臺灣博碩士論文加值系統

本試驗之目的在於利用多重式微序列法（multiplex minisequencing）分析豬隻基因型，包含肉質（CRC, RN, CAST）、繁殖（ESR1, PRLR, RBP4, LEP, BF, LEPR, LIF, ACTN1, EPOR, FSHR, GNRHR, PGK2, RLN）、抗病（FUT1, BPI）、生長（MC4R, GH）及屠體性狀（PIT1, MYOG, LXRB）等二十三個候選基因。以軟體設計專一性引子對，經過PCR反應增殖標的DNA後，將產物分別用PCR-RFLP、微序列法與多重式微序列法進行基因型鑑定。結果顯示，我們以多重式微序列法分別開發出能於單一試管內同時鑑定多個SNPs標記之DNA檢測系統。分析台灣地區杜洛克、藍瑞斯、LD及黑毛豬各基因之基因型與交替基因頻率分佈，結果發現MYOG、CAST、BPI（AvaII）、BF、ACTN1與PGK2基因在上述豬種中，皆以有利之交替基因居多，而GH、PIT1、MC4R、PRKAG3、FUT1、BPI（HpaII）、LEPR、LIF、EPOR、GNRHR及RLN基因則是存在著改進空間。本試驗所開發之SNPs檢測系統能達到快速、正確、低成本及大量分析等優勢，期望實際應用於台灣種豬基因篩選，更迅速選拔出最優良的種畜群。

The aim of this study was to establish DNA marker detection systems for single nucleotide polymorphisms associated with economic traits in breeding pigs and genotyping by multiplex minisequencing method, which include genes (CRC, RN and CAST) for meat quality, reproduction (ESR1, PRLR, RBP4, LEP, BF, LEPR, LIF, ACTN1, EPOR, FSHR, GNRHR, PGK2 and RLN), disease resistance (FUT1 and BPI), growth (MC4R and GH), and carcass (PIT1, MYOG and LXRB). Specific primer pairs were designed by GCG sequence analysis software and each of the target genes was amplified. The PCR-RFLP, minisequencing and multiplex-minisequencing methods were used for genotyping. The results showed that the multiplex-minisequencing method was able to unambiguously and simultaneously genotype many SNP loci in different animals. Genotypic and allelic frequencies of each gene in Duorc, Landrace, LD and black pigs were analyzed. It was found there were existence of alleles are favored in MYOG, CAST, BPI (AvaII), BF, ACTN1, PGK2 genes, GH, PIT1, MC4R, PRKAG3, FUT1, BPI (HpaII), LEPR, EPOR, and GNRHR genes still remained room of the genetic improvement in these pig breeds. We have successfully developed a rapid, cost-effective, and high throughput assay for genotyping and can be applied to genotype these loci in the large scale pig breeding programs.

中文摘要
英文摘要
前言 1
第一章　文獻檢討　--------------------------------------------------------------- 2
一、豬生長、屠體、肉質及抗病性狀候選基因之介紹　------------------ 2
（一）生長激素（growth hormone, GH）　----------------------------- 2
（二）肌細胞生成素（myogenin, MYOG）　----------------------------- 2
（三）腦垂體轉錄因子1（pituitary transcription factor 1, PIT1）　---- 3
（四）黑色素皮質素受體4（melanocortin-4 receptor, MC4R）　------ 4
（五）肝臟X受體（liver X receptor, LXRs）　--------------------------- 4
（六）腺嘌呤核苷單磷酸活化γ3蛋白質激酶（protein kinase AMP-activated γ 3 subunit, PRKAG3）　----------------------------- 5
（七）鈣蛋白酶抑制蛋白（calpastatin, CAST）　----------------------- 5
（八）α1-岩藻糖轉移酶（α1-fucosyltransferase, FUT1）　------------- 6
（九）殺菌/通透性增加蛋白（bactericidal/permeability-increasing protein, BPI）　---------------------------------------------------------- 7
（十）備解素（complement factor protein B, BF）　-------------------- 7
（十一）瘦體素受體（leptin receptor, LEPR）　------------------------- 8
（十二）白血病抑制因子（leukemia inhibitory factor, LIF）　--------- 8
（十三）輔肌動蛋白酶1（actinin 1, ACTN1）　------------------------- 9
（十四）紅血球細胞生成素受體（erythropoietin receptor, EPOR）　 9
（十五）激濾泡素受體（follicle-stimulating hormone receptor, FSHR）　----------------------------------------------------------------- 10
（十六）激性腺素釋素受體（gonadotropin-releasing hormone receptor, GNRHR）　---------------------------------------------------- 10
