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研究生:洪雅玲
論文名稱:酵母菌Rad51與Rad54拮抗DNA錯誤配對修復系統 以促進同種雜交子代可孕性
論文名稱(外文):Yeast Rad51 and Rad54 antagonize the mismatch repair system to promote intraspecies hybrid fertility
指導教授:王廷方
指導教授(外文):WANG, TING-FANG
口試委員:薛一蘋張永龍王廷方
口試委員(外文):HSUEH, YI-PINGCHANG, YUNG-LUNGWANG, TING-FANG
口試日期:2023-12-06
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2024
畢業學年度:112
語文別:中文
論文頁數:63
中文關鍵詞:減數分裂同源重組交叉干擾錯誤配對修復系統
外文關鍵詞:MeiosisHomologous recombinationCrossover interferenceMismatch repair(MMR)system
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多數真核生物的生殖細胞或合子進行減數分裂時,先產生雙股DNA斷裂、經同源染色體間配對、重組與聯會等機制,修補破損雙股DNA,使得同源染色體間DNA序列發生互換,最終形成重組產物,產生遺傳變異。過去研究顯示同源染色體間重組產物有兩種類型,第一型「交差重組產物」需要參與聯會的ZMM蛋白質。第二型「非交差重組產物」需要DNA內切酵素Mms4-Mus81。第一次染色體分離時,第一型「交差重組物」產會形成「染色體交叉(chiasma)」,確保紡錘體能將成對同源染色體平均分離。過多或不足的交差重組產物都會造成染色體不正常分離。異種雜交因親代基因體序列不完全相同,進行同源染色體配對與重組反應時,經常產生鹼基錯誤配對。後者被錯誤配對修復系統辨識後,會抑制同源染色體配對重組反應進行,造成不同物種間生殖隔離。本論文利用酵母菌SK1與S288c同種不同品系酵母菌株的雜交合子為研究模式,探討「同源染色體配對重組反應」與「錯誤配對修復系統」在減數分裂的關聯性。因爲同種,SK1與S288c的雜交合子可完成減數分裂同源染色體配對重組反應,產生可存活或具生育能力的有性孢子。我們比較純品系(SK1/SK1)合子與雜交品系(SK1/S288)合子的孢子存活率、交差重組產物發生機率與交差重組產物間干擾等特性,從而發現DNA重組酵素Rad51與其輔助蛋白Rad54在同種雜交合子能專一性的拮抗錯誤配對修復系統,抑低過多的交差重組產物,提升雜交合子的生育力。
During meiosis, various mechanisms such as homologous pairing, homologous recombination, and chromosome synapsis occur to repair programmed DNA double-strand DNA breaks. Ultimately, the exchanges of DNA sequences between homologous chromosomes result in genetic variation. Previous studies revealed two types of interhomolog (IH) crossing-over recombination products: the class I IH-crossover (CO) requires the involvement of ZMM proteins which are also required for chromosome synapsis; the class II IH-CO products necessitate the DNA endonuclease Mms4-Mus81. The class I IH-CO products can form "chiasmata" to ensure proper segregation of paired homologous chromosomes during the first meiotic nuclear division. Insufficient or excessive class I IH-CO products often result in unfaithful chromosome segregation, leading to infertility. Previous studies had revealed that the mismatch repair (MMR) system is the major barrier to genetic recombination in interspecies hybrid zygotes, leading to hybrid infertility. Notably, MMR is not an absolute barrier for DSB repair in intraspecies hybrid zygotes (e.g., budding yeast Saccharomyces cerevisiae SK1/S288c diploid), even though there is a high degree of sequence heterogeneity between the haploid genome sequences of SK1 and S228c. Through comparison of different characteristics between intraspecies pure and hybrid diploid strains, including spore viability, CO occurrence frequency, and CO interference characteristics, we report that DNA recombinase Rad51 and its auxiliary protein Rad54 specifically antagonize the MMR system in intraspecies hybrid zygotes to prevent superfluous IH-CO products, thereby enhancing their fertility.
目錄 I
圖目錄 IV
縮寫表 V
中文摘要 VII
英文摘要 VIII
第一章 : 緒論 1
1.1 減數分裂同源重組反應簡介 1
1.2 減數分裂同源重組產物在遺傳學與分子生物學的重要性 4
1.3 減數分裂對物種演化的重要性 6
1.4 為何同種雜交酵母菌株雖具高量DNA序列差異卻仍能產生數量可觀的活孢子 8
第二章 : 實驗材料與方法 9
2.1 酵母菌轉型 9
2.2 同步化酵母菌減數分裂週期 10
2.3 DAPI染色法定量完成減數分裂第一次染色體分離百分比 12
2.4 酵母菌螢光孢子拍攝方法 13
2.5 DiBAC4(5)染色法與流式細胞儀定量產孢率 13
2.6 利用減數分裂四分體分離法分離單一孢子與定量孢子存活率 14
2.7 定量減數分裂交叉發生頻率與交叉干擾 14
2.8 實驗菌株 16
2.9 實驗培養液 20
2.10 統計方式 21
第三章 : 結果 22
3.1 比較純品系二倍體SK1/SK1與雜交品系二倍體SK1/S288c孢子存活率 22
3.2 喪失拮抗DNA錯誤配對修復系統生物活性不影響產孢率 25
3.3 拮抗DNA錯誤配對修復系統生物活性與完成減數分裂第一次染色體分離百分比關聯性 26
3.4 拮抗DNA錯誤配對修復系統基因活性對交叉發生頻率與交叉干擾的影響 27
3.5 拮抗DNA錯誤配對修復系統基因活性不會造成第一次減數分裂染色體不正常分離 30
第四章 : 討論 31
第五章 : 結論 33
第六章 : 參考資料 43

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