- Email: [email protected]
Analysis of transcriptome profiling from the brain at maturation and regression phases in starry flounder (Platichthys stellatus)
Gene Reports 4 (2016) 45–52
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Analysis of transcriptome profiling from the brain at maturation and regression phases in starry flounder (Platichthys stellatus) Bo Wang a, Jiang Zhou b, Hongzhan Liu c, Fengrong Zheng a,⁎ a b c
First Institute of Oceanography, SOA, Qingdao 266061, PR China College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing 210095, PR China Marine College of Shandong University, Weihai 264209, PR China
a r t i c l e
i n f o
Article history: Received 13 July 2015 Received in revised form 9 November 2015 Accepted 10 March 2016 Available online 14 March 2016 Keywords: Starry flounder Reproductive regulation Transcriptome Analysis qRT-PCR
a b s t r a c t This study was to investigate the regulatory mechanisms of reproduction and endocrinology in starry flounder (Platichthys stellatus). Total RNA from the mixed sample of pituitary gland and hypothalamus of brain at the maturation and regression phases was isolated. The RNA sequencing (RNA-Seq) library was prepared and sequenced on Illumina Hiseq 2000 platform. A total of 53,567,296 and 75,630,680 short fragments were obtained from the samples of maturation and regression phases, respectively. After splicing, 30,640 unigene sequences were obtained based on the blasting results from National Center for Biotechnology Information (NCBI) database. Homogonous sequences of 24,128 unigenes (78.8%) were found and analyzed with eggNOG and KEGG (Kyoto Encyclopedia of Genes and Genomes). All 24,128 unigenes with eggNOG annotations were divided into 26 categories that are mostly associated with physiological processes, including signaling transduction, translation mechanism, etc. Based on KEGG analysis, all unigenes at the maturation phase were involved in 98 metabolic pathways, with 135 up-regulated sequences, while the unigenes at the regression phase were related to 192 metabolic pathways, with 648 up-regulated sequences. Moreover, 334 differentially expressed genes (DEGs) were up-regulated at the maturation phase, while 987 up-regulated DEGs were found at the regression phase. Total 408 unigenes were associated with the regulation of reproduction and endocrinology. The mRNA expression of some reproduction-associated DEGs including growth hormone releasing hormone (GnRH), neurokinin B (NKB), gonadotropin (GtH), follicle stimulating hormone (FSH), kisspeptin (Kiss) and prolactin-releasing peptide receptor (PrRPR) in the hypothalamus and pituitary gland at two phases was verified by qRT-PCR. Except FSH, all other 5 DEGs showed higher expression at the regression phase. This study provides valuable data for understanding the transcriptome profiles of starry flounder brain and improving the technologies of artificial breeding. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Starry flounder, Platichthys stellatus, is subordinated to Osleichthyes, Pleuronectiformes, Pleuronectidae, and Platichthys and is widely distributed in the north central coast of Yellow Sea of China. Due to its characteristics of high growth potential, strong fertility, delicious taste, suitable for intensive aquaculture, high nutritional value and high resistance to diseases, starry flounder has become one of the most important farm-raised marine fishes (Liu et al., 2009a,b). In 2006, artificial Abbreviation: RNA-Seq, RNA sequencing; NCBI, National Center for Biotechnology Information; KEEG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes; GnRH, growth hormone releasing hormone; NKB, neurokinin B; GtH, gonadotropin; FSH, follicle stimulating hormone; Kiss, kisspeptin; PrRPR, prolactinreleasing peptide receptor; qRT-PCR, quantitative real time PCR. ⁎ Corresponding author at: Ecology Laboratory of the First Institute Oceanography SOA, 6 Xianxialing Rd., Qingdao City, Shandong Province 266061, PR China. Tel.: +86 532 88893571; fax: +86 532 88893571. E-mail address: zhengfr@fio.org.cn (F. Zheng).
http://dx.doi.org/10.1016/j.genrep.2016.03.001 2452-0144/© 2016 Elsevier Inc. All rights reserved.
reproduction of starry flounder succeeded in China, so its feeding increased rapidly in recent years (Wang et al., 2006). Previous studies mainly focused on the growth (Campana and Neilson, 1982, Lee et al., 2003), biological status (Ma et al., 2006; Qi et al., 2008), ecological distribution and nutritional value (Liu et al., 2009a,b) of starry flounder. Moreover, the genetic, physiological and biochemical characteristics of wildlife starry flounder have been reported (You et al., 2007; Park and Kijma, 1991, Borsa et al., 1997). The fish gametogenesis and gonadal maturation are regulated by the neuroendocrine system through hypothalamic-pituitary-gonadal axis (HPG), so the understanding to the neuroendocrine regulation in the hypothalamus is helpful to investigate the regulation of reproduction in starry flounder and improve the technology of artificial reproduction (Lin, 2011). However, the regulatory mechanisms of gonadal development and endocrinology in starry flounder are largely unknown. The Illumina RNA sequencing has been widely used to revolutionize gene expression profiling and transcriptome analysis in non-model organisms, such as, Nilaparvata lugens and pufferfish (Hampton et al.,
46
B. Wang et al. / Gene Reports 4 (2016) 45–52
2011; Hua et al., 2011; Bao et al., 2012; Zheng et al., 2013; Gao et al., 2013). At present, the transcriptome sequencing technology has gradually replaced the gene chip technology and becomes the mainstream method for studying gene expression profiling at the level of whole genome. To investigate the genetic characteristics of starry flounders, this study applied high-performance sequencing technology to investigate the gene expression profiling of starry flounder brain at gonadal maturation and regression phases. This study may provide valuable theoretical data for improving the efficiency and quality of artificial breeding, establishing genetic linkage map, and protecting the germplasm of starry flounder as well. 2. Materials and methods 2.1. Sampling of starry flounders and total RNA extraction Healthy female starry flounders (n = 10) weighing 1000 ± 50 g were purchased from the Breeding Station of Rizhao Marine Aquatic Resources (Shandong, China) in April and May 2014 and kept in a 250 L seawater tank at 25 °C. The samples (n = 10) of brain from gonadal maturation and regression phases were collected and frozen in liquid nitrogen for RNA extraction. Total RNA was isolated by Trizol reagent according to the standard protocol and the quality of RNA was assessed by 1% agarose gel electrophoresis and ultraviolet spectrophotometer. 