（十七）磷酸甘油酸激酶2（phosphoglycerate kinase 2, PGK2）　--- 10
（十八）鬆弛素（relaxin, RLN）　---------------------------------------- 11
二、單一核苷酸多態性之簡介　-------------------------------------------- 12
三、單一核苷酸多態性之鑑定技術　-------------------------------------- 14
（一）限制酶切割　---------------------------------------------------------- 14
（二）單股DNA構型　------------------------------------------------------ 14
（三）特異交替基因雙向PCR增殖　------------------------------------- 14
（四）突變點拆離式聚合酶連鎖反應　---------------------------------- 14
（五）多重式微序列法　--------------------------------------------------- 15
（六）寡核苷酸晶片　------------------------------------------------------ 15
（七）寡核苷酸連接分析　------------------------------------------------- 15
（八）TaqMan　--------------------------------------------------------------- 15
（九）直接序列分析　-------------------------------------------------------- 16
第二章　材料與方法　------------------------------------------------------------ 17
一、試驗動物來源　---------------------------------------------------------- 17
二、全血DNA之萃取　------------------------------------------------------ 17
三、組織DNA之萃取　------------------------------------------------------- 18
四、標的DNA擴增與電泳確認　------------------------------------------- 18
五、PCR產物之純化　-------------------------------------------------------- 19
六、限制酶切割與電泳分析基因型　-------------------------------------- 19
七、序列分析　---------------------------------------------------------------- 19
八、單一微序列反應　-------------------------------------------------------- 20
九、自動化毛細管電泳與軟體分析基因型　------------------------------ 20
十、標的DNA擴增與電泳確認　-------------------------------------------
20
十一、multiple PCR產物之純化　------------------------------------------ 21
十二、多重式微序列反應　-------------------------------------------------- 21
十三、自動化毛細管電泳與軟體分析基因型　--------------------------- 21
第三章　結果與討論　------------------------------------------------------------ 25
一、單一基因之基因型鑑定　----------------------------------------------- 25
（一）生長激素　----------------------------------------------------------- 25
1. 定義GH基因之多態性　------------------------------------------ 25
2. 杜洛克、藍瑞斯、LD及黑毛豬GH基因型與交替基因頻率分析　--------------------------------------------------------------------　 25
（二）肌細胞生成素　----------------------------------------------------- 30
1. 定義MYOG基因之多態性　-------------------------------------- 30
2. 杜洛克、藍瑞斯、LD及黑毛豬MYOG基因型與交替基因頻率分析　-----------------------------------------------------------------　 30
（三）腦垂體轉錄因子1　------------------------------------------------- 35
1. 定義PIT1基因之多態性　----------------------------------------- 35
2. 杜洛克、藍瑞斯、LD及黑毛豬PIT1基因型與交替基因頻率分析　-------------------------------------------------------------------- 35
（四）黑色素皮質素受體4　---------------------------------------------- 40
1. 定義MC4R基因之多態性　-------------------------------------- 40
2. 杜洛克、藍瑞斯、LD及黑毛豬MC4R基因型與交替基因頻率分析　----------------------------------------------------------------- 40
（五）肝臟X受體　--------------------------------------------------------- 45
1. 定義LXRB基因之多態性　--------------------------------------- 45
2. 杜洛克、藍瑞斯、LD及黑毛豬LXRB基因型與交替基因頻率分析　----------------------------------------------------------------- 45
（六）腺嘌呤核苷單磷酸活化γ3蛋白質激酶　--------------------------- 50
1. 定義PRKAG3基因之多態性　------------------------------------ 50
