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研究生:陳子翰
研究生(外文):Tzu-Han Chen
論文名稱:台灣土雞肝臟差異表現基因GST-CL3、Otrf、FASN、INSIG1、apoVLDLⅡ及apoB基因單一核苷酸多態性之研究
論文名稱(外文):Single Nucleotide Polymorphisms of GST CL3, Otrf, FASN, INSIG1, apoVLDLⅡ, and apoB Genes Differentially Expressed in Liver of Taiwan Country Chickens
指導教授:黃木秋黃木秋引用關係
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:2008
畢業學年度:96
語文別:中文
論文頁數:136
中文關鍵詞:肝臟單一核苷酸多態性台灣土雞
外文關鍵詞:LiverSNPTaiwan country chickens
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本試驗之目的為利用土雞肝臟組織差異表現基因來探討差異表現基因之基因多態性。以PCR單股構形多態性分析法(single-strand conformation polymorphisms, PCR-SSCP)找出差異表現基因之基因態樣。麩胱苷肽轉移酶CL3次單元(glutathion S-transferase CL3 subunit, GST CL3)、卵運鐵蛋白酶(ovotransferrin, Otrf)、脂肪酸合成酶(fatty acid synthase, FASN)、胰島素誘導基因1(insulin induced gene 1, INSIG1)、脫輔基蛋白Ⅱ(apolipoprotein VLDLⅡ, apoVLDLⅡ)及脫輔基蛋白B(apoprotein B, apoB)等基因於聚丙烯醯胺膠片中皆呈現基因多態性。利用序列分析法偵測差異表現基因之SNP點。以PCR-RFLP及微序列分析法(minisequencing method)進行SNP之確認,所得結果一致。利用微序列法分析6個差異表現基因之SNP基因型並計算基因型頻率。發現Otrf基因在L2品系(G70C)、(T106C)、(C161T)及(A269G)變異皆為GG/TT/CC/GG之純合型。FASN基因(C41T)及apoVLDLⅡ基因(C93T)SNP點在L2與B品系皆以CC型最多。apoB1基因(T42C)在L2品系II型較ID及DD型為多,顯示這些結果為產蛋優勢之基因型。進一步以基因型與性狀資料進行統計分析之結果顯示,L2品系土雞之GST CL3 基因(T221C)之TT型雞之總產蛋數顯著高於TC及CC兩型者(P < 0.05)。apoVLDLⅡ基因(C93T),其CC型雞在2及4周齡體重高於CT型者(P < 0.01)。apoB1基因II型對之平均初產日高於ID及DD型者(P < 0.05)。以上試驗結果可作為土雞育種上候選基因之參考。
The aim of this study was to inverstigate the single nucleotide polymorphisms of the different expression genes in Taiwan country chicken livers. PCR-SSCP was used to find the gene polymorphisms and mutations. Glutathione S-transferase CL3 subunit (GST-CL3), ovotransferrin (Otrf), fatty acid synthase (FASN), insulin induced gene 1 (INSIG1), apolipoprotein VLDLⅡ (apoVLDLⅡ), and apoprotein B (apoB) genes were all found polymorphisms by PCR-SSCP. Sequencing was performed to find the SNPs. The genotypes obtained from PCR-RFLP were the same as minisequencing. The SNPs genotyping and frequency analysis of six differentially expressed genes in livers were carried out. The genotypes of Otrf (G70C), (T106C), (C161T) and (A269G) gene in L2 line were all GG/TT/CC/GG homozygotes. Most CC genotype of FASN (C41T) and apoVLDLⅡ (C93T) can be detected in both L2 and B lines. The frequency of genotype II of apoB1 (T42C) gene was more than the genotype ID and DD in the L2 line. It revealed that the genotype II of apoB1 (T42C) was more advantage for the reproductive traits especially significant on the 1st egg day. The relationship between genotypes and traits were investigated. The results showed that the total number eggs of TT type of GST CL3 (T221C) gene were higher than the TC and CC types in L2 line significantly (p < 0.05). The body weight at 2 and 4 wks of CC type of apoVLDLⅡ (C93T) gene was heavier than the type CT (p < 0.01). The age at 1st egg of apoB1 type II was significant early than the type ID and DD (p < 0.05). These results could provide the information for Taiwan country chicken selection in Taiwan.
