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A versatile zero background T-vector system for gene cloning and functional genomics.

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WOS被引频次:197
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成果类型:
期刊论文
作者:
Songbiao Chen;Pattavipha Songkumarn;Jianli Liu;Guo-Liang Wang
通讯作者:
Wang, Guo-Liang
作者机构:
[Guo-Liang Wang; Jianli Liu; Pattavipha Songkumarn; Songbiao Chen] Ohio State Univ, Dept Plant Pathol, Columbus, OH 43210 USA.
[Guo-Liang Wang] Hunan Agr Univ, Hunan Prov Key Lab Crop Germplasm Innovat & Utili, Changsha 410128, Hunan, Peoples R China.
通讯机构:
[Wang, Guo-Liang] Ohio State Univ, Dept Plant Pathol, Columbus, OH 43210 USA.
语种:
英文
期刊:
Plant physiology
ISSN:
0032-0889
年:
2009
卷:
150
期:
3
页码:
1111-1121
文献类别:
WOS:Article
所属学科:
ESI学科类别:植物学&动物科学;WOS学科类别:Plant Sciences
入藏号:
基金类别:
National Science Foundation-Plant Genome Research Program [0605017]
机构署名:
本校为其他机构
摘要:
With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics. © 2009 American Society of Plant Biologists.

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