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研究生:趙蓓娜
研究生(外文):Pratthana Jamwan
論文名稱:不同品種豬隻H-FABP,RN,CRC,CA3,ESR與PRLR基因型之比較
論文名稱(外文):Comparisons of H-FABP, RN, CRC, CA3, ESR and PRLR Genotypes among Different Breeds of Pigs
指導教授:黃木秋黃木秋引用關係
指導教授(外文):Mu-Chiou Huang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:動物科學系所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:90
中文關鍵詞:H-FABPRNCRCCA3ESRPRLR基因型分析
外文關鍵詞:H-FABPRNCRCCA3ESRPRLRgenotypingpigs
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本試驗之目的為利用微序列分析、PCR-RFLP 進行泰國與台灣豬隻心臟型脂肪酸結合蛋白(heart fatty acid binding protein, H-FABP)、酸肉(rendement napole, RN)、鈣離子釋放通道接受體(calcium release channel, CRC)、碳酸酐酶3(carbonic anhydrase, CA3)、動情素接受體(estrogen receptor, ESR)與泌乳素受體(prolactin receptor, PRLR)基因型比較。H-FABP基因是肌間脂肪含量的候選基因,試驗結果發現基因型HH-6之豬隻以台灣杜洛克(0.646)、HL-5型者以泰國比利華(0.200)、HL-4型者以台灣藍瑞斯(0.694)、HL-3型者以泰國2x dam line(0.650)、HL-2型者以泰國大白豬(0.400)、HL-1型者以泰國黑豬(0.380)及LL-0型者以台灣黑豬(0.161)所出現的頻率最高。RN基因為酸肉的候選基因,基因型為rn+/rn+之豬隻其肉質是正常的。本試驗結果顯示除了泰國藍瑞斯(0.950)、台灣杜洛克(0.994)、台灣黑豬之(0.935)非rn+/rn+型者外,其他所有豬種皆為rn+/rn+型(即基因型頻率為1.000)。CRC基因為水樣肉及緊迫症狀的候選基因,AA基因型為正常者。供試之豬隻中,泰國與台灣的大白豬、藍瑞斯、杜洛克皆全為AA型(1.000),而泰國2x sire line則以AB型出現的頻率較高(0.579)。B交替基因僅微可見於泰國比利華、泰國2x dam line及泰國與台灣黑豬中。CA3基因(C/T變異)是瘦肉及肌內脂肪相關候選基因,試驗結果顯示CC型者以泰國比利華(0.900)、CT型者以泰國藍瑞斯(0.650)、TT型者以泰國大白豬(0.800)出現頻率最高。CA3基因之A/G變異為腿肉量相關的候選基因,試驗結果顯示所有豬種皆為AA型(1.000)。ESR基因為繁殖表現的相關候選基因。AA型者以在台灣杜洛克(1.000)、AB型者以泰國2x dam line(0.480)、BB型者以泰國大白豬(0.650)所出現的頻率最高。PRLR基因亦為繁殖表現的相關候選基因之一。AA型者以台灣黑豬(0.624)、AB型者以泰國大白豬(0.800)、BB型者以泰國比利華豬(1.00)所出現的頻率最高。本試驗之基因檢測結果可提供豬遺傳改良之參考
The aim of this study was to comparisons of H-FABP, RN, CRC, CA3, ESR, and PRLR genotypes in Thai and Taiwan pigs using minisequencing and PCR-RFLP. The H-FABP gene is a candidate gene for intramuscular fat (IMF) content. The highest frequencies of HH-6 (0.646) in Taiwan Duroc, HL-5 (0.200) in Thai Pietrain, HL-4 (0.694) in Taiwan Landrace, HL-3 (0.650) in Thai 2x dam line, HL-2 (0.400) in Thai Large White, HL-1 frequency (0.380) in Thai Black pig, and LL-0 (0.161) in Taiwan Black pig were found. The RN gene is a candidate gene for acid meat. The phonotype of rn+/rn+ is normal. All genotypes of RN gene in tested animals were rn+/rn+ (1.000) other than Thai Landrace (0.950), Taiwan Duroc (0.994) and Taiwan Black pig (0.935). The CRC gene is candidate gene for PSE and PSS, all AA type (normal) (1.000) was observed in Large White, Landrace and Duroc pigs in both of Thai and Taiwan. The highest frequency of AB type (0.579) was found in 2x sire line of Thai pigs. There was still minor B allele in Pietrain, 2x dam line (Thai) and Black pigs (both of Thai and Taiwan). The CA3 (C/T) gene is a candidate gene for lean cuts (LC) and visible intermuscular fat (VIF). The highest frequencies of CC (0.900) in Thai Pietrain, CT (0.650) in Thai Landrace and TT (0.800) in Thai Large white were found. The CA3 (A/G) is a candidate gene for percentage of ham. All tested animals genotypes are AA (1.000). The ESR is a candidate gene for reproduction. The highest frquencies of AA (1.000) found in Taiwan Duroc, AB (0.480) in 2x dam line, and BB (0.650) in Thai Large White. The PRLR gene also is a candidate gene for reproduction. The highest frequencies of AA (0.624) in Taiwan Black pig, AB (0.800) in Thai Large White and BB (1.000) in Thai Pietrain were found. These results might provide the information for genetic improvement in pig breeding.
List of contents