2.2. Total RNA pretreatment and sequencing The RNA sequencing (RNA-Seq) library was constructed according to the instructions of mRNASeq Sample Preparation Kit (Illumina, USA) (McKenna et al., 2010). Briefly, mRNA was isolated from total RNA using oligo (dT) beads and fragmented into 155 bp short fragments with fragmentation buffer by chemical reagent and high temperature method. The cleaved mRNA fragments were used as templates to synthesize first-strand cDNA using random hexamer primers, and then transformed into double-stranded cDNA using RNase H and DNA polymerase I. A paired-end library was constructed from the synthesized cDNA using Genomic Sample Prep Kit (Illumina). The short fragments in desirable length were purified by using QIAquick PCR Extraction Kit (Qiagen), end repaired and linked with sequencing adapters (Margulies et al., 2005). After removing the unsuitable fragments with AMPureXP beads, the sequencing library was constructed with PCR amplification, checked with Pico green staining and fluorospectrophotometry, quantified with Agilent 2100 and sequenced using Illumina Hiseq 2000 platform (Shanghai Personal Biotechnology Co., Ltd.). For sequencing, the raw reads were filtered by removing the adaptors with polyA and low quality reads. The cleaned reads were assembled into transcripts, contigs and unigenes using Velvet and Oases (http://www.ebi.ac.uk/~zerbino/ velvet; http://www.ebi.ac.uk/~zerbino/oases) Grabherr et al., 2011; Radakovits et al., 2012). 2.3. Sequence alignment, function annotation, analyses of gene expression and associated signaling pathways −5
To obtain the best annotation (E-value b 10 ), the alignment analyses for obtained sequences were carried out by using SwissProt, nr and Non-redundant database. All sequences were also analyzed by eggNOG database (E-value b 10− 5) to obtain NOG functional annotation and classification of expressed sequences, and to collect and establish gene homologous groups, including the COG and KOG data (http://www. ncbi.nlm.nih.gov/COG, http://eggnog.embl.de/) (Tatusov et al., 2003). RPKM (reads per kilobase per million mapped reads) (Mortazavi et al., 2008) was used to normalize the abundance of transcripts. Twofold differential expression and P-value b 0.05 were used to identify differentially expressed genes (DEGs) between two physiological phases. All DEGs were analyzed by KEGG pathway database to obtain the
metabolic pathways and the specific position information (http:// www.genome.jp/kegg/) (Kanehisa et al., 2008; Minoru et al., 2004). 2.4. Verification of representative DEGs by quantitative real-time PCR (qRT-PCR) Total RNA from 10 pituitary and hypothalamus samples at different phases was transcribed into first strand cDNA by Prime Script TM II short Kit (TaKaRa) following the manufacturer's instructions. qRT-PCR was performed according to the instructions of FastStart Universal SYBR Green Master (ROX) (Roche). The specific primers for GnRH, NKB, GtH, Kiss and LHR are listed in Table 1. All samples were analyzed in three duplicates and the general housekeeping gene β-actin was used as internal reference to normalize the target genes. The gene expression levels were calculated as the ratio between the mRNA level of the target genes and the reference gene using the comparative 2− ΔCt method (Livak and Schmittgen, 2001). Target Ct values were normalized to the endogenous gene β-actin. The PCR reaction conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 60 s, and 72 °C for 1 min. 3. Results 3.1. Preparation of RNA sequencing library Results from agarose gel electrophoresis and ultraviolet spectrophotometric test showed high purity and quality of RNA in all samples (Fig. 1). Total RNA samples were treated by Agilent DNA 1000 Kit, and the size of sample library ranged from 280 to 320 bp that meets the requirements of the sequencing and analysis. 3.2. Sequencing and assembling According the data from Illumina Hiseq 2000, a total of 53,567,296 and 75,630,680 raw reads were obtained from hypothalamus and pituitary at the gonadal maturation and regression phases, respectively. The average length of raw reads was 100 nucleotides. After trimming and quality check, 46,155,608 and 71,505,792 high-quality reads were recorded for the gonadal maturation and regression phases, respectively. All high-quality reads were assembled to obtain 176,002 sequences using Oases software (http://www.ebi.ac.uk/~zerbino/oases). In all sequences, the size of 160,790 sequences was bigger than 200 bp, the size of 27,728 sequences was bigger than N50, with the average length of 1854 bp (Table 2A, Fig. 2A). The transcription of samples was commonly performed using cluster analysis according to the similarity. To select the longest transcript as unigene sequences, 30,640 sequences were obtained through clustering unit, with a maximum length of
Table 1 Sequences of PCR primers. Primer
Sequence (5′-3′)
β-actin
F5′-CAACTGGGATGACATGGAGAAG -3′ R5′-TTGGCTTTGGGGTTCAGG -3′ F5′-AGAGGTATGGGAAGAGGTCCAGT -3′ R5′-ACTGTGGAAGAGTGTCTGTGCTTT -3′ F5′-CCTGGGACCATCATCAATTACA -3′ R5′-AGGCGGAGTGGTGTCTCTTC -3′ F5′-TCTTTGTCCTTTTCCAGTCGTTC -3′ R5′-GCGTTGTTATTGCCTTCC -3′ F5′-AGCCACATTTGAGGATTAGCAAC -3′ R5′-TCTGGAGGAGGGGTTATGGA -3′ F5′-AGGTGAAGCACATTAACGGATG -3′ R5′-GAGAGSGAGAGTGGAGGGAGGA -3′ F5′-CCTTCTCCTGCCTGTTCCTG -3′ R5′-GGGCTCTGGGGATGTTGTT -3′
NKB GtH Kiss PrRPR FSH GnRH
B. Wang et al. / Gene Reports 4 (2016) 45–52
47
other nutrients, hormone synthesis and release, and intracellular cell and a series of signal response mechanism, which is associated with the regulation of gonadal development and regression in starry flounder. KEGG (Kyoto Encyclopedia of Genes and Genomes) database has been widely used to analyze gene functions and gene network in the cells. To further understand the biological functions of DEGs and their associated metabolic pathways, we analyzed all DEGs by KEGG. We found that 334 up-regulated DEGs at the maturation phase involved 98 metabolic pathways, while 987 up-regulated DEGs at the regression phase involved 192 metabolic pathways (Fig. 6). Most of metabolic pathways are associated with immune system, CNS system, endocrine system, signaling molecules and interaction and signal transduction, which is consistent with GO enrichment analysis. Moreover, among all DEGs, 408 unigenes are involved in the regulation of reproduction and endocrinology. The classification of all DEGs is shown in Table 3. The major signaling molecules included calmodulins (98), phosphatases (534), protein kinases (798) and cyclic adenosine monophosphates (87).
