Efficient and high-throughput vector construction and Agrobacterium-mediated transformation of Arabidopsis thaliana suspension-cultured cells for functional genomics

被引：39

作者：

Ogawa, Yoichi ^{[1
]}

Dansako, Tomoko ^{[1
]}

Yano, Kentaro ^{[1
]}

Sakurai, Nozomu ^{[1
]}

Suzuki, Hideyuki ^{[1
]}

Aoki, Koh ^{[1
]}

Noji, Masaaki ^{[2
]}

Saito, Kazuki ^{[1
,3
]}

Shibata, Daisuke ^{[1
]}

机构：

[1] Kazusa DNA Res Inst, Chiba 2920818, Japan

[2] Tokushima Bunri Univ, Fac Pharmaceut Sci, Tokushima 7708514, Japan

[3] Chiba Univ, Grad Sch Pharmaceut Sci, Chiba 2638522, Japan

来源：

PLANT AND CELL PHYSIOLOGY | 2008年 / 49卷 / 02期

关键词：

Agrobacterium-mediated transformation; Arabidopsis thaliana; functional genomics; gateway cloning system; high-throughput; suspension-cultured cells;

D O I：

10.1093/pcp/pcm181

中图分类号：

Q94 [植物学];

学科分类号：

071001 ;

摘要：

We established a large-scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.

引用

页码：242 / 250

页数：9

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