荧光显微镜筛选和新一代测序：参与细胞器完整性的基因鉴定的有用工具
Summary
在细胞生物学的一个基本任务是定义机制的基础，使真核细胞的细胞器的身份。在这里，我们提出了一个方法，以确定该基因的植物细胞器，利用荧光显微镜和下一代测序工具的形态和功能的完整性负责。
Abstract
本协议描述了拟南芥幼苗的荧光显微镜为基础的筛选，并介绍如何将隐性突变，改变了一个特定的标记分泌途径中的荧光标记的亚细胞分布。 拟南芥是一种强大的生物遗传研究模型，因为它的基因组大小，一代的时间，并节约王国之间的分子机制。作为一种方法来映射替代传统的方法，基于分子标记的突变基因分型阵列是有利的，因为它是相对较快，可能允许在一个非常短的时间框架的几个突变的映射。这种方法允许蛋白质的鉴定，可以影响任何在植物细胞器的完整性。在这里，作为一个例子，我们提出了一个重要的内质网（ER）的完整性的地图基因的屏幕。然而，我们的做法，可以很容易地扩展到其他植物细胞器（例如，见1,2），因此代表了对理解其他亚细胞结构的分子基础的重要一步。
Protocol
1。 EMS处理 拟南芥种子的诱变诱变剂乙基甲烷磺酸盐（EMS），3,4，这将导致基因组的C-T的变化的C / G / A突变5-7使用。 权衡0.8Ğ拟南芥种子携带的细胞器荧光标记（特别是，在这项研究中，ssGFPHDEL（信号序列的GFP-HDEL四肽）已作为ER标记）（〜40,000种子）。 在50毫升的猎鹰管转移的种子，然后加入25毫升蒸馏水。 添加0.2％（V / V）乙基…
Discussion
在这里，我们描述了共聚焦显微镜为基础的筛选为内膜突变体鉴定。这种方法可以很容易地扩展到其他细胞的细胞器，为特定的荧光蛋白标记。基于屏幕的鉴定表明，无论是在目标器官或细胞器，不应该包含标记荧光标记的异常分布的突变。 ，这些突变体分别代表人群中受到损害的器官的能力subcompartimentalize标记或突变体不能正常转运细胞器之间的标记。在筛选中，我们注意到，一些突变表明，?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们承认，化学，地质和生物科学部，办公室基础能源科学，科学办公室，美国能源部（奖DE-FG02-91ER20021）和国家科学基金会（MCB 0948584）（FB）的支持。我们感谢编辑手稿女士卡伦鸟的。
Materials
| Name of the reagent | Company | Catalogue number |
| Ethylmethane sulfonate | Sigma | M0880 |
| NaOH | J.T Baker | 3722-05 |
| Murashige skoog basal medium w gamborg vitamins | Phyto technolog laboratorie | M404 |
| Phytagel | Sigma | P8169-1Kg |
| RNeasy mini kit | Qiagen | 74104 |
| Master pure plant leaf DNA purification kit | Epicentre | MPP92100 |
| Bioprime DNA labeling system | Invitrogen | 18094-011 |
| Alcohol 200 proof | Decan laboratories inc. | 2716 |
| NaOAc | J.T Baker | |
| Gene chip Arabidopsis ATH1 genome array | Affymetrix | 900385 |
| Falcon tubes 50 mL | corning | 430290 |
| Eppendorf tubes 1.5 mL | ||
| Filter paper 90mm | Whatman | 1001090 |
| Analytical Balance | Mettler Toledo AB54-S | n.a |
| Nutating (wave) shaker | Heidolph polymax 1040 | n.a |
| Centrifuge | Eppendorf 5417-R | n.a |
Referências
- Marti, L. A missense mutation in the vacuolar protein GOLD36 causes organizational defects in the ER and aberrant protein trafficking in the plant secretory pathway. The Plant journal : for cell and molecular biology. 63, 901-913 (2010).
- Stefano, G., Renna, L., Moss, T., McNew, J., Brandizzi, F. Arabidopsis the spatial and dynamic organization of the endoplasmic reticulum and Golgi apparatus is influenced by the integrity of the c-terminal domain of RHD3, a non-essential GTPase. The Plant Journal. , (2011).
- Kim, Y., Schumaker, K. S., Zhu, J. K. EMS mutagenesis of Arabidopsis. Methods in molecular biology. 323, 101-103 (2006).
- Maple, J., Moller, S. G. Mutagenesis in Arabidopsis. Methods in molecular biology. 362, 197-206 (2007).
- Greene, E. A. Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genética. 164, 731-740 (2003).
- Krieg, D. R. Ethyl methanesulfonate-induced reversion of bacteriophage T4rII mutants. Genética. 48, 561-580 (1963).
- Kovalchuk, I., Kovalchuk, O., Hohn, B. Genome-wide variation of the somatic mutation frequency in transgenic plants. The EMBO journal. 19, 4431-4438 (2000).
- Boulaflous, A., Faso, C., Brandizzi, F. Deciphering the Golgi apparatus: from imaging to genes. Traffic. 9, 1613-1617 (2008).
- Borevitz, J. Genotyping and mapping with high-density oligonucleotide arrays. Methods in molecular biology. 323, 137-145 (2006).
- Konieczny, A., Ausubel, F. M. A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. The Plant journal : for cell and molecular biology. 4, 403-410 (1993).
- Bell, C. J., Ecker, J. R. Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics. 19, 137-144 (1994).
- Hazen, S. P., Kay, S. A. Gene arrays are not just for measuring gene expression. Trends in Plant Science. 8, 413-416 (2003).
- Hazen, S. P. Rapid array mapping of circadian clock and developmental mutations in Arabidopsis. Plant Physiology. 138, 990-997 (2005).
- Bentley, D. R. Accurate whole human genome sequencing using reversible terminator chemistry. Nature. 456, 53-59 (2008).
- Weigel, D., Glazebrook, J. Arabidopsis. A Laboratory Manual. , (2002).
- Voelkerding, K. V., Dames, S., Durtschi, J. D. Next generation sequencing for clinical diagnostics-principles and application to targeted resequencing for hypertrophic cardiomyopathy: a paper from the 2009 William Beaumont Hospital Symposium on Molecular Pathology. J. Mol. Diagn. 12, 539-551 (2010).
Tags
Fluorescence-microscopy ScreeningNext-generation SequencingIdentification Of GenesOrganelle IntegrityArabidopsis SeedlingsRecessive MutationsSubcellular DistributionTagged Fluorescent MarkerSecretory PathwayGenetic StudiesGenome SizeGeneration TimeConservation Of Molecular MechanismsArray GenotypingAlternative Mapping MethodMolecular MarkersProteins Influencing Organelle IntegrityEndoplasmic Reticulum (ER)Plant Cell OrganellesMolecular Basis
Citar este artigo
Stefano, G., Renna, L., Brandizzi, F. Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity. J. Vis. Exp. (62), e3809, doi:10.3791/3809 (2012).