Fig. 1. The total RNA from hypothalamus and pituitary gland during different gonad development periods. M: DL 2000 Marker; 1: the total RNA of brain during gonadal maturity; 2: the total RNA of brain during gonadal involution.
30,459 bp and the average length of 2696 bp. About 20,055 unigene sequences (65.50%) were mainly distributed in the range of 200– 3000 bp (Table 2B, Fig. 2B).
3.5. Verification of PrRPR, Kiss, GtH, NKB, FSH and GnRH expression at gonadal development and regression phases by qRT-PCR The expression of reproduction- and endocrinology-related genes including GnRH, GtH, FSH, PrRPR, neurokinin and Kiss was significantly different in brain between two developmental phases. The expression of GnRH, NKB, GtH, Kiss and PrRPR mRNAs (Fig. 7) in the brain from the regression phase was 5-fold higher than that from the maturation phase, while the expression level of FSH mRNA in the brain from the maturation phase was significantly higher than that from the regression phase (Fig. 6).
3.3. Sequences annotation and analysis of unigenes A total of 30,640 unigene sequences were compared to NCBI database, 24,128 sequences (78.75%) were found homologous annotation (E-value b 10− 5). All these 24,128 unigenes were analyzed by eggNOG and divided into 26 categories according to different functions, which mainly included signal transduction mechanism (26.81%), basic function (11.75%) and translation mechanism (11.18%) (Fig. 3). The expression of 24,128 unigenes at the gonadal maturation and regression phases was analyzed by the cutoff parameters of P b 0.05 and 2-fold difference, the results are shown in Fig. 4. The expression of 337 unigenes was up-regulated at the gonadal maturation phase, while the expression of 987 unigenes was up-regulated at gonadal regression phase (Fig. 4). 3.4. Analysis of differential expression of unigenes GO has been widely used to analyze biological transcriptome data (Ashburner et al., 2000), which is divided into 3 major functional categories: molecular functions, cellular components and biological processes. All 1321 DEGs between the gonadal maturation and regression phases were analyzed by GO database and the results are shown in Fig. 5. We found that the ratio of DEGs was very similar in the categories of molecular functions (ion binding), cell components (intracellular) and biological processes (biosynthetic process) between gonadal maturation and regression phases. According to GO functional enrichment analysis, DEGs are mostly involved in the accumulation of protein and
4. Discussion Since the artificial breeding of starry flounder was successful in 2006 in China, it has become one of the most important and widely farmraised marine fishes. However, the data for regulatory mechanisms of gonadal development and endocrinology, reproductive cycle and natural ovulation have been rarely studied. In this study, we investigated the transcriptome profiling in the brain of starry flounder at the phases of gonadal maturation and regression by using high-performance sequencing technology. This study may provide valuable theoretical data for understanding of natural ovulation and fertilization and protecting wild resource of starry flounders. It is well-established that the development of gonads is controlled by the hypothalamus–pituitary-gonadal axis, in which GnRH from hypothalamus will promote the production of GtH from pituitary gland, then GtH will promote the secretion of reproduction hormones from gonads, such as ovary. These reproduction hormones control follicle development and ovulation, but they also contribute to keep the balance of GtH hormone through negatively mediating GnRH and GtH secretion from HPG axis (Lin, 2011). Thus, the HPG axis plays a central role in reproduction and endocrinology. Whole-transcriptome analysis in hypothalamus and pituitary gland from the maturation and regression phases provides us with comprehensive insights into biological pathways and molecular mechanisms that regulate gonadal development and endocrinology in starry flounder (Grabherr et al., 2011). Using Illumina Hiseq 2000 technology, we obtained 30,640 unigenes, 24,128 of them with homologous annotation.
Table 2A Assembly statistic by Oases (Transcript). Total length (bp)
Locus no.
Transcript no.
N200 bp transcript
Max. length (bp)
Ave length (bp)
N50
NN50 reads no.
GC%
326,382,179
83,726
176,002
160,790
30,459
1854
3776
27,728
42.28
48
B. Wang et al. / Gene Reports 4 (2016) 45–52
Fig. 2. A: Transcript length distribution of the assembled in hypothalamus and pituitary of P. stellatus. Horizontal axis: the length of unigenes; vertical axis: the number of unigenes. B: Unigene length distribution of the assembled in hypothalamus and pituitary of P. stellatus. Horizontal axis: the length of unigenes; vertical axis: the number of unigenes.
After further analyses by GO and KEGG databases, many unigenes were related to reproduction regulation and involved in many metabolic signaling pathways that regulate gonadal maturation and regression. Starry flounder exhibits multiple spawning and asynchronous ovarian development. There are at least two major follicle populations at different developmental phases, after first ovulation, there are certain population of premature follicles. Based on our transcriptome profiling, before first ovulation (maturation phase), the up-regulated DEGs mostly included glucose transporters, protein phosphatases, tubulin, peroxidases, fructose-bisphosphate aldolases, glutamyl endopeptidases,
Table 2B Unigene statistic. Total length (bp)
Unigene no.
Max. length (bp)
Ave. length (bp)
N50
NN50 reads no.
GC%
82,619,097
30,640
30,459
2696
3969
6,897
48.94
transferrin, dihydropteridine reductases, etc. These genes are mostly involved in the metabolisms of protein, fat, calcium and phosphorus, while the expression of genes that regulate the expression of GnRH and GtH is not dramatically changed. These data indicate that ovarian development requires a large amount of nutrients and trace elements. After ovulation (regression phase), the up-regulated DEGs mostly included GnRHR, NPY, IGF, voltage-gated calcium (Ca2+) channels, progesterone receptor, etc., which are associated with the ovulation. These data suggest that the production of reproduction hormones is gradually increased during ovarian development in starry flounder. Previous studies on goldfish and carp reported that the secretion of GtH by GnRH is dependent on the availability of extracellular calcium ions, our study also revealed the important role of calcium metabolism in gonadal development (Richards et al., 2008; Liu et al., 2008; Bobe and Goetz, 2000, 2001). To verify the transcriptome data, we selected some representative DEGs that are involved in the regulation of reproduction. We observed that FSH mRNA expression in the brain tissues from the maturation phase was significantly higher compared with the regression phase,
B. Wang et al. / Gene Reports 4 (2016) 45–52
49
Fig. 3. eggNOG function classification of unigenes.