目錄
頁次
中文摘要
英文摘要
壹、前言……………………………………………………………………1
貳、文獻檢討……………………………………………………………………2
一、雞麩胱苷肽轉移酶CL3次單元基因…………………2
二、雞卵運鐵蛋白酶基因…………………………………………………3
三、雞脂肪酸合成酶基因…………………………………………………3
四、雞胰島素誘導基因1…………………………………………………5
五、雞脫輔基蛋白VLDLⅡ基因…………………………………………5
(一)apoVLDLⅡ之基因之特性…………………………………5
(二)apoVLDLⅡ基因之SNP與生長、骨骼以及體組成之關係6
(三)檢測apoVLDLⅡ基因之方法…………………………………6
六、雞脫輔基蛋白B基因………………………………………………7
(一)脫輔基蛋白B基因之特性……………………………………7
(二)脫輔基蛋白B基因之SNP與生長、肥胖性狀之關係…8
(三)檢測脫輔基蛋白B基因之方法……………………………….8
七、遺傳標記在家禽育種方面之應用…………………………………8
八、常用之SNP檢測技術…………………………………………………8
(一)單股構形多態性………………………………………………9
(二)限制片段長度多態性………………………………………9
(三)序列分析法……………………………………………………9
(四)SNaPshot……………………………………………………….9
(五)Multiplex-PCR.10
(六)多重式微序列法………………………………………………10
參、材料與方法…………………………………………………………………11
一、土雞血液樣本採集……………………………………………………11
二、血液基因組DNA之萃取…………………………………………….11
(一)血液及血塊之前處理………………………………………11
(二)血球細胞之消化分解………………………………………11
(三)DNA之萃取……………………………………………………11
(四)DNA之沉澱析出……………………………………………11
(五)DNA定量……………………………………………………12
三、GST CL3 基因之基因型分析…………………………………………14
(一)PCR-SSCP態樣分析…………………………………………14
(二)序列分析與比對………………………………………………15
(三)PCR-RFLP分析基因型……………………………………….16
(四)微序列法分析基因型……………………………………….16
四、Ovotransferrin(Otrf)基因之基因型分析…………………….20
(一)PCR-SSCP態樣分析………………………………………….20
(二)序列分析與比對………………………………………………21
(三)PCR-RFLP 分析基因型……………………………………22
(四)微序列法分析基因型………………………………………22
五、Fatty acid synthase(FASN)基因之基因型分析…………………27
(一)PCR-SSCP態樣分析………………………………………….27
(二)序列分析與比對………………………………………………28
(三)微序列法分析基因型………………………………………29
六、Insulin induced gene 1(INSIG1)基因之基因型分析………………33
(一)PCR-SSCP態樣分析………………………………………….33
(二)序列分析與比對………………………………………………34
(三)微序列法分析基因型………………………………………35
七、Apolipoprotien VLDLⅡ(apoVLDLⅡ)基因之基因型分析……39
(一)PCR-SSCP態樣分析………………………………………….39
(二)序列分析與比對……………………………………………40
(三)PCR-RFLP 分析基因型……………………………………41
(四)微序列法分析基因型………………………………………41
八、Apoprotien B(apoB)基因之基因型分析…………………………….45
(一)PCR-SSCP態樣分析………………………………………….45
(二)序列分析與比對………………………………………………47
(三)微序列法分析基因型………………………………………48
九、基因型頻率及性狀分析………………………………………………54
肆、結果與討論…………………………………………………………………55
一、麩胱苷肽轉移酶CL3次單位基因型分析…………………………….55
(一)定義GST CL3基因之多態性…………………………………55
(二)土雞麩胱苷肽轉移酶CL3次單位之基因型頻率分析….55
(三)土雞麩胱苷肽轉移酶CL3次單位之基因型與性狀分析.56
二、卵運鐵蛋白酶基因型分析…………………………………………66
(一)定義Otrf基因之多態性……………………………………….66
(二)土雞卵運鐵蛋白酶之基因型頻率分析………………………67
三、脂肪酸合成酶(FASN)基因型分析…………………………………76
(一)定義FASN基因之多態性……………………………………76
(二)土雞脂肪酸合成酶之基因型頻率與交替基因頻率分析……76
(三)土雞脂肪酸合成酶之基因型與性狀分析……………………76
四、胰島素誘導基因(INSIG1)基因型分析……………………….86
(一)定義INSIG1基因之多態性………………………………….86
