十字花科黑腐病菌中参与5S rRNA剪切的核糖核酸酶的鉴定

发布时间：2018-07-04 22:34

  本文选题：十字花科黑腐病菌 + 5S　； 参考：《广西大学》2017年硕士论文

【摘要】：核糖体RNA(ribosomal RNA,简称rRNA)是所有生物体细胞中含量最多的一种RNA分子。rRNA与核糖体蛋白一起组成蛋白质合成的机器—核糖体,是维持生命必不可少的分子。细菌细胞中的rRNA包括23S rRNA、16S rRNA和5S rRNA。之前的研究发现,在大肠杆菌等细菌中,编码rRNA的基因成簇存在,成熟的23S、16S和5S rRNA由一个共同的rRNA前体分子通过核糖核酸酶(Ribonuclease,简称RNase)的剪切加工形成。基因组测序和基因注释结果显示,在十字花科黑腐病菌(Xanthomonas campestrs pv.campestris,Xcc 8004菌株(以下简称Xcc8004)基因组中,有2个rRNA基因簇。因此,Xcc8004成熟的rRNA是由这两个基因簇转录产生的rRNA前体经过RNase的剪切加工形成。但是,Xcc rRNA转录后加工过程以及参与rRNA剪切加工的RNase还不清楚。本研究的目的是鉴定参与Xcc8004 5S rRNA剪切加工的RNase。我们首先用Northern杂交的方法检测了 Xcc8004的成熟5S rRNA及其剪切加工产物。Northern杂交结果显示,在Xcc8004的总RNA中,除了一条119nt的成熟5SrRNA主带外,还检测到几条小于119nt的次条带,其中一条大约为100nt的剪切产物带信号最强,我们称之为“5S rRNA主剪切产物”。我们推测这个主剪切产物是由成熟的5S rRNA经RNase的剪切而来。为了鉴定参与5S rRNA剪切的RNase,我们用Northern杂交检测了 20个RNase基因(Xcc8004基因组共有22个RNase基因)的突变体中5S rRNA的剪切产物。结果发现,编码核糖核酸酶D(RNaseD)的基因(rnD)突变体中,≈1OOnt的主剪切产物带消失,说明RNaseD是这个主剪切产物的产生有密切关系。接着,我们构建了 rnD突变体的互补菌株以及rnD的过量表达菌株。Northern杂交结果显示,在互补菌株中,5S rRNA的主剪切产物得到了恢复;而在过量表达菌株中,5S rRNA的剪切明显加强了。这些结果证明,RNaseD在5SrRNA的剪切中起重要作用。为了明确RNaseD是否直接参与了 5S rRNA的剪切,我们分别纯化了RNase D蛋白和5S rRNA,并在体外检测了 RNase D蛋白对5S rRNA的剪切作用。结果表明,RNaseD能直接剪切5S rRNA。由于之前的研究表明,RNase D是细菌tRNA加工的关键酶,为此,我们在体外检测了 RNase D蛋白对来自Xcc8004的两个Met-tRNA(XC4335和XC4339)的剪切作用。结果显示RNase D能剪切这两种tRNA。说明Xcc8004的RNase D既能剪切5S rRNA也能剪切tRNA。为了弄清rnD基因的生物学功能,我们检测和比较了野生型菌株、rnD突变体和rnD突变体的互补菌株的胞外多糖产生、胞外酶活性、细胞运动能力、生物膜形成能力和致病力等表型。结果发现,rnD突变导致胞外多糖产量下降,而这一表型能被rnD基因反式互补;证明rnD基因与Xcc8004的胞外多糖的产生有关。本研究首次发现RNase D具有剪切5S rRNA的功能,拓展了人们对RNase D功能的认识。
[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，

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