Abstract
BackgroundExplant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration. Considerable work has examined the physiological mechanisms underlying Phalaenopsis leaf explant browning, but the molecular mechanisms of browning remain elusive. In this study, we used whole genome RNA sequencing to examine Phalaenopsis leaf explant browning at genome-wide level.Methodology/Principal FindingsWe first used Illumina high-throughput technology to sequence the transcriptome of Phalaenopsis and then performed de novo transcriptome assembly. We assembled 79,434,350 clean reads into 31,708 isogenes and generated 26,565 annotated unigenes. We assigned Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, and potential Pfam domains to each transcript. Using the transcriptome data as a reference, we next analyzed the differential gene expression of explants cultured for 0, 3, and 6 d, respectively. We then identified differentially expressed genes (DEGs) before and after Phalaenopsis explant browning. We also performed GO, KEGG functional enrichment and Pfam analysis of all DEGs. Finally, we selected 11 genes for quantitative real-time PCR (qPCR) analysis to confirm the expression profile analysis.Conclusions/SignificanceHere, we report the first comprehensive analysis of transcriptome and expression profiles during Phalaenopsis explant browning. Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis. Genes involved in photosynthesis and ATPase activity have been found to be changed at transcription level; these changes may perturb energy metabolism and thus lead to the decay of plant cells and tissues. This study provides comprehensive gene expression data for Phalaenopsis browning. Our data constitute an important resource for further functional studies to prevent explant browning.
Highlights
Explant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration
Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis
Based on the homologs in Arabidopsis thaliana, we found multiple differentially expressed genes (DEGs) involved in biosynthesis of secondary metabolites, nucleotide metabolism, carbohydrate metabolism, and amino acids metabolism, such as: NAD (P)- binding rossmann-fold superfamily protein, nucleoside diphosphate kinase family protein, protein kinase superfamily protein, glucose-6-phosphate dehydrogenase 2, caffeoyl-CoA 3-O-methyltransferase
Summary
Plant materialLeaves of Phalaenopsis sp. plants were cut into 0.5 cm × 0.5 cm segments and cultured on Murashige & Skoog medium (Murashige & Skoog, 1962) solidified with 0.8% agar (w/v) at pH 5.8 and supplied with 3 mg/L 6-BA. Plants were cut into 0.5 cm × 0.5 cm segments and cultured on Murashige & Skoog medium (Murashige & Skoog, 1962) solidified with 0.8% agar (w/v) at pH 5.8 and supplied with 3 mg/L 6-BA. Explants were collected from leaves cultured for 0, 3, and 6 d, respectively. All explants were immediately frozen in liquid nitrogen and stored at -80°C. RNA isolation and Illumina PE library preparation for transcriptome analysis De novo assembly of the transcriptome was performed by short-read sequencing (Illumina). Total RNAs were isolated from explants that were cultured for 0, 3, and 6 d, and mixed for cDNA library construction. TRIzol Reagent (Invitrogen, CA, USA) was used to isolate total RNA following the manufacturer’s protocol. RNA quality was assessed using the Agilent 2100 Bioanalyzer with 260/280 and 260/230 absorbance ratios
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