R500 were digested by enzyme HpaII and ScrFI, The polymorphism of digested fragments by HpaII,were observed three genotypes AA, Aa and aa respectively and digested by ScrFI were observed three genotypes BB, Bb and bb respectively. Two heterozygous（A/a and B/b）have higher frequencies of patterns. This part of the research for chicken H-FABP gene is presently in investigation and require more particulary study patterns.

中文摘要………………………………………………………..Ι
英文摘要………………………………………………………ΙΙI
誌謝…………………………………………………………….V
目錄…………………………………………………………..VII
圖次索引……………………………………………………...IX
表次索引……………………………………………………...XI
附圖索引……………………………………………………..XII
壹、前言……………………………………………………….1
貳、文獻探討………………………………………………….2
一、 篩選高肉質基因之緣起………………………………….2
（一）心臟脂肪酸結合蛋白（H-FABP）基因之發現……...2
（二）H-FABP基因與肌肉內脂肪含量之關係……………..3
（三）發展具本土特性並篩選H-FABP基因雞隻品系之
重要性………………………………………………….4
二、家畜禽數量性狀之改良………………………………….5
（一） 家畜禽重要之經濟性狀……………………………….5
（二） 數量性狀基因座……………………………………….6
（三） 家禽基因組之發展…………………………………….7
三、DNA指紋技術及應用……………………………………8
（一）DNA指紋技術…………………………………………8
（二）逢機複製多態性DNA技術之原理及應用…………...12
參、試驗部分…………………………………………………..16
一、試驗一. 雞之RAPD指紋多態性分析…………………..17
（一）試驗動物………………………………………………..17
（二）材料與方法……………………………………………..17
（三）結果與討論……………………………………………..20
（四）結論……………………………………………………..21
二、試驗二. 雞心臟脂肪酸結合蛋白基因型多態性之檢測…….36
（一）試驗動物………………………………………………..36
（二）材料與方法……………………………………………..36
（三）結果與討論……………………………………………..50
（四）結論……………………………………………………..52
肆、總結………………………………………………………..53
伍、參考文獻…………………………………………………..57
陸、附圖………………………………………………………..66
柒、作者簡介…………………………………………………..82
圖次索引
圖1、.肉雞 ×蛋雞雜交對於肌肉性狀之QTL圖譜…….9
圖2、..RAPD形成之過程………………………………..14
圖3、利用RAPD片段多態性於品系-品種及個體
遺傳變異之鑑定實例………………………..…..15
圖4、雞隻RAPD指紋分析與H-FABP基因
之RFLP多態性檢測……………………………16
圖5、不同雞隻品種以OPAB09引子所擴增出
之RAPD指紋圖譜………………………………24
圖6、中興大學土雞L1品系以OPAB09引子
擴增出之RAPD指紋圖譜………………………25
圖7、中興大學土雞辰和品系以OPAB09引子
擴增出之RAPD指紋圖譜………………………26
圖8、中興大學土雞新福品系以OPAB09引子
擴增出之RAPD指紋圖譜………………………27
圖9、屏科大土雞國鋒品系以OPAB09引子
擴增出之RAPD指紋圖譜………………………28
圖10、屏科大土雞國鈞品系以OPAB09引子
擴增出之RAPD指紋圖譜………………………29
圖11、高雄種畜繁殖場土雞辰竹品系以OPAB09
引子擴增出之RAPD指紋圖譜………………..30
圖12、高雄種畜繁殖場土雞瑞昌品系以OPAB09
引子擴增出之RAPD指紋圖譜…………………31
圖13、高雄種畜繁殖場土雞聯合品系以OPAB09
引子擴增出之RAPD指紋圖譜…………………32
圖14、高雄種畜繁殖場土雞豐輝品系以OPAB09
引子擴增出之RAPD指紋圖譜…………………33
圖15、高雄種畜繁殖場土雞金山品系以OPAB09
引子擴增出之RAPD指紋圖譜…………………34
圖16、高雄種畜繁殖場土雞國民品系以OPAB09
引子擴增出之RAPD指紋圖譜…………………35
圖17、雞隻基因組DNA以引子F26-R462進行
最適煉合溫度之測試……………………………39
圖18、雞隻基因組DNA以引子F26-R500進行
最適煉合溫度之測試……………………………40
圖19、雞隻基因組DNA以引子F132-R462進行
最適煉合溫度之測試……………………………41
圖20、雞隻基因組DNA以引子F132-R500進行
最適煉合溫度之測試……………………………42
圖21、雞H-FABP基因部分片段序列圖………………54
表次索引
表1、各種分子標記系統之特性………………………11
表2 、RAPD與RFLP之特性………………………...13
表3、雞隻基因組DNA應用OPAA系列逢機引子
擴增所得之RAPD產物………………………..22
表4、雞隻基因組DNA應用OPAB系列逢機引子
擴增所得之RAPD產物………………………..23
表5、Buffer QXΙ添加需要量…………………………47
表6、QIAEX ΙΙ的使用量……………………………..48
表7、靜置溶解DNA所需的時間…………………….49
表8、雞H-FABP基因以HpaII及ScrFI切割之
態樣頻率………………………………………..56
附圖索引
附圖1、中興大學土雞辰和品系以OPAA04引子
擴增出之RAPD指譜…………………………66
附圖2、中興大學土雞L1品系以OPAB04引子
擴增出之RAPD指紋圖譜……………………67
附圖3、中興大學土雞辰和品系以OPAB04引子
擴增出之RAPD指紋圖譜……………………68
附圖4、中興大學土雞新福品系以OPAB04引子
擴增出之RAPD指紋圖譜……………………69
附圖5、屏科大土雞國鋒品系以OPAB04引子擴增
出之RAPD指紋圖譜…………………………70
附圖6、屏科大土雞國鈞品系以OPAB04引子擴增
出之RAPD指紋圖譜…………………………71
附圖7、屏科大土雞國鋒品系以OPAB06引子擴增
出之RAPD指紋圖譜…………………………72
附圖8、屏科大土雞國鈞品系以OPAB06引子擴增
出之RAPD指紋圖譜…………………………73
附圖9、高雄種畜繁殖場土雞辰竹品系以OPAA16
引子擴增出之RAPD指紋圖譜………………74
附圖10、高雄種畜繁殖場土雞瑞昌品系以OPAA16
引子擴增出之RAPD指紋圖譜………………75
附圖11、高雄種畜繁殖場土雞辰竹品系以OPAA03
引子擴增出之RAPD指紋圖譜………………76
附圖12、高雄種畜繁殖場土雞瑞昌品系以OPAA03
引子擴增出之RAPD指紋圖譜………………77
附圖13、高雄種畜繁殖場土雞聯合品系以OPAA03
引子擴增出之RAPD指紋圖譜………………78
附圖14、高雄種畜繁殖場土雞豐輝品系以OPAA03
引子擴增出之RAPD指紋圖譜………………79
附圖15、高雄種畜繁殖場土雞金山品系以OPAA03
引子擴增出之RAPD指紋圖譜………………80
附圖16、高雄種畜繁殖場土雞國民品系以OPAA03
引子擴增出之RAPD指紋圖譜………………81

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