本文選題：十字花科黑腐病菌 + 5S��； 參考：《廣西大學》2017年碩士論文

【摘要】：核糖體RNA(ribosomal RNA,簡稱rRNA)是所有生物體細胞中含量最多的一種RNA分子。rRNA與核糖體蛋白一起組成蛋白質(zhì)合成的機器—核糖體,是維持生命必不可少的分子。細菌細胞中的rRNA包括23S rRNA、16S rRNA和5S rRNA。之前的研究發(fā)現(xiàn),在大腸桿菌等細菌中,編碼rRNA的基因成簇存在,成熟的23S、16S和5S rRNA由一個共同的rRNA前體分子通過核糖核酸酶(Ribonuclease,簡稱RNase)的剪切加工形成。基因組測序和基因注釋結(jié)果顯示,在十字花科黑腐病菌(Xanthomonas campestrs pv.campestris,Xcc 8004菌株(以下簡稱Xcc8004)基因組中,有2個rRNA基因簇。因此,Xcc8004成熟的rRNA是由這兩個基因簇轉(zhuǎn)錄產(chǎn)生的rRNA前體經(jīng)過RNase的剪切加工形成。但是,Xcc rRNA轉(zhuǎn)錄后加工過程以及參與rRNA剪切加工的RNase還不清楚。本研究的目的是鑒定參與Xcc8004 5S rRNA剪切加工的RNase。我們首先用Northern雜交的方法檢測了 Xcc8004的成熟5S rRNA及其剪切加工產(chǎn)物。Northern雜交結(jié)果顯示,在Xcc8004的總RNA中,除了一條119nt的成熟5SrRNA主帶外,還檢測到幾條小于119nt的次條帶,其中一條大約為100nt的剪切產(chǎn)物帶信號最強,我們稱之為“5S rRNA主剪切產(chǎn)物”。我們推測這個主剪切產(chǎn)物是由成熟的5S rRNA經(jīng)RNase的剪切而來。為了鑒定參與5S rRNA剪切的RNase,我們用Northern雜交檢測了 20個RNase基因(Xcc8004基因組共有22個RNase基因)的突變體中5S rRNA的剪切產(chǎn)物。結(jié)果發(fā)現(xiàn),編碼核糖核酸酶D(RNaseD)的基因(rnD)突變體中,≈1OOnt的主剪切產(chǎn)物帶消失,說明RNaseD是這個主剪切產(chǎn)物的產(chǎn)生有密切關(guān)系。接著,我們構(gòu)建了 rnD突變體的互補菌株以及rnD的過量表達菌株。Northern雜交結(jié)果顯示,在互補菌株中,5S rRNA的主剪切產(chǎn)物得到了恢復;而在過量表達菌株中,5S rRNA的剪切明顯加強了。這些結(jié)果證明,RNaseD在5SrRNA的剪切中起重要作用。為了明確RNaseD是否直接參與了 5S rRNA的剪切,我們分別純化了RNase D蛋白和5S rRNA,并在體外檢測了 RNase D蛋白對5S rRNA的剪切作用。結(jié)果表明,RNaseD能直接剪切5S rRNA。由于之前的研究表明,RNase D是細菌tRNA加工的關(guān)鍵酶,為此,我們在體外檢測了 RNase D蛋白對來自Xcc8004的兩個Met-tRNA(XC4335和XC4339)的剪切作用。結(jié)果顯示RNase D能剪切這兩種tRNA。說明Xcc8004的RNase D既能剪切5S rRNA也能剪切tRNA。為了弄清rnD基因的生物學功能,我們檢測和比較了野生型菌株、rnD突變體和rnD突變體的互補菌株的胞外多糖產(chǎn)生、胞外酶活性、細胞運動能力、生物膜形成能力和致病力等表型。結(jié)果發(fā)現(xiàn),rnD突變導致胞外多糖產(chǎn)量下降,而這一表型能被rnD基因反式互補;證明rnD基因與Xcc8004的胞外多糖的產(chǎn)生有關(guān)。本研究首次發(fā)現(xiàn)RNase D具有剪切5S rRNA的功能,拓展了人們對RNase D功能的認識。
[Abstract]:Ribosomal RNAs (rRNAs) are one of the most abundant RNAs in all biological cells. RRNAs, together with ribosomal proteins, constitute ribosomal ribosomes, which are essential molecules for life support. The rRNA in bacterial cells includes 23s rRNAs 16s rRNA and 5S rRNA. Previous studies have found that in bacteria such as Escherichia coli, genes encoding rRNA are clustered and mature 23s and 5s rRNA are formed by a common rRNA precursor molecule that is shredded by ribonuclease (RNase). Genome sequencing and gene annotation showed that there were two rRNA gene clusters in the genome of Xanthomonas campestrs pv. campestris Xcc 8004 (Xcc8004). Therefore, the mature rRNA of Xcc8004 is produced by the transcription of rRNA precursor from these two gene clusters through the processing of RNase. However, the post-transcriptional processing of XCC rRNA and the RNase involved in rRNA shearing are unclear. The aim of this study was to identify the RNase involved in the processing of Xcc8004 5S rRNA. We first detected the mature 5s rRNA of Xcc8004 and its shearing products by Northern blot. Northern blot analysis showed that in the total RNA of Xcc8004, in addition to one mature 5s rRNA main band of Xcc8004, several sub-bands smaller than 119nt were also detected. One of the shearing product bands about 100nt has the strongest signal, which we call the "5S rRNA main shearing product". We conjectured that the main shearing product was derived from mature 5S rRNA shearing by RNase. In order to identify the RNase involved in 5S rRNA splicing, we used Northern blot to detect the 5s rRNA shearing products in 20 mutants of RNase gene (there are 22 RNase genes in the Xcc8004 genome). It was found that the main shearing product band of 鈮，

本文編號：2097726