探討菸草幾丁質酵素基因的poly(A)內形成信號對Poly(A)形成長度和mRNA穩定度的影響

Abstract

在真核細胞中，基因的表現需要產生成熟的RNA。要產生成熟的mRNA需要先對pre-mRNA作修飾作用。當pre-mRNA的3'端被修飾時，在3'端的非轉錄區域有poly(A) signals。poly(A) signal對poly(A)的形成佔有很重要的地位。本實驗室已經發現不同的poly(A) signal會影響GUS基因蛋白質的表現量與poly(A)斷點的選擇。因此想要更進一步觀察不同的poly(A) signal是否影響GUS RNA的Poly(A)形成長度或是GUS RNA的穩定度。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。經過突變復，分別接在載體pBI121上。以農稈菌成染菸草圓片法產生五種不同菸草轉殖株。轉殖株分別是含有兩個完整poly(A) signals (ATX)、第一個Poly(A) signals破壞(AXT)、第二個poly(A) signals破壞(ATX)、兩個完整poly(A) signals均加以破壞(AXTX)、增為四個poly(A) signals (ATAT)。實驗中發現在轉殖植物中會產生三個斷點，分別在第一個poly(A) signal之前、兩個poly(A) signal之間與第二個poly(A) signal之後；而且發現不同的poly(A) signal被破壞會改變poly(A)長度的分佈。當兩個poly(A) signal都完整時，poly(A)長度最長、一個被破壞時次之，當兩個都被破壞最短。因此poly(A) signal的個數與poly(A)長度有相關性。在這個GUS基因連結到菸草幾丁質酵素基因的3'端之建構中，當GUS基因使用第二個poly(A)斷點時，在AT的GUS RNA half-life是10.48分鐘、AXT是66.8分鐘和AXTX的89.3分鐘。雖然AXT和AXTX的half-life是AT的6-8倍，但是mRNA的相對量卻是AXT和AXTX的3-7倍。因此，基因的表現是由RNA的量所決定而非RNA的half-life所決定。當GUS基因使用第三個poly(A)斷點時，RNA的表現模式亦與使用第二斷點類似。總結以上的資料認為GUS基因蛋百質的表現與RNA的量有較大相關而與half-life關係較小。

In eukaryotic cells, to produce mature mRNA needs processing of pre-mRNA. When pre-mRNA is processed, the poly(A) signals in the 3?? untranslated region are the most important for 3' processing. In our laboratory, we found that different poly(A) signals influence GUS gene expression and the polyadenylation site. Thus we propose to study the influence of the mRNA with different polyadenylation sites on its stability and the effect of the different poly(A) signals on poly(A) length. Two poly(A) signals within tobacco endochitinase gene were cloned into pBI12l vector. Five different constructs were constructed, and they are AT、AXT、ATX、AXTX and ATAT. Five transgenetic tobaccos were obtained by agrobacterium infecting tobacco leaf disc. Our study indicates that different poly(A) signals influence the length of poly(A) tail. When there are two functional poly(A) signals in the 3' untranslated region, the length of polyadenylation was improved. However, the number of functionals poly(A) signals is not related to the length of poly(A) tail. Furthermore there are two conserved poly(A) signals in the endochitinase gene. The half life of the GUS mRNA which is polyadenylation at the second site in AT is 10.48 minute compared with that of AXT 66.8 minute and that of AXTX 89.3 minute. The half life of AXT and AXTX is almost 3-9 times of AT. But the amount of AT mRNA is 3-7 time more than AXT and AXTX mRNA. Therefore, the amount of mRNA rather than its half life determine the amount of protein. The pattern of RNA half-life is almost the same in all constructs with the RNA which is polyadenylation at third polyadenylation site. We conclued that the poly(A) length is determined by the second poly(A) signal, and the expression of GUS protein is much related to the amount of mRNA present but not half-life of RNA.