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研究生:周雅鳳
論文名稱:應用性別鑑定引子辨別鸚鵡種類之研究
論文名稱(外文):Studies on the Sex-specific Primer for Parrot Species Identification
指導教授:吳建平吳建平引用關係洪炎明洪炎明引用關係
指導教授(外文):Chean-Ping WuYan-MingHorng
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:動物科學系研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2017
畢業學年度:106
語文別:中文
論文頁數:121
中文關鍵詞:性別鑑定引子分辨鸚鵡種類
外文關鍵詞:Sex-specific PrimerParrot Species Identification
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大多數鳥類無法由外觀進行性別判定,須由其他方法正確鑑定性別,才能進行配對繁殖,特別是瀕臨絕種之鳥類。目前商業上多以分子技術的方法進行性別鑑定,而Chelex 100為廣泛萃取DNA之方法,其萃取之DNA可有效供PCR使用做多種檢驗。本試驗擬探討以5 % Chelex 100萃取羽毛DNA之加熱溫度及時間的最佳處理條件及利用不同性別鑑定PCR特異引子分辨鸚鵡種類。試驗一、探討5 % Chelex 100萃取羽毛DNA之加熱溫度及時間的最佳處理條件,本試驗分三個部分,第一部分以58℃加熱4小時、60℃加熱2小時及62℃加熱1小時,探討萃取禽類羽毛DNA最適溫度條件,結果顯示,溫度上升至62℃時,萃取時間可縮短至1小時。第二部分為利用62℃加熱1至6小時,探討萃取鸚鵡羽毛DNA最適加熱時間,結果顯示,溫度固定62℃時,加熱時間越長DNA萃取濃度及純度有顯著上升。第三部分為模擬夏季高溫,利用37℃保存新鮮鴿子羽毛30天,並每3天萃取一次DNA,探討羽毛DNA之萃取濃度及純度變化。結果顯示,羽毛保存於37℃時,從第15天起DNA萃取效果降低,第30天所萃取之DNA無法有效經PCR擴增分辨環帶。試驗二、探討利用不同性別特異引子分辨鸚鵡種類,共使用9組引子(P1、P2、P4、P5、P9、P11、P23、P25及P28)對禽類進行種別鑑定。結果顯示,P1引子對經PCR擴增可分辨和尚鸚鵡(雌雄共有環帶約700 bp)、P屬鸚鵡(雌雄共有環帶約680 bp)、五色鳥及太平洋鸚鵡(雌雄共有環帶約1100 bp)、天鵝(雌雄共有環帶約590 bp)、超級鸚鵡及公主鸚鵡(雌性環帶約320 bp)、赤紅腹小太陽(雌雄共有環帶約650 bp及550 bp)、鉛色水鶇(雌性環帶約450 bp)、鴿子(雌雄共有環帶約680 bp、650 bp及雌性環帶450 bp)、巴拉望雉及孔雀(雌雄共有環帶約590 bp及雌性環帶約450 bp),而P1引子對無法擴增雌雄共有環帶,於PCR時加入18s ribosomal gene primer,需加入18s ribosomal gene primer作為對照的鳥種為月輪、亞歷山大、花頭、達摩、折衷、麒麟鸚鵡及鮮紅胸鸚鵡(雌性環帶約430 bp)。P2引子對可分辨九官鳥(雌雄共有環帶約350 bp及雌性環帶380 bp)。P5引子對可分辨和尚鸚鵡(雌性環帶略低)。P4引子對依環帶位置不同可分辨月輪、亞歷山大(雌性環帶約520 bp、690 bp)、花頭(雌性環帶約520 bp)、達摩(雌性環帶約490 bp、520 bp及590 bp),P9引子對可分辨小鸚、牡丹(雌雄共有環帶約250 bp及雌性環帶約420 bp)與天鵝(雌性環帶約810 bp)之環帶差異。P23引子對為天鵝特異片段(雌雄共有環帶約600 bp及350 bp及雌性環帶約810 bp)。P11引子對為鴿子特異片段(雌雄共有環帶約250 bp及雌性環帶約730 bp)。P25、P28引子對為雀科特異片段,P25引子對不僅能鑑定雀科(雌雄共有環帶約300 bp及雌性環帶約330 bp),還能分辨橙頭地鶇、鉛色水鶇(雌雄共有環帶及雌性環帶皆略高)。綜上所述,羽毛以5 % Chelex 100萃取DNA的最適條件為62℃加熱1小時,且加熱時間越長,DNA萃取濃度及純度顯著上升,當羽毛於37℃下保存第15天DNA萃取濃度明顯下降,至第30天則無法萃取足量DNA供PCR擴增環帶。而9組性別鑑定引子中,P1引子對可分辨鳥種範圍最廣泛,P2引子對僅九官鳥有差異,P5、P4、P9、P11、P23、P25及P28引子對為特異片段,對其以外之鳥種鑑定環帶位置不同,可由此差異分辨鳥種。
Most birds can’t be identified sex by appearance, and sex must be correctly identified by other methods for mating, especially on endangered birds. Nowadays, molecular technique is a common way for sex identification in
commercial, and Chelex 100 is a wide range of methods for DNA extraction, the extracted DNA can be used effectively for a variety of PCR experiments. This experiment intends to explore the optimum treatment conditions for the heating
temperature and time of using 5 % Chelex 100 to extract feathers DNA and use different PCR specific primers to distinguish parrot species. The first experiment explored the optimal treatment conditions for the heating temperature and time of feather DNA extracted with 5 % Chelex 100. The experiment was divided into three parts. The first part was heated at 58℃for 4 hours, heated at 60℃for 2 hours
