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研究生:李淑嫺
研究生(外文):Shu-Hsien Li
論文名稱:台灣土雞脂肪組織差異表現轉錄體多態性與生產性狀之相關性
論文名稱(外文):Association of Polymorphisms of Differentially Expressed Transcripts in Adipose Tissue of Taiwan Country Chickens with Performances
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
畢業學年度:96
語文別:中文
論文頁數:114
中文關鍵詞:脂肪組織產蛋性狀SNPs台灣土雞
外文關鍵詞:Adipose tissueLaying traitsSNPsTaiwan country chickens
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本試驗之主要目的是針對台灣土雞晶片試驗脂肪組織之高低產差異表現轉錄基因建立SNPs(single nucleotide polymorphisms),並探討其與性狀表現之相關性。首先利用單股核酸構形多態性(single strand conformation polymorphism, SSCP)檢測AFABP、apoVLDLII、CST3、CNOT8、FTH1、GOT1、GSN、ITFG1、PDIA3以及PDPK1等差異表現基因片段,發現其結果皆具不同態樣,經序列分析結果,確認十個基因共21個變異位置。進一步設計特異引子,利用微序列分析法(minisequencing)對219隻L2品系與93隻B品系台灣土雞進行基因型分析。發現每種基因皆可以利用微序列檢測出不同基因型。最後將各基因型與性狀進行統計分析發現,AFABP之C332T與G412A處分別為CC及GG基因型者,其平均初產日齡的表現上顯著地較TT及AA基因型者為低(P < 0.05)。CST3之T97G、T139G、A186G、T236C與T267C處分別為TT、TT、AA、TT及TT基因型者,其總產蛋數與產蛋率顯著高於為GG、GG、GG、CC及CC基因型者(P < 0.05),而T97G之平均停產日則較短;再者,T236C與T267C為TC雜合型者之平均產蛋日與平均窩蛋數皆顯著大於CC型者(P < 0.05);而在體重方面T139G與A186G變異點為GG基因型者之體重表現亦顯著低於TT型及AA型者(P < 0.05),T236C與T267C為TT及TC基因型者之體重表現亦顯著高於CC型者(P < 0.05);GOT1-G27A之AA基因型者在平均產蛋日、平均窩蛋數及最大窩蛋數的表現上均顯著地大於GG型及GA型者(P < 0.05)。GSN基因缺少第173-175鹼基(-/-) 及C219T-TT基因型者,其第12至36週齡體重皆顯著高於另一純合型[+/+]及CC基因型(P < 0.05)。ITFG1基因之G78A,在產蛋性狀方面,GA基因型者之最大窩蛋數顯著多於GG型者。而在PDPK1之C268T,為CC基因型者於平均初產日齡及平均停產日均顯著小於TT型者(P < 0.05)。總結而得選拔AFABP為C332T-CC與G412A-GG基因型;CST3為T97G-TT、T139G-TT及A186G-AA基因型,或T236C及T267C之交替基因為T者;GOT1為G27A- AA基因型;ITFG1為G78A-GA基因型;或PDPK1為C268T-CC型者皆有利於生產性能之提升。而選拔CST3為T139G-TT與A186G-AA基因型,以及T236C與T267C之交替基因為T者;或GSN之nt173-175 (-/-)及C219T-TT型者則有利於生長表現之提升。此研究結果可應用於土雞的育種選拔計畫,使其更有快速改良效益。
The purpose of this study is to investigate the SNPs (single nucleotide polymorphisms) genotypes of differentially expressed genes in adipose tissue of Taiwan country chickens, and their correlations with laying and growth Traits. The SSCP (single strand conformation polymorphism) analysis of AFABP, apoVLDLII, CST3, CNOT8, FTH1, GOT1, GSN, ITFG1, PDIA3 and PDPK1 genes was performed. The different types of SSCP patterns in all these genes were found. Nucleotide sequencing was carried out. Twenty-one SNPs were observed in these 10 gene sequences. Genotyping of 219 L2 and 93 B lines of Taiwan country chickens, detected were carried by minisequencing. The different genotypes of these genes were presented in chickens. The correlations between genotypes and performances of chickens were investigated. The results showed that the age of first egg (AFE) of C332T-CC and G412A-GG in AFABP, are significantly earlier than TT and AA types (P < 0.05). While the T97G, T139G, A186G, T236C and T267C of CST3 gene are TT, TT, AA, TT and TT types