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PLoS ONE:研究建立全新植物基因链接与克隆系统

  1. 克隆
  2. 基因
  3. 植物
  4. 高通量测序

来源:《PLoS综合》 2013-04-06 09:40

随着高通量测序技术的普及与基因组信息爆炸式的增长,解析基因与基因组孕藏的功能信息成为我们了解生命密码的必需步骤。功能基因研究是破解基因组信息这部天书的重要手段之一,而功能基因的研究离不开载体的构建与转基因方法。

随着高通量测序技术的普及与基因组信息爆炸式的增长,解析基因与基因组孕藏的功能信息成为我们了解生命密码的必需步骤。功能基因研究是破解基因组信息这部天书的重要手段之一,而功能基因的研究离不开载体的构建与转基因方法。传统的载体构建耗时耗力,伴随着烦琐的酶切与连接手段,成功地构建一个用于植物转化的载体往往需要几天甚至几个星期的时间,有时操作人员因为找不到适合的酶切位点而头痛不已,这些都是限制功能基因研究的瓶颈问题。

为了克服以上问题, 中国科学院昆明植物研究所胡向阳研究组的王春涛博士与博士生孔翔祥等人员,基于TA克隆技术建立了一套全新的基因连接与克隆系统。这一套系统完全克服了传统克隆过程中上述难题,而且具有操作简易、高效、稳定等特点。研究组成员将这套系统成功地运用到水稻与拟南芥的原生质转化与蛋白细胞定位方面,以及拟南芥稳定转化等功能基因研究。一系列的证据表明利用这套系统构建的载体,目标基因可以稳定高效地在拟南芥中表达,获得的转基因材料可以用于下一步的生理生化与功能分析。

上述研究结果以A series of TA-based and zero-background vectors for plant functional genomics为题发表在《公共科学图书馆-综合》(PLoS ONE)上,该研究工作得到中国科学院“百人计划”项目、国家自然基金等项目的资助。(生物谷Bioon.com)

A Series of TA-Based and Zero-Background Vectors for Plant Functional Genomics

Chuntao Wang, et al.

With the sequencing of genomes from many organisms now complete and the development of high-throughput sequencing, life science research has entered the functional post-genome era. Therefore, deciphering the function of genes and how they interact is in greater demand. To study an unknown gene, the basic methods are either overexpression or gene knockout by creating transgenic plants, and gene construction is usually the first step. Although traditional cloning techniques using restriction enzymes or a site-specific recombination system (Gateway or Clontech cloning technology) are highly useful for efficiently transferring DNA fragments into destination plasmids, the process is time consuming and expensive. To facilitate the procedure of gene construction, we designed a TA-based cloning system in which only one step was needed to subclone a DNA fragment into vectors. Such a cloning system was developed from the pGreen binary vector, which has a minimal size and facilitates construction manipulation, combined with the negative selection marker gene ccdB, which has the advantages of eliminating the self-ligation background and directly enabling high-efficiency TA cloning technology. We previously developed a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, subcellular localization analysis and promoter activity detection. Our results show that such a system is highly efficient and serves as a high-throughput platform for transient or stable transformation in plants for functional genome research.

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