Liver Transcriptome Analysis of the Large Yellow Croaker (Larimichthys crocea) during Fasting by Using RNA-Seq

Abstract

The large yellow croaker (Larimichthys crocea) is an economically important fish species in Chinese mariculture industry. To understand the molecular basis underlying the response to fasting, Illumina HiSeqTM 2000 was used to analyze the liver transcriptome of fasting large yellow croakers. A total of 54,933,550 clean reads were obtained and assembled into 110,364 contigs. Annotation to the NCBI database identified a total of 38,728 unigenes, of which 19,654 were classified into Gene Ontology and 22,683 were found in Kyoto Encyclopedia of Genes and Genomes (KEGG). Comparative analysis of the expression profiles between fasting fish and normal-feeding fish identified a total of 7,623 differentially expressed genes (P < 0.05), including 2,500 upregulated genes and 5,123 downregulated genes. Dramatic differences were observed in the genes involved in metabolic pathways such as fat digestion and absorption, citrate cycle, and glycolysis/gluconeogenesis, and the similar results were also found in the transcriptome of skeletal muscle. Further qPCR analysis confirmed that the genes encoding the factors involved in those pathways significantly changed in terms of expression levels. The results of the present study provide insights into the molecular mechanisms underlying the metabolic response of the large yellow croaker to fasting as well as identified areas that require further investigation.

Fig 1. Length statistics of contigs obtained from the two livers of large yellow croaker transcriptome libraries.

The length distribution of the transcriptome libraries is shown. Sequences with lengths of 100–500 bp were the most abundant in the two transcriptome libraries, encompassing 86.78% and 89.27% of the contigs in the transcriptome library of fasting fish and normal feeding fish, respectively.

Fig 2. Significantly differentially expressed genes identified by KEGG in the glycolysis/gluconeogenesis pathway.

Red indicates significantly upregulated genes, green indicates significantly downregulated genes, and blue indicates genes that were both up- and downregulated.

Fig 3. Significantly differentially expressed genes identified by KEGG in the pathway of fat digestion and absorption.

Red indicates significantly upregulated genes, green indicates significantly downregulated genes, and blue indicates genes that were both up- and downregulated.

Fig 4. Significantly differentially expressed genes identified by KEGG in the TCA cycle pathway.

Red indicates significantly upregulated genes, green indicates significantly downregulated genes, blue indicates genes that were both up- and downregulated.

Fig 5. Significantly differentially expressed genes identified by KEGG in the protein processing in the endoplasmic reticulum pathway.

Green indicates significantly downregulated genes, and blue indicates genes that were both up- and downregulated.

Fig 6. qPCR analysis of genes related to the glycolysis/gluconeogenesis pathway in the large yellow croaker during fasting and re-feeding.

To determine changes in the levels of expression of the following genes, 6-phosphofructokinase 1(pfkA), glucose-6-phosphatase (G6PC), hexokinase (HK), and pyruvate kinase (PK), the livers of 6 fish were collected at 0, 7, 14, 21, 28, 35, 42 days of fasting and 7 and 14 days of re-feeding, respectively. Total RNA was extracted from these livers and used in qPCR analysis. The mRNA level of each gene was normalized to that of β-actin. For each time point, values represent fold change in expression of each gene compared to that observed at 0 d fasting, which was set at 1.0. The results are expressed as means ± SD (n = 3). Independent-sample t-test using the SPSS software (Version 11.5) was conducted to determine the statistical significance of the changes in expression levels in fasting or re-feeding fish relative to that observed in 0-d fasting fish. Significant differences were considered at *0.01 < p ≦ 0.05 and ** p < 0.01.

Fig 7. qPCR analysis of the genes related to the fat digestion and absorption pathway in the large yellow croaker during fasting and re-feeding stages, namely, fatty acid-binding protein (FABP), apolipoprotein A-I (ApoA-I), phosphatidate phosphatase (PAP), Niemann-Pick C1-like protein 1 (NPC1L1), 2-acylglycerol O-acyltransferase (MGAT), microsomal triglyceride transfer protein large subunit (Mttp).

The experimental procedure was similar to that presented in Fig 6.

Fig 8. qPCR analysis of the genes related to the TCA cycle pathway in the large yellow croaker during fasting and re-feeding stages, namely, citrate synthase (CS), isocitrate dehydrogenase (icd), isocitrate dehydrogenase (IDH3), 2-oxoglutarate dehydrogenase E1 component (OGDH).

The experimental procedure was similar to that presented in Fig 6.