(二)土雞INSIG1之基因型頻率與交替基因頻率分析………….86
(三)土雞INSIG1之基因型與性狀分析……………………….86
五、脫輔基蛋白VLDL Ⅱ(apoVLDLⅡ)基因型分析…………………….96
(一)定義apoVLDLⅡ基因之多態性………………………………96
(二)土雞apoVLDLⅡ之基因型頻率與交替基因頻率分析……….96
(三)土雞apoVLDLⅡ之基因型與性狀分析……………………….96
六、脫輔基蛋白B基因(apoB)3’端側翼區域(apoB1)及exon 26(apoB2)之基因型分析………………………………………………………….107
(一)定義apoB基因之多態性………………………………………107
(二)土雞apoB1 T42C之基因型頻率與交替基因頻率分析…108
(三)土雞apoB2 C289T、G611A及C656A基因型頻率分析108
(四)土雞apoB1 T42C之基因型與性狀分析……………………108
(五)土雞apoB2 C289T、G611A及C656A基因型與性狀分析…109
伍、結論………………………………………………………………………….126
陸、參考文獻……………………………………………………………………127




表次
頁次
表 1.增殖土雞GST-CL3基因引子、限制酶切割片段長度及基因型……19
表 2.增殖土雞Otrf基因引子、限制酶切割片段長度及基因型………….26
表 3.增殖土雞FASN基因片段之引子及PCR片段長度…………………32
表 4.增殖土雞INSIG1基因片段之引子及PCR片段長度……………….38
表 5.增殖土雞apoVLDLⅡ基因引子、限制酶切割片段長度及基因型.44
表 6.增殖土雞apoB基因3’端終止密碼區外之側翼序列(apoB1)片段之引子及PCR片段長度…………………………………………….51
表 7.增殖土雞apoB基因exon 26(apoB2)片段之引子及PCR片段長度………………………………………………………………………53
表 8.以多重式微序列分析法檢測土雞GST CL3基因3個SNPs位置之鹼基及波峰顏色………………………………………………………62
表 9.土雞GST CL3基因3個突變鹼基組合之基因型頻率…………….63
表 10.土雞GST CL3基因(T178C)、(T221C)及(C265T)基因型與產蛋性狀之關係………………………………………………………64
表 11.L2品系土雞CL3基因(T178C)、(T221C)及(C265T)基因型與40週齡體重相關係……………………………………………….65
表 12.以多重式微序列分析法檢測土雞Otrf基因4個SNPs位置之鹼基及波峰顏色……………………………………………………………74
表 13.土雞Otrf基因4個突變鹼基組合之基因型頻率……………75
表 14.以微序列分析法檢測土雞FASN基因1個SNP位置之鹼基及波峰顏色……………………………………………………………………82
表 15.土雞FASN基因之基因型及交替基因頻率…………………83
表 16.土雞FASN基因C41T基因型與產蛋性狀之關係………………….84
表 17.L2品系土雞FASN基因(C41T)基因型與40週齡體重之關係………………………………………………………………………85
表 18.以微序列分析法檢測土雞INSIG1基因1個SNP位置之鹼基及波峰顏色…………………………………………………………………92
表 19.土雞INSIG1基因之基因型及交替基因頻率………………………93
表 20.土雞INSIG1基因(G81A)基因型與產蛋性狀之相關分析…………94
表 21.L2品系土雞INSIG1基因(G81A)基因型與40週齡體重之關係………………………………………………………………………95
表 22.以微序列分析法檢測土雞apoVLDLⅡ基因1個SNP位置之鹼基及波峰顏色……………………………………………………………103
表 23.土雞apoVLDLⅡ基因之基因型及交替基因頻率……………104
表 24.土雞apoVLDLⅡ基因(C93T)基因型與產蛋性狀之關係…….105
表 25.L2品系土雞apoVLDLⅡ基因(C93T)基因型與40週齡體重之關係………………………………………………………………………106
表 26.以微序列分析法檢測土雞apoB1基因1個插入/缺失SNP位置之鹼基及波峰顏色………………………………………………………114
表 27.土雞apoB1基因之基因型及交替基因頻率…………………………115
表 28.土雞apoB1基因(T42C)基因型與產蛋性狀之關係………………116
表 29.L2品系土雞apoB1基因(T42C)基因型與40週齡體重之相關分析………………………………………………………………………117
表 30.以多重式微序列分析法檢測土雞apoB基因exon 26 3個SNPs位置之鹼基及波峰顏色…………………………………………………122
表 31.土雞apoB基因exon26 3個突變鹼基組合之基因型頻率………….123
表 32.土雞apoB基因exon26(C289T)、(G611A)及(C656A)基因型與產蛋性狀之關係……………………………………………………124
表 33.L2品系土雞apoB基因exon26(C289T)、(G611A)及(C656A)基因型與40週齡體重之關係……………………………………….125


圖次
圖 1.試驗流程圖…………………………………………………………….13
