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利用形态发生基因通过农杆菌介导的顽拗型玉米自交系未成熟胚转化

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes.

作者信息

Masters Alicia, Kang Minjeong, McCaw Morgan, Zobrist Jacob D, Gordon-Kamm William, Jones Todd, Wang Kan

机构信息

Department of Agronomy, Iowa State University; Department of Applied Science and Technology, Corteva Agriscience.

Department of Agronomy, Iowa State University; Crop Bioengineering Center, Iowa State University; Interdepartmental Plant Biology Major, Iowa State University.

出版信息

J Vis Exp. 2020 Feb 14(156). doi: 10.3791/60782.

Abstract

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

摘要

本文展示了一种使用形态发生基因“婴儿潮”(Bbm)和“沃氏2”(Wus2)通过农杆菌介导对玉米自交系进行遗传转化的详细方案。Bbm由玉米磷脂转移酶基因(Pltp)启动子调控,Wus2受玉米生长素诱导型(Axig1)启动子控制。携带这些形态发生基因的农杆菌菌株位于转移DNA(T-DNA)上,且带有额外拷贝的农杆菌毒性(vir)基因,用于感染玉米未成熟胚外植体。体细胞胚在受感染胚的盾片上形成,可通过除草剂抗性进行筛选并萌发成植株。构建在DNA构建体中的热激活cre/loxP重组系统可在转化过程的早期阶段从玉米基因组中去除形态发生基因。使用该方案,W22、B73和Mo17的转化频率分别可达约14%、4%和4%(每100个受感染胚的独立转基因事件数)。

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