A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts.
A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts.
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DOI：
10.1186/1746-4811-2-13
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发表时间：
2006-06-29
期刊：
影响因子：
5.1
通讯作者：
Ronald PC
中科院分区：
文献类型：
--
作者：
Bart R;Chern M;Park CJ;Bartley L;Ronald PC
使用原生质体的瞬时分析是处理来自高通量分析的大量遗传数据的理想选择。以前，通常从双子叶组织或细胞悬浮培养物中制备原生质体，但缺乏一个良好的系统来分离和操纵水稻原生质体。我们已经建立了一个水稻幼苗原生质体系统，旨在快速鉴定大量基因。我们报道了从7-14天黄化水稻幼苗中分离原生质体的优化方法。我们发现，报告基因GL2和GUS在聚乙二醇(PEG)介导的原生质体转化后约20小时达到最高表达。此外，我们还发现转化效率随质粒大小的不同而显着不同。5微克4.5kb的质粒转化效率为60-70%。相反，使用50μg的12kb质粒，我们获得了最大25-30%的效率。我们还表明，短干扰RNA(SiRNAs)可以快速有效地沉默外源基因。靶向荧光素酶的siRNA仅在3小时后就导致了显著的沉默水平，并且表达下降了83%。我们还从全绿色组织制备的细胞中分离出了原生质体。这些绿色组织衍生的原生质体可以转化为表达高水平的荧光素酶活性，并将用于检测光敏细胞过程。我们报道了一种分离、转化和基因沉默黄化水稻叶和茎来源的原生质体的系统。此外，我们还将这项技术扩展到从全绿色组织中分离出的原生质体。原生质体系统将在高通量分析和功能生物学之间架起一座桥梁，因为它可以用于快速研究大量功能未知的基因。
Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking. We have established a rice seedling protoplast system designed for the rapid characterization of large numbers of genes. We report optimized methods for protoplast isolation from 7–14 day old etiolated rice seedlings. We show that the reporter genes luciferase GL2 and GUS are maximally expressed approximately 20 h after polyethylene glycol (PEG)-mediated transformation into protoplasts. In addition we found that transformation efficiency varied significantly with plasmid size. Five micrograms of a 4.5 kb plasmid resulted in 60–70% transformation efficiency. In contrast, using 50 μg of a 12 kb plasmid we obtained a maximum of 25–30% efficiency. We also show that short interfering RNAs (siRNAs) can be used to silence exogenous genes quickly and efficiently. An siRNA targeting luciferase resulted in a significant level of silencing after only 3 hours and up to an 83% decrease in expression. We have also isolated protoplasts from cells prepared from fully green tissue. These green tissue-derived protoplasts can be transformed to express high levels of luciferase activity and should be useful for assaying light sensitive cellular processes. We report a system for isolation, transformation and gene silencing of etiolated rice leaf and stem-derived protoplasts. Additionally, we have extended the technology to protoplasts isolated from fully green tissue. The protoplast system will bridge the gap between hi-throughput assays and functional biology as it can be used to quickly study large number of genes for which the function is unknown.
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影响因子：
7.2
作者：
Sugimoto, K;Takeda, S;Hirochika, H
通讯作者：
Hirochika, H
影响因子：
7.4
作者：
Pih, KT;Yi, MJ;Son, D
通讯作者：
Son, D
DOI：
10.1073/pnas.96.26.15348
发表时间：
1999-12-21
影响因子：
11.1
作者：
Hobo, T;Kowyama, Y;Hattori, T
通讯作者：
Hattori, T
影响因子：
1.6
作者：
An, CI;Sawada, A;Kobayashi, A
通讯作者：
Kobayashi, A
影响因子：
64.8
作者：
Asai, T;Tena, G;Sheen, J
通讯作者：
Sheen, J