and heated at 62℃for 1 hour to investigate the optimal temperature conditions for extracting feather DNA. The results showed that the extraction time could be shortened when the temperature increased to 62℃ to 1 hour. The second part is the use of 62℃ heating 1 to 6 hours to explore the extraction of feather DNA optimal heating time, the results show that the longer the heating time, DNA extraction concentration and purity increased significantly. The third part is to simulate the hot summer conditions. The fresh Columba livia feathers were stored at 37℃ for 30 days. DNA was extracted after 3 days and stored at 37℃ to investigate the extraction concentration and purity of feather DNA. The second experiment explored the use of different sex-specific primers to distinguish parrot species. The avians were identified using nine primer sets (P1, P2, P4, P5, P9, P11,
P23, P25 and P28). The results showed that P1 primer can be distinguished by PCR amplification Myiopsitta monachus (male and female common band 700 bp), Pionus (male and female common band 680 bp), Megalaima nuchalis and Forpus
coelestis (male and female common band 1100 bp), Cygnus atratus (female specific band 590 bp), Polytelis swainsonii and Polytelis alexandrae (female specific band 320 bp), Pyrrhura perlata (male and female common band 650 bp
and 550 bp), Rhyacornis fuliginosa (female specific band 450 bp), Columba livia(male and female common band 680 bp,650 bp and female specific band 450 bp),Polyplectron napoleonis and Pavo cristatus (male and female common band 590 bp and female specific band 450 bp), 18s ribosomal gene primer was added during PCR, whereas 18s ribosomal gene primer was added as control in Psittacula krameri, Psittacula eupatria, Psittacula cyanocephala, Psittacula alexandri, Eclectus roratus, Tanygnathus megalorynchos and Neophema splendida (female specific band 430 bp). The P2 primer pair distinguishes Gracula religiosa (male and female common band 350 bp and female specific band 380 bp). P5 primer pairs distinguish Myiopsitta monachus (female specific band slightly lower). P4 primer pair according to the ring position can be resolved Psittacula krameri, Psittacula eupatria (female specific band 520 bp, 690 bp), Psittacula cyanocephala (female specific band 520 bp), Psittacula alexandri (female specific band 490 bp, 520 bp and 590 bp). The P9 primer pair distinguishes the difference between the bands of Agapornis roseicollis, Agapornis personatus (male and female common band 250 bp and female specific band 420 bp) and Cygnus atratus (female specific band 810 bp). The P23 primer pair is a Cygnus atratus specific fragment (male and female common band 600 bp, 350 bp and female specific band 810 bp). The P11 primer pair is a specific fragment of Columba livia (male and female common band 250 bp and female
specific band 730 bp). The P25 and P28 primer pairs were Passeridae specific fragments. The P25 primer pair identified Passeridae (male and female common band 300 bp and female specific band330 bp), as well as Zoothera citrina and Rhyacornis fuliginosa (two bands slightly higher). In summary, the optimal conditions for feather DNA extraction with 5 % Chelex 100 were 62℃for 1 hour. When the heating time is longer, DNA extraction concentration and purity