Fig. 4. Transcript abundance of unigenes of two different physiological stages. Nao1: The transcriptome library of gonad maturation of the brain. Nao2: The transcriptome library of gonad degenerate of the brain. Horizontal axis is the express different multiples, the vertical axis is the significant difference in expression, 2 times the difference threshold. Horizontal line of P b 0.05 threshold. Blue dots represent genes with differential expression conditions, yellow dots represent those that don't fit the difference expression of conditional gene in the diagram.
50 B. Wang et al. / Gene Reports 4 (2016) 45–52 Fig. 5. GO classification of starry flounder unigenes. The unigenes were classified into three main categories (cellular component, biological process and molecular function) and 40 subcategories. The x-axis indicates the percentage of unigenes. The y-axis indicates the significance of enrichment degree.
B. Wang et al. / Gene Reports 4 (2016) 45–52
51
Fig. 6. KEGG pathway classification of starry flounder unigenes.
while the expression of GtH, PrRPR, NKB, GnRH and Kiss mRNAs at the regression phase was significantly higher than that at the maturation phase. These data are consistent with those in transcriptome profiling. Levavi et al. (2009) reported that FSH plays a key role in the early development of gonads but not ovulation in salmonoids, while LH is a key player in gamete maturation and ovulation. Rocha et al. (2007) found that FSHR is strongly expressed in the eggs during vitellogenesis but not in mature eggs from sea bass, while the expression of LHβ
transcripts is highly expressed in the eggs from gametogenesis and ovulation (Gen et al., 2003). All data indicate that the role of GtHs in the regulation of bony fish reproduction is species-specific with some complexity. In summary, we reported the transcriptome profiling of hypothalamus and pituitary gland from the maturation and regression phases of starry flounder. Most of DEGs are involved in the regulation of reproduction and endocrinology. This study provides comprehensive and valuable data for understanding the regulatory mechanisms of reproductive cycle in starry flounder and improving the technology for artificial breeding.
Table 3 The genes involved in procreation regulation.
Acknowledgments Classification of genes
Number
Prolactin Thyroid hormone Endothelial growth factor Aromatase Estrone Estradiol Growth hormone Growth hormone releasing hormone Gonadotropin releasing hormone Estrogen Androgen Somatostatin Preprosomatostatin Kisspeptin Prolactin-releasing peptide receptor Follicle-stimulating hormone Neurokinin B and neuropeptide Catecholamine Dopamine Tachykinin Insulin-like growth factor 5-Hydroxytryptamine Activin Inhibin Bombesin Cholecystokinin Galanin Ghrelin Testosterone Ghrelin
13 11 89 1 1 8 24 6 12 24 8 9 1 4 6 1 81 1 14 9 18 25 20 7 1 4 6 1 3 1
This work was supported by the National High Technology Research and Development Program of China (863 Program, 2012AA10A413), the Fundamental Research Funds for the Central Public Research Institutes (2012G30) and the National Marine Public Welfare Research Project (201205020).
Fig. 7. The expression of GnRH, NKB, FSH, GtH, Kiss, and PrRPR during gonad mature stage and gonad degradation stage. N1: The pituitary gland and hypothalamus of gonad maturation. N2: The pituitary gland and hypothalamus of gonad degenerate.
52
B. Wang et al. / Gene Reports 4 (2016) 45–52
References Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M., Sherlock, G., 2000. Gene Ontology: tool for the unification of biology [J]. Nat. Genet. 25, 25–29. Bao, Y.Y., Wang, Y., Wu, W.J., Zhao, D., Xue, J., Zhang, B.Q., Shen, Z.C., Zhang, C.X., 2012. De novo intestine-specific transcriptome of the brown planthopper Nilaparvata lugens revealed potential functions in digestion, detoxification and immune response. Genomics 99 (4), 256–264. Bobe, J., Goetz, F.W., 2000. A tumor necrosis factor decoy receptor homologue is upregulated in the brook trout (Salvelinus fontinalis) ovary at the completion of ovulation [J]. Biol. Reprod. 62 (2), 420–426. Bobe, J., Goetz, F.W., 2001. Molecular cloning and expression of a TNF receptor and two TNF ligands in the fish ovary [J]. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 129 (2/3), 475–481. Borsa, P., Blanquer, A., Berrebi, P., 1997. Genetic structure of the flounders Platichthys flesus and P. stellatus at different geographic scales [J]. Mar. Biol. 129, 233–246. Campana, S.E., Neilson, J.D., 1982. Daily growth increments in otoliths of starry flounder (Platichthys stellatus) and the influence of some environmental variables in their production. Can. J. Fish. Aquat. Sci. 39 (7), 937–942. Gao, Y., Zhang, H., Gao, Q., Wang, L., Zhang, F., Siva, V.S., Zhou, Z., Song, L., Zhang, S., 2013. Transcriptome analysis of artificial hybrid pufferfish Jiyan-1 and its parental species: implications for pufferfish heterosis. PLoS One 8 (3), e58453. Gen, K., Yamaguchi, S., Okuzawa, K., Kumakura, N., Tanaka, H., Kagawa, H., 2003. Physiological roles of FSH and LH in red seabream, Pagrus major [J]. Fish Physiol. Biochem. 28 (1), 77–80. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B.W., Nusbaum, C., Lindblad-Toh, K., Friedman, N., Regev, A., 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome [J]. Nat. Biotechnol. 29, 644–652. Hampton, M., Melvin, R.G., Kendall, A.H., Kirkpatrick, B., Peterson, N., Andrews, M.T., 2011. Deep sequencing the transcriptome reveals seasonal adaptive mechanisms in a hibernating mammal. PLoS One 6 (10), 1–13, e27021. Hua, W.P., Zhang, Y., Song, J., Zhao, L.J., Wang, Z.Z., 2011. Denovo transcriptome sequencing in Salvia miltiorrhiza to identify genes involved in the biosynthesis of active ingredients. Genomics 98 (4), 272–279. Kanehisa, M., Araki, M., Goto, S., Hattori, M., Hirakawa, M., Itoh, M., Katayama, T., Kawashima, S., Okuda, S., Tokimatsu, T., Yamanishi, Y., 2008. KEGG for linking genomes to life and the environment [J]. Nucleic Acids Res. 36, D480–D484. Lee, S.M., Lee, J.H., Kim, K.D., 2003. Effect of dietary essential fatty acids on growth, body composition and blood chemistry of juvenile starry flounder (Platichthys stellatus). Aquaculture 225 (1), 269–281. Levavi, Sivan B., Bogerd, J., Mananos, E.L., Gomez, Z., Lareyre, J.J., 2009. Perspectives on fish gonadotropins and their receptors [J]. Gen. Comp. Endocrinol. 165 (3), 412–437. Lin, H.R., 2011. Fish Physiology. Zhongshan University Press. Liu, S.L., Wang, Bo, Liu, Z.H., Xie, L.P., 2009a. Analysis and evaluation of the nutrition of Platichthys stellatus. Prog. Fish. Sci. 30 (6), 18–30.
