概要: Cowpea (Vigna unguiculata) is an important protein source in Sub-Saharan Africa. Optimizing resilience and productivity through genetic engineering in cowpea has been slow due in part to a lack of defined species-specific regulatory elements and difficulty testing gene function within the native system. In many plant species, Agrobacterium-mediated transient gene expression is widely used to validate constructs before investing in transgenic lines, but its implementation in legumes has been challenging. In this study, we optimized an in planta agroinfiltration assay in trifoliate cowpea leaves using a betalain reporter. To demonstrate the “intact plant” aspect of this system, we used this assay to characterize drought-inducible promoters by challenging cowpea plants with drought stress. Subsequently, to identify and broaden the pool of native promoters known in cowpea, we developed a user-friendly web application, CowPEAsy, allowing users to interrogate gene expression from our canopy-level, developmental-series RNA-Seq dataset. Finally, using CowPEAsy, we identified six promoters that showed constitutive expression across all conditions and verified these promoters with our transient system. This work provides an in vivo platform for preliminary validation of regulatory elements in cowpea and other legumes and enhances current genetic resources by identifying a suite of physiologically relevant promoters of varying strengths.
项目整体设计: Leaf tissue punches for RNA-Seq were collected from greenhouse grown IT97K-499-35 cowpea plants at bottom, middle, and top canopy positions and at developmental stages R1 (5.5 weeks after planting) and R3 (6.5 weeks after planting).
Cowpea were planted in-ground in a Plant Care Facility greenhouse at the University of Illinois Urbana-Champaign. Seeds were planted with 3 inches between plants and 30 inches between rows. Plant maintenance involved irrigation, weekly fertilizer application, and supplemental lighting (14h/10h photoperiod, 1000 umol/m^2*s).
处理方案:
Leaf tissue punches for RNA-Seq were collected from greenhouse grown IT97K-499-35 cowpea plants at bottom, middle, and top canopy positions and at developmental stages R1 (5.5 weeks after planting) and R3 (6.5 weeks after planting).
提取方案:
RNA was purified using the MagMAX kit for RNA extraction (Thermo Fisher Scientific, cat# AM1830).
建库方案:
Libraries were constructed using the Kapa HyperPrep mRNA kit (Roche, cat# KK8581).
