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Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development
Published on: March 5, 2017
RNA-Seq-based transcriptome analysis of stem development and dwarfing regulation in Agapanthus praecox ssp.
Di Zhang1, Li Ren1, Jian-Hua Yue1
1Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
Abstract:
Agapanthus praecox is a monocotyledonous ornamental bulb plant. Generally, the scape (inflorescence stem) length can develop more than 1m, however application 400 mg·L(-1) paclobutrazol can shorten the length beyond 70%. To get a deeper insight into its dwarfism mechanism, de novo RNA-Seq technology has been employed, for the first time, to describe the scape transcriptome of A. praecox. We got 71,258 assembled unigenes, and 45,597 unigenes obtained protein functional annotation. Take the above sequencing results as a reference gene set, using RNA-seq (quantification) technology analyzed gene expression profiles between the control and paclobutrazol-treated samples, and screened 2838 differentially expressed genes. GO, KEGG and MapMan pathway analyses indicated that these differentially expressed genes were significantly enriched in response to stimulus, hormonal signaling, carbohydrate metabolism, cell wall, cell size, and cell cycle related biological process. To validate the expression profiles obtained by RNA-Seq, real-time qPCR was performed on 24 genes selected from key significantly enriched pathways. Comprehensive analysis suggested that paclobutrazol blocks GA signal that can effectively inhibit scape elongation; the GA signal interact with other hormonal signals including auxin, ethylene, brassinosteroid and cytokinins, and trigger downstream signaling cascades leading to metabolism, cell wall biosynthesis, cell division and the cycle decreased obviously, and finally induced dwarfism trait. Furthermore, AP2/EREBP, bHLH, C2H2, ARR, WRKY and ARF family's transcription factors were involved in the regulation of scape development in A. praecox. This transcriptome dataset will serve as an important public information platform to accelerate research on the gene expression and functional genomics of Agapanthus.
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