Liu, S.L., Wang, B., Liu, Z.H., Xie, L.P., Jiang, M.J., 2009b. Nutrition analysis and evaluation of starry flounder. Prog. Fish. Sci. 30 (6), 18–24. Liu, Z.L., Shimada, M., Richards, J.S., 2008. The involvement of the toll-like receptor family in ovulation [J]. J. Assist. Reprod. Genet. 25 (6), 223–228. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCt method. Methods 25 (4), 402–408. Ma, A.J., Zhuang, Z.M., Li, Chen, Sun, X.L., Wang, X.A., J.L., Lei, 2006. The biologic characteristics of starry flounder (Platichthy stelltus Pallas). Mar. Fish. Res. 27 (5), 91–95. Margulies, M., Egholm, M., Aitman, W.E., Attiya, S., Bader, J.S., 2005. Genome sequencing in microfabricated highdensity picolitre reactors. Nature 437, 376–380. McKenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A., Garimella, K., Altshuler, D., Gabriel, S., Daly, M., 2010. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20 (9), 1 297–1 303. Minoru, K., Susumu, Goto, Kawashima, C.S., Yasushi, O., Masahiro, H., 2004. The KEGG resource for deciphering the genome [J]. Nucleic Acids Res. 32 (Database issue), D277–D280 (January 1). Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L., Wold, B., 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq [J]. Nat. Methods 5, 621–628. Park, J.Y., Kijma, A., 1991. Genetic variability and differentiation within and between the stone flounder (Kareius bicoloratus) and the starry flounder (Platichthys stellatus) [J]. Tohoku J. Agric. Res. 41 (3–4), 69–82. Qi, G.S., Li, D., Chen, S.Q., Zhuang, Z.M., Liu, C.L., Song, Z.C., Deng, Y.S., 2008. Morphological characteristics and internal structure of Platichthys stellatus Pallas. J. Fish. Sci. China 15 (1), 1–11. Radakovits, R., Jinkerson, R.E., Fuerstenberg, S.I., Tae, H., Settlage, R.E., Boore, J.L., Posewitz, M.C., 2012. Draft genome sequence and genetic transformation of the oleaginous alga: Nannochloropsis gaditana [J]. Nat. Commun. 21 (3), 686. Richards, J.S., Liu, Z.L., Shimada, M., 2008. Immune-like mechanisms in ovolution [J]. Trends Endocrinol. Metab. 19 (6), 191–196. Rocha, A., Gomez, A., Zanuy, S., Cerdá-Reverter, J.M., Carrillo, M., 2007. Molecular characterization of two sea bass gonadotropin receptors: cDNA cloning, expression analysis, and functional activity [J]. Mol. Cell. Endocrinol. 272 (1–2), 63–76. Tatusov, R.L., Fedorova, N.D., Jackson, J.D., Jacobs, A.R., Kiryutin, B., Koonin, E.V., Krylov, D.M., Mazumder, R., Mekhedov, S.L., Nikolskaya, A.N., Rao, B.S., Smirnov, S., Sverdlov, A.V., Vasudevan, S., Wolf, Y.I., Yin, J.J., Natale, D.A., 2003. The COG database: an updated version includes eukaryotes [J]. BMC Bioinf. 4, 41 (11). Wang, B., Wang, Z.L., Sun, P.X., Liu, Z.H., Wang, S.X., 2006. The development prospect of the breeding conditions of Platichthys stellatus. Fish. Modernization 5, 16–18. You, F., Wu, Z.H., Li, J., Wang, B., Xu, D.D., Ni, J., Yu, D.D., Zhu, X.P., Zhang, P.J., Xu, Y.L., 2007. RAPD analysis of genetic diversity in cultured stock of Platichthys stellatus. Adv. Mar. Sci. 25 (1), 73–78. Zheng, M.G., Tian, J.H., Yang, G.P., Zheng, L., Chen, G.G., Chen, J.L., Wang, B., 2013. Transcriptome sequencing, annotation and expression analysis of Nannochloropsis sp. at different growth phases. Gene 523 (2), 117–121.
