[讨论交流] 探讨下植物病毒与试验技术

donggua

来我们这个版块的大部分是研究植物病毒或与植物病毒相关的同行。 希望能和大家探讨下植物病毒与实验技术，换句话说目前应用到植物病毒研究的技术。

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Microscopy: Electron microscopy, confocal microscopy, living cell imaging, cryo-microscopy, etc.


各种电镜技术，从刚刚开始的电镜 （electron microscopy) 到现在各式各样的先进的电镜。 越来越多的应用到植物病毒的研究中来，让病毒研究更加直观，可视化。比如下面两篇文献：
Wei, T., C. Zhang, et al. (2010). "Formation of Complexes at Plasmodesmata for Potyvirus Intercellular Movement Is Mediated by the Viral Protein P3N-PIPO." Plos Pathogens: -.
        Intercellular transport of viruses through cytoplasmic connections, termed plasmodesmata (PD), is essential for systemic infection in plants by viruses. Previous genetic and ultrastructural data revealed that the potyvirus cyclindrical inclusion (CI) protein is directly involved in cell-to-cell movement, likely through the formation of conical structures anchored to and extended through PD. In this study, we demonstrate that plasmodesmatal localization of CI in N. benthamiana leaf cells is modulated by the recently discovered potyviral protein, P3N-PIPO, in a CI: P3N-PIPO ratio-dependent manner. We show that P3N-PIPO is a PD-located protein that physically interacts with CI in planta. The early secretory pathway, rather than the actomyosin motility system, is required for the delivery of P3N-PIPO and CI to PD. Moreover, CI mutations that disrupt virus cell-to-cell movement compromise PD-localization capacity. These data suggest that the CI and P3N-PIPO complex coordinates the formation of PD-associated structures that facilitate the intercellular movement of potyviruses in infected plants.

Wei, T., T. Huang, et al. (2010). "Sequential Recruitment of the Endoplasmic Reticulum and Chloroplasts for Plant Potyvirus Replication." Journal of Virology: 799-809.
        The replication of positive-strand RNA viruses occurs in cytoplasmic membrane-bound virus replication complexes (VRCs). Depending on the virus, distinct cellular organelles such as the endoplasmic reticulum (ER), chloroplast, mitochondrion, endosome, and peroxisome are recruited for the formation of VRC-associated membranous structures. Previously, the 6,000-molecular-weight protein (6K) of plant potyviruses was shown to be an integral membrane protein that induces the formation of 6K-containing membranous vesicles at endoplasmic reticulum (ER) exit sites for potyvirus genome replication. Here, we present evidence that the 6K-induced vesicles predominantly target chloroplasts, where they amalgamate and induce chloroplast membrane invaginations. The vesicular transport pathway and actomyosin motility system are involved in the trafficking of the 6K vesicles from the ER to chloroplasts. Viral RNA, double-stranded RNA, and viral replicase components are concentrated at the 6K vesicles that associate with chloroplasts in infected cells, suggesting that these chloroplast-bound 6K vesicles are the site for potyvirus replication. Taken together, these results suggest that plant potyviruses sequentially recruit the ER and chloroplasts for their genome replication.
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本帖最后由 donggua 于 2011-12-7 22:42 编辑


PCR.

http://en.wikipedia.org/wiki/Variants_of_PCR
application:

Cloning
Detection
Mutagenesis
Expression.

这是一个在普通不过的技术了，不过文章从低档次到science ，nature，cell 这么高档次的文章绝大多数都用到它，也说明了这个技术的重要性。PCR从第一次应用到现在为止已经已经有N年之多，有人只是去应用现有的技术，有人想着去改变，也就出现了后来的各式各样的PCR，见上面的链接。

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Northern blot
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample。

RNA silencing experiments, mRNA and siRNA detection.

Transgene plant virus gene RNA detection.

etc.
Northern blot技术
http://bbs.virology.com.cn/viewt ... ght=northern%2Bblot

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WESTERNBLOT

The western blot (sometimes called the protein immunoblot) is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

http://en.wikipedia.org/wiki/Western_blot

Western Blot详解(原理、分类、试剂、步骤及问题解答)
http://bbs.virology.com.cn/viewt ... ight=western%2Bblot
westernblot经验
http://bbs.virology.com.cn/viewt ... ight=western%2Bblot

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ELISA
Enzyme-linked immunosorbent assay (ELISA), is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay (EIA) to detect the presence of a substance in a liquid sample or wet sample.
种类：
"Indirect" ELISA
Sandwich ELISA  DAS-ELISA， TAS-ELISA
Competitive ELISA
Multiple and Portable ELISA (M&P ELISA)


ELISA操作技巧
http://bbs.virology.com.cn/thread-4072-1-1.html