跳躍子flea插入let-7 complex基因座可能導致mir-100之低表現量，進而造成果蠅存活率降低和發育遲緩

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林伯駿(Bo-Chun Lin) 查詢紙本館藏

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生命科學系

論文名稱

跳躍子flea插入let-7 complex基因座可能導致mir-100之低表現量，進而造成果蠅存活率降低和發育遲緩
(Hypo-expression of mir-100, reduced survival rate, and delayed development in Drosophila melanogaster may result from the insertion of flea transposon in let-7 complex locus)

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摘要(中)

跳躍子flea插入let-7 complex 基因座可能導致mir-100之低表現量，進而造成果蠅存活率降低和發育遲緩

摘要

　　miRNA是小片段的RNA分子，約含21至23個鹼基，可藉由與mRNA的互補配對，抑制蛋白質轉譯。而miRNA cluster，可藉由poly-cistronic轉錄方式使miRNA表現。在let-7 cluster裡包含miR-100、let-7、miR-125這三個miRNA，這個cluster在前人的研究中被證實會在果蠅幼蟲轉變成蛹時表現。但是，一個以microarray的方式偵測miRNA表現的研究中，發現Canton S (CS-UCI)品系中miR-100在前蛹期表現量較其它miRNA或其它品系miR-100表現量較低之現象。本研究以該研究所使用之品系為對象，並以Oregon R (OR-UCI)和得自於長庚大學皮海薇老師實驗室的Canton S (CS-PI)探討miR-100表現量異於同cluster之其他miRNA的原因。經由一系列PCR實驗，發現在CS-UCI的mir-100與let-7區間有一段插入的片段，定序後進行BLAST序列比對，發現這個插入片段有98%的序列與flea跳躍因子相似，因此推測flea可能造成miR-100的低表現量。在不同溫度的蛹存活率測試中，觀察CS-UCI的蛹存活率，於高溫環境下顯著較低，因此推測miR-100可能具有影響蛹於高溫環境中耐受力的功能，這一個獨特的現象，讓我們可以了解miR-100在果蠅生長中扮演的角色。

關鍵詞：miRNA、cluster、mir-100、let-7 complex、果蠅發育

摘要(英)

Hypo-expression of mir-100, reduced survival rate, and delayed development in Drosophila melanogaster may result from the insertion of flea transposon in let-7 complex locus

ABSTRACT

　　MicroRNAs (miRNAs) are 21~23nt long RNAs, which regulate gene expression at post-transcriptional level by inhibiting the translation of target mRNAs through complementary pairing. Clustered miRNAs, such as let-7 cluster, are suggested to exhibit coordinate expression via a poly-cistronic transcription or inhibit translation. Let-7 cluster, consisting of miR-100, let-7, and miR-125, are increasingly expressed during larva-to-pupa transition in Drosophila melanogaster. However, the expression level of miR-100 was show to be lower than those of let-7 and miR-125 in Canton S strain as well as those of miR-100 in other strains in previous studies. In this study, we investigated the cause and the effects of low expression level of miR-100 in Canton S strain from UCI (CS-UCI) by using Oregon R (OR) strain and Canton S strains from Dr. Hai-wei Pi’s lab (CS-PI) as control strains. In qRT-PCR experiments, the expression level of miR-100 was confirmed to be low in CS-UCI but high in CS-PI and OR. A series of PCR experiments in genomic DNA revealed the presence of a flea element between mir-100 and let-7 in CS-UCI but not in CS-PI, suggesting the lower expression level of miR-100 might result from this insertion. Base on our preliminary tests, CS-UCI pupae showed significantly lower survival rate at 30℃ than the other two strains, suggesting miR-100 might involve in buffering heat stress during pupal development. The detail molecular mechanism on low expression level of miR-100 and its phenotypic effects in Drosophila development require further investigation.

Key words： microRNAs、miRNA cluster、mir-100、let-7 Complex、Drosophila development

關鍵字(中)

★ miRNA
★ cluster
★ mir-100
★ let-7 complex
★ 果蠅發育

關鍵字(英)

★ microRNAs
★ miRNA cluster
★ mir-100
★ let-7 Complex
★ Drosophila development

論文目次

第一章、緒論 1
1-1 MicroRNA的簡介 1
1-1-1 MicroRNA在生物體中的功能 1
1-1-2 最早發現的miRNA以及它的功能 1
1-1-3 MicroRNA的生合成 2
1-2　miRNA cluster的簡介 3
1-2-1 miRNA cluster在生物體的定義 3
1-2-2 miRNA cluster表現的調控方式 3
1-2-3 miRNA cluster如何演化而來 4
1-3　let-7 cluster的介紹與調節功能 5
1-3-1 let-7 cluster的簡介 5
1-3-2 let-7 cluster在果蠅中的功能 5
1-3-3 miR-100在其他物種的功能 6
第二章、材料方法 7
2-1 果蠅品系與培養環境 7
2-2　檢測各果蠅品系let-7-C的區段序列 7
2-2-1 總核酸 (Total DNA)萃取 7
2-2-2引子設計 8
2-2-3 聚合?鏈鎖反應放大 let-7-C的區段序列 8
2-2-4 DNA片段的純化與序列分析 9
2-3　檢測果蠅let-7-C轉錄成的primary RNA 9
2-3-1 總核糖核酸的萃取 9
2-3-2 let-7 cluster pri-miRNA的片段組成 10
2-3-3 miR-100的相對表現量 10
2-3-4 qPCR實驗數據的統整與分析 11
2-4　miR-100功能性分析 11
2-4-1 蛹期的存活率 11
2-4-2 miR-100對發育時間所造成的影響 12
2-4-3統計分析方法 13
第三章、結果 14
3-1　在CS-UCI中mir-100與let-7間插入了flea跳躍因子 14
3-2　Primary miRNA在各品系的合成差異 16
3-3　miR-100在不同品系果蠅中前蛹期的表現量 17
3-4　在果蠅中miR-100的功能預測 18
3-4-1 在不同溫度中蛹的存活率 18
3-4-2 在25℃果蠅的存活率與發育時間 19
第四章、討論 21
4-1 flea可能影響CS-UCI裡的miR-100表現量 21
4-2 在果蠅中miR-100的功能 23
第五章、參考文獻 25
圖表 28
附錄一、檢測在let-7-C的區段大小 39
附錄二、定序mir-100與let-7之間的區段 40
附錄三、RT-PCR 區段 (B)的定序結果 41
附錄四、比對分析RT-PCR區段(B) 42

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指導教授

葉淑丹(Shu-Dan Yeh)

審核日期

2017-1-20

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