increased significantly. Feather was stored at 37℃ for 15 days and the DNA extraction concentration was significantly reduced. By the 30th day, enough DNA was not extracted for PCR amplification. 9 sex identification primer, the P1 primer pair has the wide range of distinguishable birds. P2 primers differ only for Gracula religiosa. The P5, P4, P9, P11, P23, P25 and P28 primer pairs are specific fragments. Different species of birds can be identified outside the band location, which can distinguish between bird species.
【圖次】.............3
【表次】.............7
中文摘要.............8
英文摘要.............10
壹、前言.............12
貳、文獻探討..........14
一、 比較不同 DNA 萃取方法 ........14
(一) Chelex 100 法 .............14
(二) 苯酚-氯仿法(Phenol-chloroform)...........15
(三) 固相萃取技術(solid phase extraction)..........15
(四) 商業試劑組(Commercial investigator kit)........17
二、 Chelex 100 之概述....................22
(一) Chelex 之分類 ......................22
(二) Chelex100 之簡介 .....................22
(三) Chelex100 之特性 ...................23
(四) Chelex 100 DNA 萃取之相關技術研究.................25
1. 從人類萃取 DNA .....................................25
2. 從禽類萃取 DNA ................................27
三、鳥類性別鑑定相關研究技術 ..............................28
(一) 泄殖腔辦別法(vent sexing)...........................28
(二) 腹腔鏡手術法(laparoscopy)..........................29
(三) 性類固醇鑑定法(sex steroid hormone analysis)........29
(四) 核型分析法(karyotyping analysis)....................30
(五) DNA 分子技術檢驗法(DNA molecular marker analysis) ....30
1. CHD 基因.............................................30
2. EE0.6 基因 .........................................31
3. 逢機增殖多態性 DNA ..........................31
四、鳥之概述 ...............................35
(一) 鳥之演化.................................35
(二) 鳥之分類...................................35
參、試驗一 探討 Chelex 100 萃取禽類羽毛 DNA 之條件 .........37
一、前言 .........................37
二、材料與方法..................37
1. 試驗動物.....................................37
2. 試驗流程............................37
3. 試驗動物羽毛收集與 DAN 萃取:.........................40
4. 基因組 DNA 檢測濃度與吸光值.............................42
5. PCR 分析 ......................................45
6. 備製 2 %瓊脂糖膠以及電泳分析 PCR 產物....................46
三、結果與討論 ...............................47
肆、試驗二 探討以不同引子分辨鳥種 ............................65
一、前言 .......................................65
二、材料與方法 ...................65
1. 試驗動物................................65
2. 試驗流程......................................65
3. 試驗動物羽毛樣本採集以及 DNA 萃取.........................65
4. PCR 分析 .......................................69
5. 2 %瓊脂糖膠備製以及電泳分析 PCR 產物......................69
三、結果與討論 ..............................71
伍、結論................................................102
陸、參考文獻............................................103
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