Chinese Abstract
English Abstract
Chapter I. Introduction-------------------------------------------------------------------------- 1
Chapter II. Literature review------------------------------------------------------------------- 2
1. Important genes effect on meat quality traits in pigs ---------------------------------- 2
1.1 Heart fatty acid-binding protein (H-FABP) gene---------------------------------- 2
1.1.1 Introduction of heart fatty acid-binding protein (H-FABP)------------------ 3
1.1.2 The association between heart fatty acid-binding protein (H-FABP) and meat quality -------------------------------------------------------------------------
4
1.1.3 Detection of heart fatty acid-binding protein (H-FABP)---------------------- 5
1.2 Rendenment napole (RN) gene ------------------------------------------------------ 6
1.2.1 Introduction of rendenment napole (RN) gene-------------------------------- 6
1.2.2 The association between rendenment napole (RN) gene and meat quality 7
1.2.3 Detection of rendenment napole (RN) gene ----------------------------------- 9
1.3 Calcium release channel (CRC) gene ------------------------------------------------ 9
1.3.1 Introduction of calcium release channel (CRC) gene ------------------------- 9
1.3.2 The association between calcium release channel (CRC) gene and meat quality --------------------------------------------------------------------------------
10
1.3.3 Detection of calcium release channel (CRC) gene ---------------------------- 11
1.4 Carbonic anhydrase 3 (CA3) gene --------------------------------------------------- 11
1.4.1 Introduction of carbonic anhydrase 3 (CA3) gene ----------------------------- 11
1.4.2 Detection of carbonic anhydrase 3 (CA3) gene -------------------------------- 12
2. Important genes effect on reproductive traits in pigs --------------------------------- 12
2.1 Estrogen receptor (ESR) gene ------------------------------------------------------- 14
2.1.1 Introduction of estrogen receptor (ESR) gene --------------------------------- 14
2.1.2 The association between estrogen receptor (ESR) gene and reproduction trait -----------------------------------------------------------------------------------
15
2.1.3 Detection of estrogen receptor (ESR) gene ------------------------------------- 15
2.2 Prolactin receptor (PRLR) gene ----------------------------------------------------- 15
2.2.1 Introduction of prolactin receptor (PRLR) gene ------------------------------- 15
2.2.2 The association between prolactin receptor (PRLR) gene an reproduction trait ------------------------------------------------------------------------------------
16
2.2.3 Detection of prolactin receptor (PRLR) gene ----------------------------------- 16
3. Application of molecular biotechnology ------------------------------------------------ 16
3.1 Introduction of a single nucleotide polymorphism -------------------------------- 16
3.2 Identification of the single nucleotide polymorphisms techniques ------------- 17
3.2.1 Restriction fragment length polymorphism (RFLP) ------------------------- 17
3.2.2 Single-strand conformation polymorphism (SSCP) ------------------------- 18
3.2.3 Taqman ---------------------------------------------------------------------------- 18
3.2.4 Arrayed primer extension (APEX) -------------------------------------------- 19
3.2.5 Multiplex minisequencing ------------------------------------------------------ 19
3.2.6 Pyrosequencing ------------------------------------------------------------------ 20
3.2.7 Oligonucleotide ligation assay (OLA) ---------------------------------------- 20
3.2.8 Invader® assay -------------------------------------------------------------------- 21
3.2.9 Sequence analysis ---------------------------------------------------------------- 21
3.2.10 Multiple-polymerase chain reaction (Multiplex PCR) --------------------- 22
Chapter III. Materials and methods ---------------------------------------------------------- 23
1. DNA extraction from whole blood ------------------------------------------------------ 24
2. DNA extraction from tissue -------------------------------------------------------------- 24
3. Heart-type fatty acid binding protein genotype analysis ----------------------------- 25
4. Rendenment napole (RN) genotype analysis ----------------------------------------- 31
5. Calcium release channel (CRC) genotype analysis ----------------------------------- 35
6. Carbonic anhydrase 3 (CA3) gene genotype analysis -------------------------------- 37
7. Estrogen receptor (ESR) genotype analysis ------------------------------------------- 43
8. Prolactin receptor (PRLR) gene genotype analysis ----------------------------------- 45
Chapter IV. Results and discussion ----------------------------------------------------------- 50
1. Heart fatty acid-binding protein (H-FABP) genotype analysis ---------------------- 50
1.1 Heart fatty acid-binding protein (H-FABP) gene polymorphism ---------------- 50
1.2 Heart fatty acid-binding protein (H-FABP) gene genotyping frequencies analysis ---------------------------------------------------------------------------------------
56
2. Rendenment napole (RN) genotype analysis ------------------------------------------ 59
2.1 Rendenment napole (RN) gene polymorphism ----------------------------------- 59
2.2 Rendenment napole (RN) gene genotyping frequencies analysis -------------- 62
3. Calcium release channel (CRC) genotype analysis ----------------------------------- 63
3.1 Calcium release channel (CRC) gene polymorphism ----------------------------- 63
3.2 Calcium release channel (CRC) gene genotyping frequencies analysis -------- 63
4. Carbonic anhydrase 3 (CA3) genotype analysis --------------------------------------- 66
4.1 Carbonic anhydrase 3 (C/T) genotype analysis ------------------------------------ 66
4.1.1 Carbonic anhydrase 3 (C/T) gene polymorphism ---------------------------- 66
4.1.2 Carbonic anhydrase 3 (C/T) gene genotyping frequencies analysis------- 67
4.2 Carbonic anhydrase 3 (A/G) genotype analysis ----------------------------------- 70
4.2.1 Carbonic anhydrase 3 (A/G) gene polymorphism --------------------------- 70
4.2.2 Carbonic anhydrase 3 (A/G) gene genotyping frequencies analysis ------ 71
5. Estrogen Receptor Gene (ESR) genotype analysis ------------------------------------ 74
5.1 Estrogen receptor (ESR) gene polymorphism ------------------------------------- 74
5.2 Estrogen receptor (ESR) gene genotyping frequencies analysis ---------------- 75
6. Prolactin receptor gene (PRLR) genotype analysis ----------------------------------- 78
6.1 Prolactin receptor (PRLR) gene polymorphism ----------------------------------- 78
6.2 Prolactin receptor (PRLR) gene genotyping frequencies analysis -------------- 79
Chapter V. Conclusion --------------------------------------------------------------------------- 83
References ---------------------------------------------------------------------------------------- 84