respectively, the number of total eggs (Eggs) and hen day rate (HDR) are significantly higher than GG, GG, GG, CC and CC types (P < 0.05); Whereas, the mean pause day (MPD) of T97G-TT is significantly shorter than it of T97G-GG; Furthermore, when the T236C and T267C of CST3 are TC heterozygote, the mean lay day (MLD) and mean clutch egg (MCE) are significantly higher than that are CC genotype (P < 0.05); Body weight (BW) of T139G-TT and A186G-AA, T236C-TT/TC and T267C-TT/TC genotypes in CST3 are significantly higher than that of the other homozygotes. MLD, MCE and max clutch egg of G27A-AA type in GOT1 are significantly higher than those of GG and GA types (P < 0.05). The chickens with nt173-175 (-/-) and C219T-TT genotypes in GSN are significantly heavier than the chickens with nt173-175 (+/+) and C219T-CC genotypes for BW at 12-36 weeks (P < 0.05). Chickens with G78A-GA genotype of ITFG1 are significantly higher than those with GG type for max clutch egg (P < 0.05). Chickens with C268T-CC genotype of in PDPK1 are significantly shorter than those with TT type for AFE and MPD (P < 0.05). We conclude that C332T-CC and G412A-GG of AFABP; T97G-TT, T139G-TT and A186G-AA or allele T of T236C and T267C in CST3; G27A- AA of GOT1; G78A-GA of ITFG1; and C268T-CC of PDPK1, can be used in a selection program for develop of egg production. Furthermore, T139G-TT, A186G-AA and allele T of T236C and T267C in CST3; nt173-175 (-/-) and C219T-TT of GSN, acted in dominant fashion on growth performance of chickens. The results could be applied these genotype loci in the large scale chickens breeding improvement programs.
中文摘要

英文摘要
前言 …………………………………………………………………………………… 1
壹、文獻檢討 ………………………………………………………………………… 2
一、土雞脂肪高低產差異表現基因之簡述 ……………………………………... 2
(一)Fatty acid binding protein 4 (FABP4;AFABP) …………………..…… 2
(二)Apolipoprotein-very low density lipoprotein II (apo-VLDL-II) ………. 2
(三)Cystatin (CST3)………………………………………………..….…… 3
(四)CCR4-NOT transcription complex, subunit 8 (CNOT8) ………......….. 5
(五)Ferritin, heavy polypeptide 1(FTH1) ………………………………….. 5
(六)Glutamic-oxaloacetic transaminase 1, soluble (aspartate aminotransferase 1)(GOT1; AST1) ………………..…………………. 6
(七)Gelsolin (GSN) ………………………………………………...………. 6
(八)Integrin alpha FG-GAP repeat containing 1 (ITFG1)….…………......... 7
(九)Protein disulfide isomerase family A, member 3 (PDIA3)….…………. 8
(十)3-phosphoinositide dependent protein kinase-1 (PDPK1)……………... 8
二、遺傳標記(genetic markers)於家禽上的應用………………………….…… 9
三、單一核苷酸多態性………………………………………………………….… 9
四、單一核苷酸多態性檢測技術原理………………………………….………… 10
五、單一核苷酸多態性檢測技術 ………………………………………...……… 10
(一)單股核酸構形多態性 ………………………………………………… 10
(二)直接序列分析 ………………………………………………...………… 11
(三)SNaPshot 微序列法………………………………………...………..... 11
貳、材料與方法 ……………………………………………………………..………… 12