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Analysis of transcriptome profiling from the brain at maturation and regression phases in starry flounder (Platichthys stellatus) Bo Wang a, Jiang Zhou b, Hongzhan Liu c, Fengrong Zheng a,⁎ a b c
First Institute of Oceanography, SOA, Qingdao 266061, PR China College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing 210095, PR China Marine College of Shandong University, Weihai 264209, PR China
a r t i c l e
i n f o
Article history: Received 13 July 2015 Received in revised form 9 November 2015 Accepted 10 March 2016 Available online 14 March 2016 Keywords: Starry flounder Reproductive regulation Transcriptome Analysis qRT-PCR
a b s t r a c t This study was to investigate the regulatory mechanisms of reproduction and endocrinology in starry flounder (Platichthys stellatus). Total RNA from the mixed sample of pituitary gland and hypothalamus of brain at the maturation and regression phases was isolated. The RNA sequencing (RNA-Seq) library was prepared and sequenced on Illumina Hiseq 2000 platform. A total of 53,567,296 and 75,630,680 short fragments were obtained from the samples of maturation and regression phases, respectively. After splicing, 30,640 unigene sequences were obtained based on the blasting results from National Center for Biotechnology Information (NCBI) database. Homogonous sequences of 24,128 unigenes (78.8%) were found and analyzed with eggNOG and KEGG (Kyoto Encyclopedia of Genes and Genomes). All 24,128 unigenes with eggNOG annotations were divided into 26 categories that are mostly associated with physiological processes, including signaling transduction, translation mechanism, etc. Based on KEGG analysis, all unigenes at the maturation phase were involved in 98 metabolic pathways, with 135 up-regulated sequences, while the unigenes at the regression phase were related to 192 metabolic pathways, with 648 up-regulated sequences. Moreover, 334 differentially expressed genes (DEGs) were up-regulated at the maturation phase, while 987 up-regulated DEGs were found at the regression phase. Total 408 unigenes were associated with the regulation of reproduction and endocrinology. The mRNA expression of some reproduction-associated DEGs including growth hormone releasing hormone (GnRH), neurokinin B (NKB), gonadotropin (GtH), follicle stimulating hormone (FSH), kisspeptin (Kiss) and prolactin-releasing peptide receptor (PrRPR) in the hypothalamus and pituitary gland at two phases was verified by qRT-PCR. Except FSH, all other 5 DEGs showed higher expression at the regression phase. This study provides valuable data for understanding the transcriptome profiles of starry flounder brain and improving the technologies of artificial breeding. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Starry flounder, Platichthys stellatus, is subordinated to Osleichthyes, Pleuronectiformes, Pleuronectidae, and Platichthys and is widely distributed in the north central coast of Yellow Sea of China. Due to its characteristics of high growth potential, strong fertility, delicious taste, suitable for intensive aquaculture, high nutritional value and high resistance to diseases, starry flounder has become one of the most important farm-raised marine fishes (Liu et al., 2009a,b). In 2006, artificial Abbreviation: RNA-Seq, RNA sequencing; NCBI, National Center for Biotechnology Information; KEEG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes; GnRH, growth hormone releasing hormone; NKB, neurokinin B; GtH, gonadotropin; FSH, follicle stimulating hormone; Kiss, kisspeptin; PrRPR, prolactinreleasing peptide receptor; qRT-PCR, quantitative real time PCR. ⁎ Corresponding author at: Ecology Laboratory of the First Institute Oceanography SOA, 6 Xianxialing Rd., Qingdao City, Shandong Province 266061, PR China. Tel.: +86 532 88893571; fax: +86 532 88893571. E-mail address: zhengfr@fio.org.cn (F. Zheng).
http://dx.doi.org/10.1016/j.genrep.2016.03.001 2452-0144/© 2016 Elsevier Inc. All rights reserved.
reproduction of starry flounder succeeded in China, so its feeding increased rapidly in recent years (Wang et al., 2006). Previous studies mainly focused on the growth (Campana and Neilson, 1982, Lee et al., 2003), biological status (Ma et al., 2006; Qi et al., 2008), ecological distribution and nutritional value (Liu et al., 2009a,b) of starry flounder. Moreover, the genetic, physiological and biochemical characteristics of wildlife starry flounder have been reported (You et al., 2007; Park and Kijma, 1991, Borsa et al., 1997). The fish gametogenesis and gonadal maturation are regulated by the neuroendocrine system through hypothalamic-pituitary-gonadal axis (HPG), so the understanding to the neuroendocrine regulation in the hypothalamus is helpful to investigate the regulation of reproduction in starry flounder and improve the technology of artificial reproduction (Lin, 2011). However, the regulatory mechanisms of gonadal development and endocrinology in starry flounder are largely unknown. The Illumina RNA sequencing has been widely used to revolutionize gene expression profiling and transcriptome analysis in non-model organisms, such as, Nilaparvata lugens and pufferfish (Hampton et al.,
46
B. Wang et al. / Gene Reports 4 (2016) 45–52
2011; Hua et al., 2011; Bao et al., 2012; Zheng et al., 2013; Gao et al., 2013). At present, the transcriptome sequencing technology has gradually replaced the gene chip technology and becomes the mainstream method for studying gene expression profiling at the level of whole genome. To investigate the genetic characteristics of starry flounders, this study applied high-performance sequencing technology to investigate the gene expression profiling of starry flounder brain at gonadal maturation and regression phases. This study may provide valuable theoretical data for improving the efficiency and quality of artificial breeding, establishing genetic linkage map, and protecting the germplasm of starry flounder as well. 2. Materials and methods 2.1. Sampling of starry flounders and total RNA extraction Healthy female starry flounders (n = 10) weighing 1000 ± 50 g were purchased from the Breeding Station of Rizhao Marine Aquatic Resources (Shandong, China) in April and May 2014 and kept in a 250 L seawater tank at 25 °C. The samples (n = 10) of brain from gonadal maturation and regression phases were collected and frozen in liquid nitrogen for RNA extraction. Total RNA was isolated by Trizol reagent according to the standard protocol and the quality of RNA was assessed by 1% agarose gel electrophoresis and ultraviolet spectrophotometer. 