2. 杜洛克、藍瑞斯、LD及黑毛豬PRKAG3基因型與交替基因頻率分析　-------------------------------------------------------------- 50
（七）鈣蛋白酶抑制蛋白　------------------------------------------------ 55
1. 定義CAST基因之多態性　--------------------------------------- 55
2. 杜洛克、藍瑞斯、LD及黑毛豬CAST基因型與交替基因頻率分析　----------------------------------------------------------------- 55
（八）α1-岩藻糖轉移酶　-------------------------------------------------
64
1. 定義FUT1基因之多態性　---------------------------------------- 64
2. 杜洛克、藍瑞斯、LD及黑毛豬FUT1基因型與交替基因頻率分析　----------------------------------------------------------------- 64
（九）殺菌/通透性增加蛋白　-------------------------------------------- 69
1. 定義BPI基因之多態性　------------------------------------------ 69
2. 杜洛克、藍瑞斯及黑毛豬BPI基因型與交替基因頻率分析　- 69
（十）備解素　-------------------------------------------------------------- 78
1. 定義BF基因之多態性　------------------------------------------- 78
2. 杜洛克、藍瑞斯、LD及黑毛豬BF基因型與交替基因頻率分析　-------------------------------------------------------------------- 78
（十一）瘦體素受體　------------------------------------------------------ 83
1. 定義LEPR基因之多態性　--------------------------------------- 83
2. 杜洛克、藍瑞斯、LD及黑毛豬LEPR基因型與交替基因頻率分析　----------------------------------------------------------------- 83
（十二）白血病抑制因子　------------------------------------------------ 88
1. 定義LIF基因之多態性　------------------------------------------ 88
2. 杜洛克、藍瑞斯、LD及黑毛豬LIF基因型與交替基因頻率分析　-------------------------------------------------------------------- 88
（十三）輔肌動蛋白酶1　------------------------------------------------- 93
1. 定義ACTN1基因之多態性　------------------------------------- 93
2. 杜洛克、藍瑞斯、LD及黑毛豬ACTN1基因型與交替基因頻率分析　-------------------------------------------------------------- 93
（十四）紅血球細胞生成素受體　--------------------------------------- 98
1. 定義EPOR基因之多態性　--------------------------------------- 98
2. 杜洛克、藍瑞斯、LD及黑毛豬EPOR基因型與交替基因頻率分析　----------------------------------------------------------------- 98
（十五）激濾泡素受體　--------------------------------------------------- 103
1. 定義FSHR基因之多態性　--------------------------------------- 103
2. 杜洛克、藍瑞斯、LD及黑毛豬FSHR基因型與交替基因頻率分析　----------------------------------------------------------------- 103
（十六）激性腺素釋素受體　--------------------------------------------- 108
1. 定義GNRHR基因之多態性　------------------------------------- 108
2. 杜洛克、藍瑞斯、LD及黑毛豬GNRHR基因型與交替基因頻率分析　-------------------------------------------------------------- 108
（十七）磷酸甘油酸激酶2　----------------------------------------------
113
1. 定義PGK2基因之多態性　--------------------------------------- 113
2. 杜洛克、藍瑞斯及LD PGK2基因型與交替基因頻率分析　--　 113
（十八）鬆弛素　----------------------------------------------------------- 118
1. 定義RLN基因之多態性　----------------------------------------- 118
2. 杜洛克及黑毛豬RLN基因型與交替基因頻率分析　--------- 118
二、多基因之基因型鑑定　-------------------------------------------------- 122
（一）同時檢測五個生長屠體候選基因（GH、MYOG、PIT1、MC4R、LXRB）--------------------------------------------------------- 122
（二）同時檢測九個商業用候選基因（CRC、FUT1、ESR1、RBP4、PRLR、CAST249、CAST638、MC4R、RN）　--------------- 122
（三）同時檢測十個繁殖候選基因（ESR1、RBP4、PRLR、LEP、BF、LEPR、LIF、EPOR、FSHR、GNRHR）　---------------------- 123
第四章　結論　--------------------------------------------------------------------- 134
參考文獻　-------------------------------------------------------------------------- 135

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