探討菸草幾丁質酵素基因的poly(A)形成信號對poly(A)形成位置和mRNA穩定度的影響

Abstract

在真核細胞中，各種生理代謝作用是由基因先經轉錄作用產生pre-mRNA，pre-mRNA經修飾成為成熟的mRNA後，再經轉譯作用所產生的蛋白質來進行調控。而基因要形成成熟的mRNA，大多需要位元在基因3'非轉譯區域的poly(A) signal來指引polyadenylation的進行。由於本實驗室先前已發現poly(A) signal對蛋白質的表現量確有影響，因此對於植物中的poly(A) signal對mRNA的polyadenylation及穩定度的影響這兩方面做進一步的研究。 先將含有兩個poly(A) signals的菸草幾丁質酵素基因，構築在載體上，並經突變後，再接在載體pBI121上，以農桿菌感染菸草葉圓片的方式，產生五種不同的菸草轉殖植株，包括含有菸草幾丁質基因的兩個完整poly(A) signals (p100)、第一個poly(A) signal被破壞（pAxT）、第二個poly(A) signal被破壞（pATx）、兩個poly(A) signals均加以破壞（pAxTx）、增為四個poly(A) signals (paATAT)的不同轉殖植株。 在poly(A) signal對mRNA進行polyadenylation的影響上，發現在二個或二個以上的poly(A) signals之使用上，mRNA進行polyadenylation時似乎較偏好使用第一個poly(A) signal；但poly(A) signal的序列會進一步影響poly(A) site位置使用的頻率，研究結果發現若在二個或以上的poly(A) signals之使用上，序列完全符合AAUAAA的poly(A) signal會使得其後的poly(A) site的使用頻率大為提高。 在mRNA穩定度的研究結果發現，五種轉殖植株中，p100、pAxT轉殖株的GUS基因mRNA半衰期最長，可達三個小時左右；其他pATx、pAxTx、paATAT三種轉殖株的GUS基因的mRNA半衰期則明顯偏低，約一個多小時左右；這結果與本實驗室先前所做蛋白質短暫表現的情況相符。因此結合poly(A) sites的分佈實驗結果，我們推測mRNA越長，也就是poly(A) sites在越遠的地方形成，mRNA越穩定；而當mRNA越穩定，蛋白質的表現量就越高。

In eukaryotic cells, the mature mRNAs are produced by the processing of pre-mRNAs. To become mature mRNAs, the poly(A) signals in the 3' untranslated region are needed to direct the polyadenylation. In our laboratory, we have found that the poly(A) signals have influences on the gene expression. The purpose of this study is to analyze the effects of the poly(A) signals on the mRNA stability and polyadenylation in plants. Two poly(A) signals within tobacco endochitinase gene are mutated and cloned into pBI121 vectors. Five different transgenic tobaccos, including p100, pAxT, pATx, pAxTx, and paATAT, are generated by using Agrobacterium to infect leaf disks. Our study indicates that most poly(A) sites are formed at the downstream of the first poly(A) signal. Also, the poly(A) sites are affected by the sequence of the poly(A) signals if there are two or more than two poly(A) signals in the 3' untranslated region. The efficiency of the polyadenylation is improved when the sequence of the poly(A) signal is AAUAAA. The half-life of the GUS mRNA in the p100 and pAxT transgenic plants is almost three hours, which is much longer than the other tested transgenic plants whose half-life is almost one hour. Based on the position of the poly(A) sites, we conclude that the mRNA stability is controlled by the length of the mRNA, which is determined by the position of the poly(A) sites. Furthermore, the mRNA stability may be a major factor to affect the amount of GUS expression from the study of transient expression of GUS gene.