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pHairyRed: A Novel Binary Vector Containing the DsRed2 Reporter Gene for Visual Selection of Transgenic Hairy Roots

摘要T We developed a new plant transformation vector, pHairyRed, for enabling high throughput, non-destructive selection of Agrobacterium rhizogenes-mediated 'hairy-root' transformation. pHairyRed allows easy in planta visualization of transgenic tissue with minimal disturbance to the plant. The DsRed2 reporter gene, encoding a red fluorescent protein, was cloned to yield pHairyRed (harbouring a multiple cloning site), which was used with the highly efficient K599 A. rhizogenes strain to infect soybean (G/ycine max L. Merrill) plants. DsRed2 fluorescence was easily detected in planta for the duration of a 5-week study with negligible levels of background autofluorescence. This enabled visual selection of transformed roots and subsequent excission of non-transformed roots. pHairyRed-transformed roots nodulated normally when inoculated with Bradyrhizobium japonicum. Within the nodule, DsRed2 fluorescence was plant-specific, being absent in the bacteroid-dominated nodule infected zone. To test the reliability of pHairyRed as a high-fidelity binary vector reporter system, the gene encoding the soybean Nod factor receptor, GmNFR1α, was cloned into the vector for use in a complementation study with a non-nodulating nfr1α mutant of soybean. Complementation was achieved and, without exception, DsRed2 fluorescence was detected in all hairy roots that successfully formed nodules (100%, n = 34).We anticipate broad application of this reporter system for the further analysis of root-related events in soybean and related legumes.

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作者单位 Australian Research Council Centre of Excellence for Integrative Legume Research, The University of Queensland, St Lucia, Brisbane, QLD, 4072, Australia [1]
分类号 Q946.8
栏目名称 METHOD DEVELOPMENT
DOI 10.1093/mp/ssq084
发布时间 2011-09-08
基金项目
Financial support was provided by the Australian Research Council the University of Queensland Strategic Fund
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分子植物(英文版)

分子植物(英文版)

2011年04卷3期

537-545页

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