De novo sequencing and analysis of the transcriptome during the browning of fresh-cut Luffa cylindrica 'Fusi-3' fruits

Abstract

Fresh-cut luffa (Luffa cylindrica) fruits commonly undergo browning. However, little is known about the molecular mechanisms regulating this process. We used the RNA-seq technique to analyze the transcriptomic changes occurring during the browning of fresh-cut fruits from luffa cultivar 'Fusi-3'. Over 90 million high-quality reads were assembled into 58,073 Unigenes, and 60.86% of these were annotated based on sequences in four public databases. We detected 35,282 Unigenes with significant hits to sequences in the NCBInr database, and 24,427 Unigenes encoded proteins with sequences that were similar to those of known proteins in the Swiss-Prot database. Additionally, 20,546 and 13,021 Unigenes were similar to existing sequences in the Eukaryotic Orthologous Groups of proteins and Kyoto Encyclopedia of Genes and Genomes databases, respectively. Furthermore, 27,301 Unigenes were differentially expressed during the browning of fresh-cut luffa fruits (i.e., after 1-6 h). Moreover, 11 genes from five gene families (i.e., PPO, PAL, POD, CAT, and SOD) identified as potentially associated with enzymatic browning as well as four WRKY transcription factors were observed to be differentially regulated in fresh-cut luffa fruits. With the assistance of rapid amplification of cDNA ends technology, we obtained the full-length sequences of the 15 Unigenes. We also confirmed these Unigenes were expressed by quantitative real-time polymerase chain reaction analysis. This study provides a comprehensive transcriptome sequence resource, and may facilitate further studies aimed at identifying genes affecting luffa fruit browning for the exploitation of the underlying mechanism.

Fig 1. Total phenol content in luffa fruit slices at different browning time points.

The vertical bars represent the standard error of triplicate experiments.

Fig 2. Unigenes annotated by a BLASTX search of databases (E < 10⁻⁵).

Fig 3. Unigenes with matches in the NCBInr database and homologs in 10 plant species.

Fig 4. Histogram of the KOG classifications.

Fig 5. Summary of the GO classification of Unigenes.

Fig 6. Pathway assignments based on the KEGG database.

Fig 7. Differentially expressed genes in fresh-cut luffa fruits at various time points.

Fig 8. Clustering and classification of 27,301 differentially expressed genes.

The numbers in the top corner of each panel represent the identification number of the 26 profiles and the number of identified genes in the cluster, respectively.

Fig 9. Clustering of 15 browning-related genes.

The color scale represents FPKM-normalized log₂-transformed gene expression levels. Different columns represent different samples, and different lines represent different genes.

Fig 10. Quantitative real-time polymerase chain reaction analyses of the expression levels of selected Unigenes during the browning of luffa fruits.

18S rRNA was used as the internal control. The error bars represent the standard error of three biological replicates.

Fig 11. Correlation analysis of fold-change values based on RNA-seq and qRT-PCR data.

The RNA-seq fold-change refers to the ratio of FPKM values from 0 to 6 h. The qRT-PCR fold-change refers to the expression levels at 1, 3, and 6 h normalized against the expression level at 0 h. **significant correlation (P < 0.01).