2.2. Total RNA pretreatment and sequencing The RNA sequencing (RNA-Seq) library was constructed according to the instructions of mRNASeq Sample Preparation Kit (Illumina, USA) (McKenna et al., 2010). Briefly, mRNA was isolated from total RNA using oligo (dT) beads and fragmented into 155 bp short fragments with fragmentation buffer by chemical reagent and high temperature method. The cleaved mRNA fragments were used as templates to synthesize first-strand cDNA using random hexamer primers, and then transformed into double-stranded cDNA using RNase H and DNA polymerase I. A paired-end library was constructed from the synthesized cDNA using Genomic Sample Prep Kit (Illumina). The short fragments in desirable length were purified by using QIAquick PCR Extraction Kit (Qiagen), end repaired and linked with sequencing adapters (Margulies et al., 2005). After removing the unsuitable fragments with AMPureXP beads, the sequencing library was constructed with PCR amplification, checked with Pico green staining and fluorospectrophotometry, quantified with Agilent 2100 and sequenced using Illumina Hiseq 2000 platform (Shanghai Personal Biotechnology Co., Ltd.). For sequencing, the raw reads were filtered by removing the adaptors with polyA and low quality reads. The cleaned reads were assembled into transcripts, contigs and unigenes using Velvet and Oases (http://www.ebi.ac.uk/~zerbino/ velvet; http://www.ebi.ac.uk/~zerbino/oases) Grabherr et al., 2011; Radakovits et al., 2012). 2.3. Sequence alignment, function annotation, analyses of gene expression and associated signaling pathways −5
To obtain the best annotation (E-value b 10 ), the alignment analyses for obtained sequences were carried out by using SwissProt, nr and Non-redundant database. All sequences were also analyzed by eggNOG database (E-value b 10− 5) to obtain NOG functional annotation and classification of expressed sequences, and to collect and establish gene homologous groups, including the COG and KOG data (http://www. ncbi.nlm.nih.gov/COG, http://eggnog.embl.de/) (Tatusov et al., 2003). RPKM (reads per kilobase per million mapped reads) (Mortazavi et al., 2008) was used to normalize the abundance of transcripts. Twofold differential expression and P-value b 0.05 were used to identify differentially expressed genes (DEGs) between two physiological phases. All DEGs were analyzed by KEGG pathway database to obtain the
metabolic pathways and the specific position information (http:// www.genome.jp/kegg/) (Kanehisa et al., 2008; Minoru et al., 2004). 2.4. Verification of representative DEGs by quantitative real-time PCR (qRT-PCR) Total RNA from 10 pituitary and hypothalamus samples at different phases was transcribed into first strand cDNA by Prime Script TM II short Kit (TaKaRa) following the manufacturer's instructions. qRT-PCR was performed according to the instructions of FastStart Universal SYBR Green Master (ROX) (Roche). The specific primers for GnRH, NKB, GtH, Kiss and LHR are listed in Table 1. All samples were analyzed in three duplicates and the general housekeeping gene β-actin was used as internal reference to normalize the target genes. The gene expression levels were calculated as the ratio between the mRNA level of the target genes and the reference gene using the comparative 2− ΔCt method (Livak and Schmittgen, 2001). Target Ct values were normalized to the endogenous gene β-actin. The PCR reaction conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 60 s, and 72 °C for 1 min. 3. Results 3.1. Preparation of RNA sequencing library Results from agarose gel electrophoresis and ultraviolet spectrophotometric test showed high purity and quality of RNA in all samples (Fig. 1). Total RNA samples were treated by Agilent DNA 1000 Kit, and the size of sample library ranged from 280 to 320 bp that meets the requirements of the sequencing and analysis. 3.2. Sequencing and assembling According the data from Illumina Hiseq 2000, a total of 53,567,296 and 75,630,680 raw reads were obtained from hypothalamus and pituitary at the gonadal maturation and regression phases, respectively. The average length of raw reads was 100 nucleotides. After trimming and quality check, 46,155,608 and 71,505,792 high-quality reads were recorded for the gonadal maturation and regression phases, respectively. All high-quality reads were assembled to obtain 176,002 sequences using Oases software (http://www.ebi.ac.uk/~zerbino/oases). In all sequences, the size of 160,790 sequences was bigger than 200 bp, the size of 27,728 sequences was bigger than N50, with the average length of 1854 bp (Table 2A, Fig. 2A). The transcription of samples was commonly performed using cluster analysis according to the similarity. To select the longest transcript as unigene sequences, 30,640 sequences were obtained through clustering unit, with a maximum length of
Table 1 Sequences of PCR primers. Primer
Sequence (5′-3′)
β-actin
F5′-CAACTGGGATGACATGGAGAAG -3′ R5′-TTGGCTTTGGGGTTCAGG -3′ F5′-AGAGGTATGGGAAGAGGTCCAGT -3′ R5′-ACTGTGGAAGAGTGTCTGTGCTTT -3′ F5′-CCTGGGACCATCATCAATTACA -3′ R5′-AGGCGGAGTGGTGTCTCTTC -3′ F5′-TCTTTGTCCTTTTCCAGTCGTTC -3′ R5′-GCGTTGTTATTGCCTTCC -3′ F5′-AGCCACATTTGAGGATTAGCAAC -3′ R5′-TCTGGAGGAGGGGTTATGGA -3′ F5′-AGGTGAAGCACATTAACGGATG -3′ R5′-GAGAGSGAGAGTGGAGGGAGGA -3′ F5′-CCTTCTCCTGCCTGTTCCTG -3′ R5′-GGGCTCTGGGGATGTTGTT -3′
NKB GtH Kiss PrRPR FSH GnRH
B. Wang et al. / Gene Reports 4 (2016) 45–52
47
other nutrients, hormone synthesis and release, and intracellular cell and a series of signal response mechanism, which is associated with the regulation of gonadal development and regression in starry flounder. KEGG (Kyoto Encyclopedia of Genes and Genomes) database has been widely used to analyze gene functions and gene network in the cells. To further understand the biological functions of DEGs and their associated metabolic pathways, we analyzed all DEGs by KEGG. We found that 334 up-regulated DEGs at the maturation phase involved 98 metabolic pathways, while 987 up-regulated DEGs at the regression phase involved 192 metabolic pathways (Fig. 6). Most of metabolic pathways are associated with immune system, CNS system, endocrine system, signaling molecules and interaction and signal transduction, which is consistent with GO enrichment analysis. Moreover, among all DEGs, 408 unigenes are involved in the regulation of reproduction and endocrinology. The classification of all DEGs is shown in Table 3. The major signaling molecules included calmodulins (98), phosphatases (534), protein kinases (798) and cyclic adenosine monophosphates (87).
Fig. 1. The total RNA from hypothalamus and pituitary gland during different gonad development periods. M: DL 2000 Marker; 1: the total RNA of brain during gonadal maturity; 2: the total RNA of brain during gonadal involution.
30,459 bp and the average length of 2696 bp. About 20,055 unigene sequences (65.50%) were mainly distributed in the range of 200– 3000 bp (Table 2B, Fig. 2B).
3.5. Verification of PrRPR, Kiss, GtH, NKB, FSH and GnRH expression at gonadal development and regression phases by qRT-PCR The expression of reproduction- and endocrinology-related genes including GnRH, GtH, FSH, PrRPR, neurokinin and Kiss was significantly different in brain between two developmental phases. The expression of GnRH, NKB, GtH, Kiss and PrRPR mRNAs (Fig. 7) in the brain from the regression phase was 5-fold higher than that from the maturation phase, while the expression level of FSH mRNA in the brain from the maturation phase was significantly higher than that from the regression phase (Fig. 6).
