通过核提取和分类策略,研究Arabidopsis thaliana内核复制过程中的mRNA水平的协议
Karin Rothkegel1, Alex Cabrera Núñez2, Rodrigo A Gutiérrez3
1Agencia Nacional de Investigación y Desarrollo-Millennium Science Initiative Program, Millenium Institute for Integrative Biology (iBio), Fundación Ciencia y Vida, Santiago 7500565, Chile; Center for Genome Regulation (CRG), Santiago 7510000, Chile; Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago 8331010, Chile.
STAR protocols
|January 29, 2026
概括
这项研究详细介绍了一项用于分析Arabidopsis thaliana cotyledons.植物内复制过程中mRNA变化的协议. 它使得研究与细胞生长和性变化相关的基因表达转变成为可能.
科学领域:
- 植物生物学 植物生物学
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
背景情况:
- 植物中的细胞分化和扩张通常涉及细胞循环停止和内复制.
- 内核复制 (Endoreplication) 是一种没有细胞分裂的DNA复制过程,导致性水平的增加.
- 了解内核复制过程中的基因表达变化对于植物发育至关重要.
研究的目的:
- 建立一个详细的协议,用于调查mRNA水平的变化,在内核复制在阿拉比多普西斯·塔利亚纳cotyledons.
- 提供一种基于 ploidy 的原子核隔离和分类方法.
- 为了能够在不同种类水平上进行定量基因表达分析.
主要方法:
- 从Arabidopsis thalianacotyledons. 的总核的分离.
- 根据DNA含量 (ploidy水平) 进行基于流细胞计的核的分类.
- 从排序核中提取RNA和定量实时PCR (qPCR) 用于基因表达分析.
主要成果:
- 开发了一个强大的协议,用于研究在内核复制过程中的mRNA动态.
- 该方法允许根据其性状态分离原子核.
- 家政和标志性基因的基因表达可以在不同水平上量化.
结论:
- 描述的协议有助于研究植物内复制过程中的基因表达调节.
- 这种方法对于理解细胞分化和扩张背后的分子机制非常有价值.
- 它为未来的研究提供了一个基础,用于植物中依赖于 ploidy 的基因调节.
更多相关视频
相关概念视频
02:22Regulated mRNA Transport
7.0K
In eukaryotes, transcription and translation are compartmentalized; an mRNA is first synthesized in the nucleus and then selectively transported to the cytoplasm for protein synthesis. Before transport, a pre-mRNA undergoes several steps of post-transcriptional modifications including splicing, 5' capping, and the addition of a poly-adenine tail. Various proteins bind to the pre-mRNA during these modifications. The mRNA transport takes place with the help of multiple proteins playing...
7.0K
02:01pre-mRNA Processing
57.5K
In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl...
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl...
57.5K
02:31Nuclear Export of mRNA
8.8K
Before mRNAs are exported to the cytoplasm, it is crucial to check each mRNA for structural and functional integrity. Eukaryotic cells use several different mechanisms, collectively known as mRNA surveillance, to look for irregularities in mRNAs. Irregular or aberrant mRNA are rapidly degraded by various enzymes. If a defective mRNA escapes the surveillance, it would be translated into a protein which would either be non-functional or not function properly. One of the primary irregularities in...
8.8K
02:31Nuclear Export of mRNA
5.4K
5.4K
02:51mRNA Stability and Gene Expression
6.6K
The structure and stability of mRNA molecules regulates gene expression, as mRNAs are a key step in the pathway from gene to protein. In eukaryotes, the half-life of mRNA varies from a few minutes up to several days. mRNA stability is essential in growth and development. The absence of the proteins regulating its stability, such as tristetraprolin in mice, can cause systemic issues, including bone marrow overgrowth, inflammation, and autoimmunity.
Cis-acting Elements involved in mRNA stability
Cis-acting Elements involved in mRNA stability
6.6K
02:27Nonsense-mediated mRNA Decay
11.8K
The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
11.8K