可选择标记独立转化玉米自交系 B73 和高粱 P898012 由形态发生调节剂 BABY BOOM 和 WUSCHEL2 介导。

Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2.

机构信息

Plant Transformation Core Facility, Division of Plant Sciences, University of Missouri, 1-33 Agriculture Building, Columbia, MO, 65211, USA.

Department of Cell and Molecular Biology, University of Rhode Island, 530 Liberty Lane, West Kingston, RI, 02892, USA.

出版信息

Plant Cell Rep. 2017 Sep;36(9):1477-1491. doi: 10.1007/s00299-017-2169-1. Epub 2017 Jul 5.

Abstract

Discriminatory co-expression of maize BBM and WUS transcriptional factor genes promoted somatic embryogenesis and efficient Agrobacterium -mediated transformation of recalcitrant maize inbred B73 and sorghum P898012 genotypes without use of a selectable marker gene. The use of morphogenic regulators to overcome barriers in plant transformation is a revolutionary breakthrough for basic plant science and crop applications. Current standard plant transformation systems are bottlenecks for genetic, genomic, and crop improvement studies. We investigated the differential use of co-expression of maize transcription factors BABY BOOM and WUSCHEL2 coupled with a desiccation inducible CRE/lox excision system to enable regeneration of stable transgenic recalcitrant maize inbred B73 and sorghum P898012 without a chemical selectable marker. The PHP78891 expression cassette contains CRE driven by the drought inducible maize RAB17 promoter with lox P sites which bracket the CRE, WUS, and BBM genes. A constitutive maize UBI promoter directs a ZsGreen GFP expression cassette as a reporter outside of the excision sites and provides transient, transgenic, and developmental analysis. This was coupled with evidence for molecular integration and analysis of stable integration and desiccation inducible CRE-mediated excision. Agrobacterium-mediated transgenic introduction of this vector showed transient expression of GFP and induced somatic embryogenesis in maize B73 and sorghum P898012 explants. Subjection to desiccation stress in tissue culture enabled the excision of CRE, WUS, and BBM, leaving the UBI ::GFP cassette and allowing subsequent plant regeneration and GFP expression analysis. Stable GFP expression was observed in the early and late somatic embryos, young shoots, vegetative plant organs, and pollen. Transgene integration and expression of GFP positive T plants were also analyzed using PCR and Southern blots. Progeny segregation analysis of primary events confirmed correlation between functional GFP expression and presence of the GFP transgene in T plants generated from self pollinations, indicating good transgene inheritance. This study confirms and extends the use of morphogenic regulators to overcome transformation barriers.

摘要

玉米 BBM 和 WUS 转录因子基因的歧视性共表达促进了体细胞胚胎发生和玉米自交系 B73 和高粱 P898012 基因型的农杆菌介导的高效转化,而无需使用选择标记基因。使用形态发生调节剂来克服植物转化中的障碍是基础植物科学和作物应用的革命性突破。当前的标准植物转化系统是遗传、基因组和作物改良研究的瓶颈。我们研究了玉米转录因子 BABY BOOM 和 WUSCHEL2 的共表达与干燥诱导的 CRE/lox 切除系统的差异使用,以在没有化学选择标记的情况下使稳定的转基因玉米自交系 B73 和高粱 P898012 再生。PHP78891 表达盒包含由干旱诱导的玉米 RAB17 启动子驱动的 CRE,lox P 位点框定 CRE、WUS 和 BBM 基因。组成型玉米 UBI 启动子指导 ZsGreen GFP 表达盒作为切除位点之外的报告基因,并提供瞬时、转基因和发育分析。这与分子整合的证据以及稳定整合和干燥诱导的 CRE 介导的切除分析相结合。该载体的农杆菌介导的转基因导入显示 GFP 的瞬时表达,并诱导玉米 B73 和高粱 P898012 外植体的体细胞胚胎发生。在组织培养中受到干燥胁迫会导致 CRE、WUS 和 BBM 的切除,留下 UBI:: GFP 盒,并允许随后的植物再生和 GFP 表达分析。在早期和晚期体细胞胚、幼芽、营养植物器官和花粉中观察到稳定的 GFP 表达。还使用 PCR 和 Southern 印迹分析了 GFP 阳性 T 植物的转基因整合和表达。对初级事件的后代分离分析证实了 GFP 表达与自花授粉产生的 T 植物中 GFP 转基因的存在之间的相关性,表明良好的转基因遗传。这项研究证实并扩展了形态发生调节剂的使用以克服转化障碍。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6d31/5565672/eb2a55edfd14/299_2017_2169_Fig1_HTML.jpg

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