圖 2.土雞GST-CL3基因部分核苷酸序列、引子位置、限制酶切位、及延伸引子位置………………………………………………………….18
圖 3.土雞Otrf基因部分核苷酸序列、引子位置、限制酶切位、及延伸引子位置……………………………………………………….25
圖 4.土雞FASN基因部分核苷酸序列、引子位置、及延伸引子位置……………………………………………………………………….31
圖 5.土雞INSIG1基因部分核苷酸序列、引子位置、及延伸引子位置……………………………………………………………………….37
圖 6.土雞apoVLDLⅡ基因部分核苷酸序列、引子位置、限制酶切位及延伸引子位置………………………………………………………….43
圖 7.土雞apoB基因3’端終止密碼區外之側翼序列部分核苷酸序列、引子位置及延伸引子位置……………………………………………….50
圖 8.土雞apoB基因exon 26部分核苷酸序列、引子位置及延伸引子位置……………………………………………………………………….52
圖 9.土雞GST CL3基因PCR增殖結果電泳與單股構形多態性分析圖……………………………………………………………………….57
圖 10.利用序列分析法進行土雞GST CL3基因(T178C)、(T221C)及(C265T)之基因型分析……………………………………………….58
圖 11.土雞GST-CL3基因序列比對結果……………………………………59
圖 12.土雞GST CL3基因以CNLlvCL3 F及R引子增殖單一片段(474bp),以TaqαⅠ限制酶切割(C265T)之電泳圖…………….60
圖 13.利用多重式微序列法進行土雞GST CL3基因(T178C)、(T221C)及(C265T)3點SNPs之基因型分析………………………………61
圖 14.土雞Otrf基因PCR增殖結果電泳與單股構形多態性分析圖…….68
圖 15.利用序列分析法進行土雞Otrf基因10 個SNP點之基因型分析69
圖 16.土雞Otrf基因序列比對結果………………………………………….70
圖 17.土雞Otrf基因以CNLlvOVO F及R引子增殖單一片段(405 bp),以NlaⅢ限制酶切割(G70C)之電泳圖………………………………71
圖 18.土雞Otrf基因以CNLlvOVO引子增殖單一片段(405 bp),以SfcⅠ限制酶切割(T106C)之電泳圖………………………………72
圖 19.利用多重式微序列法進行土雞Otrf基因(G70C)、(T106C)、(C161T)及(A269G) 4點SNPs之基因型分析…………………………………73
圖 20.土雞FASN基因PCR增殖結果電泳與單股構形多態性分析圖…………………………………………………………………….78
圖 21.利用序列分析法進行土雞FASN基因(C41T)之基因型分析………79
圖 22.土雞FASN基因序列比對結果………………………………………80
圖 23.利用微序列方法進行土雞FASN基因(C41T)之基因型分析………81
圖 24.土雞INSIG1基因PCR增殖結果電泳與單股構形多態性分析圖…………………………………………………………………….88
圖 25.利用序列分析法進行土雞INSIG1基因(G81A)之基因型分析…89
圖 26.土雞INSIG1基因序列比對結果…………………………………….90
圖 27.利用微序列方法進行土雞INSIG1基因(G81A)之基因型分析….91
圖 28.土雞apoVLDLⅡ基因PCR增殖結果電泳與單股構形多態性分析圖……………………………………………………………….98
圖 29.利用序列分析法進行土雞apoVLDLⅡ基因(C93T)之基因型分析.99
圖 30.土雞apoVLDLⅡ基因序列比對結果…………………………………100
圖 31.土雞apoVLDLⅡ基因以apoVLDLⅡ引子增殖單一片段(494bp),以SfcⅠ限制酶切割(C93T)之電泳圖……………………101
圖 32.利用微序列方法進行土雞apoVLDLⅡ基因(C93T)之基因型分析.102
圖 33.土雞apoB基因3’端終止密碼區側翼序列片段 PCR增殖結果電泳與單股構形多態性分析圖………………………………….110
圖 34.土雞apoB基因3’端側翼序列比對結果…………………………….111
圖 35.土雞apoB基因3’端終止密碼區側翼序列片段以c apoB1引子增殖單一片段(174bp),以3% 瓊脂糖凝膠分離具有插入/缺失不同大小片段之電泳圖…………………………………………….112
圖 36.利用微序列方法進行土雞apoB基因3端終止密碼區側翼序列片段(T42C)基因型分析………………………………………………….113
圖 37.土雞apoB基因exon26片段 PCR增殖結果電泳與單股構形多態性分析圖……………………………………………………….118
圖 38.利用序列分析法進行土雞apoB基因exon26增殖片段(C289T)、(G611A)及(C656A)之基因型分析…………………………….119
圖 39.土雞apoB基因exon26序列比對結果……………………………….120
圖 40.利用多重式微序列法進行土雞apoB基因exon26(C289T)、(G611A)及(C656A)3點SNPs之基因型分析…………………….121
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