List of figures

Figure 1. Schematic presentation of putative roles of plasmalemmal and cytoplasmic fatty acid-binding proteins in cellular metabolism of long-chain fatty-acid----
5
Figure 2. Oil red O straining of lipid droplets in adipocytes from piglet of nine different genotypes culture-------------------------------------------------------------
6
Figure 3. Metabolism of muscle----------------------------------------------------------------- 8
Figure 4. Proposed mechanism for induction of malignant hyperthermia----------------- 11
Figure 5. Flow chart of experimental------------------------------------------------------------ 23
Figure 6. The 5’ upstream region sequence of H-FAP gene in pig ------------------------- 28
Figure 7. The intron 2 sequence of H-FABP gene in pig ------------------------------------ 29
Figure 8. The sequence of RN (PRKAG3) gene in pig--------------------------------------- 33
Figure 9. The sequence of CRC gene in pig---------------------------------------------------- 37
Figure 10. The sequence of CA3 (A/G) gene in pig------------------------------------------- 40
Figure 11. The sequence of CA3 (C/T) gene in pig------------------------------------------- 42
Figure 12. The sequence of ESR gene in pig--------------------------------------------------- 45
Figure 13. The sequence of PRLR gene in pig------------------------------------------------- 48
Figure 14. Electrophoretogram of H-FABP 5’upstream region PCR products ----------- 51
Figure 15. Electrophoretogram of H-FABP intron 2 PCR products ------------------------ 51
Figure 16. Electrophoretogram of the HinfI digested PRC fragment amplified from H-FABP 5’upstream region ---------------------------------------------------------
52
Figure 17. Electrophoretogram of the HaeIII digested PCR fragment amplified from H-FABP intron 2 ----------------------------------------------------------------------
53
Figure 18. Electrophoretogram of the MspI digested PCR fragment amplified from H-FABP intron 2 ----------------------------------------------------------------------
54
Figure 19. H-FABP genotyping of H, D, and A loci in pig by minisequencing----------- 55
Figure 20. Electrophoretogram of RN (PRKAG3) gene PCR products-------------------- 59
Figure 21. Electrophoretogam of BsrBI digested PCR fragment amplified from RN (PRKAG3) gene------------------------------------------------------------------------
60
Figure 22. Genotyping of RN (PRKAG3) gene in pigs by minisequencing--------------- 61
Figure 23. Electrophoretogram of CRC gene PCR products--------------------------------- 64

