臺灣博碩士論文加值系統

中文摘要
本論文主要目的係探討台灣雨傘節中-bungarotoxin (-BuTX)之mRNAs的基因結構與特性。我們以Cap-finder cDNA synthesis方法從台灣雨傘節毒腺mRNAs中選殖出完整序列的-BuTX mRNA基因結構體：包括5’-untranslated region (5’-UTR；33bp)、signal peptide (63 bp)、protein coding region (222 bp)及3’-untranslated region (3’-UTR ；195 bp，其中含一組TGA終止密碼與AATAAA signal polyadenylation序列)，因此-BuTX mRNA的基因總長度約為530 bp，且此-BuTX 5’-UTR及3’-UTR序列與海生及陸生蛇科(Hydrophilidae及Elapidae)的蛇毒基因之5’-UTR及3’-UTR幾乎完全一致。另一方面我們亦定序出16種polymorphic -BuTX isoforms之 cDNA (定為R1至R16) 並分析其佔有率，接著我們亦分析-BuTX genomic DNA的序列，發現其蛋白質轉譯區(protein coding region)僅呈現出一種序列 (且與R1的序列相同)，並無多樣性變異的情形發生，顯示-BuTX mRNAs之多樣性是源自同一種-BuTX DNA的序列。另外我們發現，台灣雨傘節-BuTX mRNAs的變異大多數是屬於silent mutations，且常發生在-BuTX mRNAs的第78、107、129、198及201 nt的位置。而且從16種Polymorphic -BuTX mRNAs序列中可推算出10種不同-BuTX蛋白質序列，其中一種isoform已有文獻發表與証實它之存在。另外，這16種Polymorphic -BuTX isoforms之 cDNA，若以電腦程式推算這些異構蛋白質之等電點 (isoelectric point；pI)，可得到兩群不同等電點之異構物，為了証實台灣雨傘節蛇毒中具有-BuTX isoforms，我們將HPLC純化之台灣雨傘節蛇毒進行2-D電泳(two-dimentional electrophoresis)，及合併Western blotting作用，結果轉漬膜上有兩個-BuTX抗原的反應區，而這兩個抗原反應區具有相同分子量但不同等電點，且其等電點與電腦程式預測之結果相吻，因此台灣雨傘節蛇毒中確實有-BuTX isoforms 的存在。接著以PCR方法分析-BuTX DNA 與cDNA的coding region，發現台灣雨傘節之-BuTX DNA並不含intron，繼以 partial digestion 及Southern hybridization方法，偵測到-BuTX DNA是以單一模板(single copy)的方式存在於台灣雨傘節蛇毒基因體(genome)中。綜合上述結果，台灣雨傘節-BuTX mRNAs具多樣性序列，可能是由RNA editing(即Post Transcriptional RNA Editing)的結果所產生的。RNA editing可能是促使蛇類的蛇腺細胞中能有多樣化之-neurotoxins 發展的原因，亦是使台灣雨傘節蛇毒在環境的適應上具演化的優勢。

ABSTRACT
The mRNA encoding -bungarotoxin (-Butx) was prepared from the venom glands of Bungarus multicinctus by Cap-finder cDNA synthesis. The sequences of 3’- and 5’-flanking regions including a signal peptide of-Butx were almost identical with those of elapidae and hydrophiidae toxins, suggesting that they may have the same origin. Sixteen polymorphic mRNA sequences of -Butx were detected from B. multicinctus gland cells. Analysis of the genomic DNA of -Butx indicated that the polymorphic mRNA was originated from one DNA sequence. Most of the mutations in -Butx mRNA were silent and the hot-spot variations occurred at 78, 107, 129, 198 and 201 nt in -Butx mRNA. Ten distinct protein sequences of -Butx could be deduced from the polymorphic mRNA, and one of the isoforms has already been isolated. Since -Butx DNA is a single copy in the genome, the RNA polymorphism might result from post-transcriptional editing. These results indicate that the authentic -Butx is in fact derived from edited mRNAs. RNA editing may contribute a common mechanism toward the diversity of -neurotoxins in snake glands.

目 錄
頁次
臺：中文摘要--------------------------------------------------------4
貳：英文摘要--------------------------------------------------------6
參：第一章 緒論---------------------------------------------------7
肆：研究材料-------------------------------------------------------19
伍：第二章 台灣雨傘節 -BuTX cDNA Library選殖----21
1. 實驗方法與步驟---------------------------------------------- 22
2. 結果與討論-----------------------------------------------------30
3. 圖表--------------------------------------------------------------34
陸：第三章 台灣雨傘節 -BuTX RNA Editing的現象---44
1. 實驗方法與步驟-----------------------------------------------45
2. 結果與討論-----------------------------------------------------51
3. 圖表--------------------------------------------------------------55
柒：第四章 台灣雨傘節 -BuTX 異構物(Isoforms)
及重組蛋白質的表現----------------------------60
1. 實驗方法與步驟-----------------------------------------------61
2. 結果與討論-----------------------------------------------------69
3. 圖表--------------------------------------------------------------73
捌：討論--------------------------------------------------------------84
玖：總結--------------------------------------------------------------89
拾：參考文獻--------------------------------------------------------90
拾壹：附錄----------------------------------------------------------105

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