一、試驗動物 …………………………………………………………...………… 12
二、雞隻血液基因體DNA之萃取 ………………………………………..……… 12
(一)血球之清洗………………………………………………………..…… 12
(二)血球細胞之消化分解……………………………………………..…… 12
(三)DNA之萃取…………………………………………………………… 12
(四)DNA之沉澱析出……………………………………………………… 12
(五)DNA之定量…………………………………………………………… 14
三、標的DNA擴增與電泳確認…………………………………………………… 14
(一)PCR引子設計……………………………………………….……….… 14
(二)PCR增殖單一基因片段……………………………………………..… 14
(三)瓊脂糖膠體電泳 ……………………………………………………… 14
四、單股構型多態性分析………………………………………………………… 14
(一)聚丙烯醯胺凝膠之配製 ……………………………………………… 14
(二)膠體前處理 …………………………………………………………… 15
(三)樣品前處理……………………………………………..……………… 15
(四)電泳 …………………………………………………………………… 15
(五)銀染 …………………………………………………………………… 15
五、序列分析……………………………………………………………….……… 16
(一)序列分析之聚合酶連鎖反應 ………………………………………… 16
(二)純化序列分析之聚合酶連鎖反應產物 ……………………………… 16
(三)序列分析 ……………………………………………………………… 16
六、微序列法分析基因型 ………………………………………………...……… 16
(一)SNP特異引子設計 …………………………………………………… 16
(二)PCR產物之酵素消化..……………………………………………….... 17
(三)雙去氧單一鹼基延伸反應…………………………………………….. 17
(四)微序列產物之純化 …………………………………………………… 17
(五)自動化毛細管電泳與軟體分析基因型 ……………………………… 17
七、統計分析……………………………………………………………….……… 17
參、結果與討論 ………………………………………………………………..……… 21
一、脂肪型脂肪酸結合蛋白 ……………………………………………………... 21
(一)定義AFABP基因之多態性 ……………………………………….… 21
(二) L2與B品系土雞AFABP基因型與交替基因頻率分析 …………… 21
(三) L2系土雞AFABP基因型與表現性狀之相關性 …………………… 21
二、極低密度脂蛋白-載脂蛋白II ………………………………………………… 29
(一)定義apoVLDLII基因之多態性 ………………………………...… 29
(二)L2與B品系土雞apoVLDLII基因型與交替基因頻率分析………. 29
(三)L2系土雞apoVLDLII基因型與表現性狀之相關性………………... 29
三、半胱胺酸蛋白酶抑制劑C ……………………………………………….…… 37
(一)定義CST3基因之多態性 …………………………………………… 37
(二)L2與B品系土雞CST3基因型與交替基因頻率分析 …………… 37
(三) L2系土雞CST3基因型與表現性狀之相關性 ……………………... 38
四、CCR4-NOT轉錄複合體亞基8 ……………………………………………..… 48
(一)定義CNOT8基因之多態性 ………………………………………….. 48
(二) L2與B品系土雞CNOT8基因型與交替基因頻率分析 …………… 48
(三)L2系土雞CNOT8基因型與表現性狀之相關性 ………………… 49
五、含鐵蛋白重鍊多胜肽1 …………………………………………………………… 48
(一) 定義FTH1基因之多態性……………………………………….…… 48
(二)L2與B品系土雞FTH1基因型與交替基因頻率分析………….……… 48
六、麩胺酸草醯乙酸轉胺酶1 …………………………………………………..… 65
(一)定義GOT1基因之多態性………………………………………..…… 65
(二)L2與B品系土雞GOT1基因型與交替基因頻率分析 …………... 65
(三) L2系土雞GOT1基因型與表現性狀之相關性 ………………..…… 65
七、溶膠蛋白…………………………………………………………………….… 73
(一)定義GSN基因之多態性 ……………………………………………... 73
(二) L2與B品系土雞GSN基因型與交替基因頻率分析…………...…… 73
(三)L2系土雞GSN基因型與表現性狀之相關性………………...…… 73
八、整合素 α FG-GAP 重複1 …………………………………………...……… 81
(一)定義ITFG1基因之多態性 …………………………………………… 81
(二) L2與B品系土雞ITFG1基因型與交替基因頻率分析……………… 81
(三)L2系土雞ITFG1基因型與表現性狀之相關性 …………..……… 81
九、蛋白雙硫異構酶家族A3 …………………………………………...………… 89
(一)定義PDIA3基因之多態性 …………………………………...……… 89
(二)L2與B品系土雞PDIA3基因型與交替基因頻率分析 ………..… 89
(三) L2系土雞PDIA3基因型與表現性狀之相關性 ………………….… 89
十、3-磷酸肌醇依賴性蛋白激酶1 ……………………………………………..… 97
(一)定義PDPK1基因之多態性 ………………………………………..… 97
(二) L2與B品系土雞PDPK1基因型與交替基因頻率分析 …………… 97
(三) L2系土雞PDPK1基因型與表現性狀之相關性 …………………… 97
肆、結論 ……………………………………………………………………..………… 105
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