Figure 24. Electrophoretogram of the BsiHKAI digested PCR fragment amplified from CRC gene-------------------------------------------------------------------------------

64
Figure 25. Genotyping of CRC gene in pigs by minisequencing---------------------------- 65
Figure 26. Electrophoretogram of CA3 (C/T) gene PCR products-------------------------- 67
Figure 27. Genotyping of CA3 (C/T) gene in pigs by minisequencing--------------------- 68
Figure 28. Genotyping of CA3 (C/T) gene in pigs by sequencing-------------------------- 69
Figure 29. Electrophoretogram of CA3 (A/G) gene PCR products------------------------- 71
Figure 30. Genotyping of CA3 (A/G) gene in pigs by minisequencing-------------------- 72
Figure 31. Genotyping of CA3 (A/G) gene in pigs by sequencing-------------------------- 73
Figure 32. Electrophoretogram of ESR gene PCR products--------------------------------- 75
Figure 33. Electrophoretogram of the PvuII digested PCR fragment amplified from ESR gene-------------------------------------------------------------------------------
76
Figure 34. Genotyping of ESR gene in pigs by minisequencing---------------------------- 77
Figure 35. Electrophoretogram of PRLR gene PCR products------------------------------- 79
Figure 36. Electrophoretogram of the AluI digested PCR fragment amplified from PRLR gene-----------------------------------------------------------------------------
80
Figure 37. Genotyping of PRLR gene in pigs by minisequencing-------------------------- 81















List of tables

Table 1. The fatty acid-binding protein multi-gene family----------------------------------- 3
Table 2. Candidate gene with association to reproductive traits in pigs-------------------- 13
Table 3. Primers, restriction fragments and genotypes of H-FABP 5’upstream region and H-FABP intron 2 in pig------------------------------------------------------------
30
Table 4. Primers, restriction fragments and genotypes of RN (PRKAG3) gene in pig--- 34
Table 5. Primers, restriction fragments and genotypes of CRC gene in pig---------------- 38
Table 6. Primers, restriction fragments and genotypes of ESR gene in pig---------------- 48
Table 7. Primers, restriction fragments and genotypes of PRLR gene in pig-------------- 49
Table 8. The site and peak colors of H-FABP gene in pig detected by minisequencing-- 56
Table 9. Genotypic frequencies of the H-FABP gene in Thai and Taiwan pigs------------ 57
Table 10. Thai and Taiwan pigs classified genotypic frequencies--------------------------- 58
Table 11. The site and peak colors of RN (PRKAG3) gene in pig detected by minisequencing-------------------------------------------------------------------------
61
Table 12. Genotypic frequencies of RN gene in Thai and Taiwan pigs -------------------- 62
Table 13. The site and peak colors of CRC gene in pig detected by minisequencing---- 65
Table 14. Genotypic frequencies of CRC gene in Thai and Taiwan pigs ------------------ 66
Table 15. The site and peak colors of CA3 (C/T) gene in pig detected by minisequencing-------------------------------------------------------------------------
67
Table 16. Genotypic frequencies of CA3 (C/T) gene in Thai and Taiwan pigs ----------- 70
Table 17. The site and peak colors of CA3 (A/G) gene in pig detected by minisequencing ------------------------------------------------------------------------
72
Table 18. Genotypic frequencies of CA3 (A/G) gene in Thai and Taiwan pigs ----------- 74
Table 19. The site and peak colors of ESR gene in pig detected by minisequencing----- 77
Table 20. Genotypic frequencies of ESR gene in Thai and Taiwan pigs ------------------- 78
Table 21. The site and peak colors of PRLR gene in pig detected by minisequencing--- 81
Table 22. Genotypic frequencies of PRLR gene in Thai and Taiwan pigs ----------------- 82
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