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標題: | 題目:探討菸草幾丁質酵素基因3'端poly(A)形成信號對於蛋白質產生之影響 The Effects of 3' End Poly(A) Signals from Tobacco Endochitinase Gene in Protein Production |
作者: | Li-Feng Huang 黃麗芬 |
出版年 : | 1996 |
學位: | 碩士 |
摘要: | 在?核生物中,mRNA的合成,首先乃由RNA聚合酵素II於基因的?動子處開始執行轉錄的工作,當轉錄至基因3'端poly(A) signal後的10?30核甘酸時,新合成的RNA在poly(A) site被切斷,並加上poly(A) tail。在植物細胞中,部分基因的3'端並不含poly(A) signal;而另一部份基因的3'端則和一般?核細胞基因相似,含有poly(A) signal,但常含一個以上的poly(A) signals。因此植物的3'端調控機制,是一個有趣的研究課題。 經由基因銀行的搜尋,找到一個菸草幾丁質酵素基因,含有兩個poly(A) signals。使用PCR將此基因的3'端構築在pBI221SI載體之GUS基因後,最後以電穿孔法送入菸草原生質體中,觀測GUS的表現量。我們突變其中一個或二個poly(A) signals,以測試二個poly(A) signals的功能。接下來增加poly(A) signals的數目,及減少GUS基因與poly(A) signals之間的距離,觀察其對基因表現的影響。 由GUS表現?的觀測,得知二個poly(A) signals都有功能,但重要性並不相同。第二個poly(A) signal顯然較為重要。而減少GUS基因與poly(A) signals之間的距離,則會使GUS表現?明顯下降,但使用相同長度但序列不同的DNA片段取代,並不能回復GUS的表現?,顯示poly(A) signals之前含有cis-regulatory elements。至於增加poly(A) signals的數目,使總數達到四個,其GUS表現?反而降低,推測可能是蛋白質之間相互幹擾的結果。另外的數據也顯示poly(A) signals的數目越多,GUS的表現並沒有顯著的增加。 In eukaryotic cells, the 3' ends of most nascent mRNAs are processed by endonucleolytic cuts by the addition of poly(A) tail to become mature mRNAs. The functions of poly(A) tail are to enhance the stability of mRNA, increase the rate of translation efficiency, and regulate the translation of mRNA. In animal, poly(A) signals were defined by AAUAAA followed by GU-rich sequences in the 3' end of mRNA. Intensive studies have identified the functions of these poly(A) signals in mammals and yeast. In plants, less than half of known mRNAs contains AAUAAA signal, and little was known about the poly(A) signals in plant. The further identification and characterization of mRNA processing signals in plants should be analyzed. In this study, the 3' regulatory region containing two poly(A) signals from tobacco endochitinase gene was isolated by polymerase chain reaction, and inserted into the 3' regulatory region of GUS gene. The transient expression of reporter GUS gene indicates that the presence of poly(A) signal is required for significant amount of GUS expression. These two poly(A) signals function separately, and the second poly(A) signal from the stop codon is more important in gene expression than the first one. Also, when the distance between stop codon and poly(A) signals was decreased, the GUS expression was reduced. Even it was replaced with the same length fragment, the GUS expression was not restored. This may imply that cis-regulatory DNA elements in the upstream of poly(A) signal are involved in gene expression. Furthermore, GUS expression of variant with four poly(A) signals is lower than that of variant with two poly(A) signals. This result may be due to the interference of binding among processing proteins in the four poly(A) signal regions. With the series of four poly(A) signal mutants, the levels of these GUS expression implied that the more poly(A) signals do not cause the GUS expression to rise. There must be other cis-regulatory DNA elements in the 3' end region required for polyadenylation. However, the further study is required to understand the function of poly(A) signal in gene expression in detail. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76211 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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