拟南芥m60突变体的表型分析、基因克隆及基本功能预测

发布时间：2018-05-17 00:02

本文选题：拟南芥 + 根　；参考：《湖北大学》2016年硕士论文

【摘要】：核糖体生物合成是细胞中最基本的生物学过程之一,真核生物的核糖体是由47种结构蛋白和23S rRNA、5.8S rRNA、5S rRNA构成的60S亚基以及另外32种结构蛋白和18S rRNA构成的40S亚基所组成。rRNA的缺失会使得核糖体不能有效地行使翻译蛋白质的功能,从而导致一些代谢途径不能正常进行,生长发育过程即会表现出一系列的缺陷表型。本实验选用拟南芥(Arabidopsis thaliana)m60突变体植株为实验对象,逐步分析突变体表型继而对基因M60做功能预测分析。突变体m60植株较野生型有明显表型:植株生长矮小,腋芽发育生长旺盛,有性生殖发育过程迟缓;叶片形态发育异常,表现为叶面小,叶缘缺刻明显;主根生长迟缓,侧根发生明显增多,通过进一步的观察,我们发现突变体m60根尖的细胞数目明显少于野生型。经I-PCR的方法,确定该突变体中T-DNA单拷贝插入在基因M60第二个内含子上,经NCBI比对其CDS序列后,发现其是一个DEAD-boxRNA解旋酶编码基因。我们克隆出该基因的启动子区并连接报告基因构建表达载体,并结合qRT-PCR检测分析的结果,发现该基因在根和叶中都有大量表达。克隆该基因自身启动子连接基因全长构建回复载体,转基因之后能够完全回复缺陷表型,证实这些缺陷表型确实是该基因的突变所导致。真核生物的核糖体生物合成及其调控机制在芽殖酵母中的研究比较清楚,而高等植物中的相关研究却非常有限。拟南芥M60基因在酵母中同源基因是核仁定位,通过影响pre-rRNA的剪切成熟来影响核糖体60s大亚基的形成,这恰好也启示我们M60基因在拟南芥中也与核糖体发生过程相关。核糖体是细胞内蛋白合成的分子机器,既然拟南芥M60基因影响核糖体的发生,那势必会影响细胞内蛋白质的翻译水平,进而可能引起这一系列的表型。通过观测m60突变体表型,推测该基因可能与生长有关的代谢生理途径有关,所以这也为我们下一步寻找可能被影响到的下游基因奠定了重要的基础。
[Abstract]:Ribosomal biosynthesis is one of the most basic biological processes in cells. The deletion of ribosomes in eukaryotes is composed of 47 structural proteins and 5.8S rRNA5S rRNA of 23s rRNAs and 40s subunits composed of 32 other structural proteins and 18s rRNA. As a result, some metabolic pathways can not be normal, the growth and development process will show a series of defective phenotypes. In this experiment, Arabidopsis thaliana)m60 mutant plant was selected as the experiment object, the mutant surface type was analyzed step by step and the function of gene M60 was predicted. The mutant m60 has more obvious phenotype than wild type: plant growth is short, axillary bud growth is vigorous, sexual reproduction is slow, leaf morphology is abnormal, leaf surface is small, leaf edge is short, main root growth is slow. The number of cells in the root tip of the mutant m60 was significantly less than that of the wild type. By the method of I-PCR, the single copy of T-DNA was inserted into the second intron of gene M60 in this mutant. After comparing the CDS sequence of M60 with NCBI, it was found that it was a DEAD-boxRNA helicase encoding gene. We cloned the promoter region of the gene and ligated the reporter gene to construct the expression vector. Combined with the results of qRT-PCR analysis, we found that the gene was expressed in both roots and leaves. The full length of the gene promoter ligand gene was cloned to construct the response vector, and the defective phenotypes could be fully recovered after transgenic. It was proved that these defective phenotypes were caused by the mutation of the gene. The ribosomal biosynthesis of eukaryotes and its regulatory mechanisms are well understood in budding yeast, but the related studies in higher plants are very limited. The homologous gene of Arabidopsis M60 gene is nucleolus localization in yeast, which affects the formation of ribosomal 60s subunit by affecting pre-rRNA shearing maturation, which also enlightens us that M60 gene is also related to ribosomal development in Arabidopsis thaliana. Ribosome is a molecular machine for protein synthesis in cells. Since Arabidopsis M60 gene affects ribosome production, it will inevitably affect the level of protein translation in cells, which may lead to this series of phenotypes. By observing the surface type of m60 mutant, we speculate that the gene may be related to the metabolic physiological pathway related to growth, so this may also lay an important foundation for us to search for the downstream gene which may be affected next.
【学位授予单位】：湖北大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

【相似文献】