3.3. Sequences annotation and analysis of unigenes A total of 30,640 unigene sequences were compared to NCBI database, 24,128 sequences (78.75%) were found homologous annotation (E-value b 10− 5). All these 24,128 unigenes were analyzed by eggNOG and divided into 26 categories according to different functions, which mainly included signal transduction mechanism (26.81%), basic function (11.75%) and translation mechanism (11.18%) (Fig. 3). The expression of 24,128 unigenes at the gonadal maturation and regression phases was analyzed by the cutoff parameters of P b 0.05 and 2-fold difference, the results are shown in Fig. 4. The expression of 337 unigenes was up-regulated at the gonadal maturation phase, while the expression of 987 unigenes was up-regulated at gonadal regression phase (Fig. 4). 3.4. Analysis of differential expression of unigenes GO has been widely used to analyze biological transcriptome data (Ashburner et al., 2000), which is divided into 3 major functional categories: molecular functions, cellular components and biological processes. All 1321 DEGs between the gonadal maturation and regression phases were analyzed by GO database and the results are shown in Fig. 5. We found that the ratio of DEGs was very similar in the categories of molecular functions (ion binding), cell components (intracellular) and biological processes (biosynthetic process) between gonadal maturation and regression phases. According to GO functional enrichment analysis, DEGs are mostly involved in the accumulation of protein and
4. Discussion Since the artificial breeding of starry flounder was successful in 2006 in China, it has become one of the most important and widely farmraised marine fishes. However, the data for regulatory mechanisms of gonadal development and endocrinology, reproductive cycle and natural ovulation have been rarely studied. In this study, we investigated the transcriptome profiling in the brain of starry flounder at the phases of gonadal maturation and regression by using high-performance sequencing technology. This study may provide valuable theoretical data for understanding of natural ovulation and fertilization and protecting wild resource of starry flounders. It is well-established that the development of gonads is controlled by the hypothalamus–pituitary-gonadal axis, in which GnRH from hypothalamus will promote the production of GtH from pituitary gland, then GtH will promote the secretion of reproduction hormones from gonads, such as ovary. These reproduction hormones control follicle development and ovulation, but they also contribute to keep the balance of GtH hormone through negatively mediating GnRH and GtH secretion from HPG axis (Lin, 2011). Thus, the HPG axis plays a central role in reproduction and endocrinology. Whole-transcriptome analysis in hypothalamus and pituitary gland from the maturation and regression phases provides us with comprehensive insights into biological pathways and molecular mechanisms that regulate gonadal development and endocrinology in starry flounder (Grabherr et al., 2011). Using Illumina Hiseq 2000 technology, we obtained 30,640 unigenes, 24,128 of them with homologous annotation.
Table 2A Assembly statistic by Oases (Transcript). Total length (bp)
Locus no.
Transcript no.
N200 bp transcript
Max. length (bp)
Ave length (bp)
N50
NN50 reads no.
GC%
326,382,179
83,726
176,002
160,790
30,459
1854
3776
27,728
42.28
48
B. Wang et al. / Gene Reports 4 (2016) 45–52
Fig. 2. A: Transcript length distribution of the assembled in hypothalamus and pituitary of P. stellatus. Horizontal axis: the length of unigenes; vertical axis: the number of unigenes. B: Unigene length distribution of the assembled in hypothalamus and pituitary of P. stellatus. Horizontal axis: the length of unigenes; vertical axis: the number of unigenes.
After further analyses by GO and KEGG databases, many unigenes were related to reproduction regulation and involved in many metabolic signaling pathways that regulate gonadal maturation and regression. Starry flounder exhibits multiple spawning and asynchronous ovarian development. There are at least two major follicle populations at different developmental phases, after first ovulation, there are certain population of premature follicles. Based on our transcriptome profiling, before first ovulation (maturation phase), the up-regulated DEGs mostly included glucose transporters, protein phosphatases, tubulin, peroxidases, fructose-bisphosphate aldolases, glutamyl endopeptidases,
Table 2B Unigene statistic. Total length (bp)
Unigene no.
Max. length (bp)
Ave. length (bp)
N50
NN50 reads no.
GC%
82,619,097
30,640
30,459
2696
3969
6,897
48.94
transferrin, dihydropteridine reductases, etc. These genes are mostly involved in the metabolisms of protein, fat, calcium and phosphorus, while the expression of genes that regulate the expression of GnRH and GtH is not dramatically changed. These data indicate that ovarian development requires a large amount of nutrients and trace elements. After ovulation (regression phase), the up-regulated DEGs mostly included GnRHR, NPY, IGF, voltage-gated calcium (Ca2+) channels, progesterone receptor, etc., which are associated with the ovulation. These data suggest that the production of reproduction hormones is gradually increased during ovarian development in starry flounder. Previous studies on goldfish and carp reported that the secretion of GtH by GnRH is dependent on the availability of extracellular calcium ions, our study also revealed the important role of calcium metabolism in gonadal development (Richards et al., 2008; Liu et al., 2008; Bobe and Goetz, 2000, 2001). To verify the transcriptome data, we selected some representative DEGs that are involved in the regulation of reproduction. We observed that FSH mRNA expression in the brain tissues from the maturation phase was significantly higher compared with the regression phase,
B. Wang et al. / Gene Reports 4 (2016) 45–52
49
Fig. 3. eggNOG function classification of unigenes.
Fig. 4. Transcript abundance of unigenes of two different physiological stages. Nao1: The transcriptome library of gonad maturation of the brain. Nao2: The transcriptome library of gonad degenerate of the brain. Horizontal axis is the express different multiples, the vertical axis is the significant difference in expression, 2 times the difference threshold. Horizontal line of P b 0.05 threshold. Blue dots represent genes with differential expression conditions, yellow dots represent those that don't fit the difference expression of conditional gene in the diagram.
50 B. Wang et al. / Gene Reports 4 (2016) 45–52 Fig. 5. GO classification of starry flounder unigenes. The unigenes were classified into three main categories (cellular component, biological process and molecular function) and 40 subcategories. The x-axis indicates the percentage of unigenes. The y-axis indicates the significance of enrichment degree.
B. Wang et al. / Gene Reports 4 (2016) 45–52
51
Fig. 6. KEGG pathway classification of starry flounder unigenes.
while the expression of GtH, PrRPR, NKB, GnRH and Kiss mRNAs at the regression phase was significantly higher than that at the maturation phase. These data are consistent with those in transcriptome profiling. Levavi et al. (2009) reported that FSH plays a key role in the early development of gonads but not ovulation in salmonoids, while LH is a key player in gamete maturation and ovulation. Rocha et al. (2007) found that FSHR is strongly expressed in the eggs during vitellogenesis but not in mature eggs from sea bass, while the expression of LHβ
transcripts is highly expressed in the eggs from gametogenesis and ovulation (Gen et al., 2003). All data indicate that the role of GtHs in the regulation of bony fish reproduction is species-specific with some complexity. In summary, we reported the transcriptome profiling of hypothalamus and pituitary gland from the maturation and regression phases of starry flounder. Most of DEGs are involved in the regulation of reproduction and endocrinology. This study provides comprehensive and valuable data for understanding the regulatory mechanisms of reproductive cycle in starry flounder and improving the technology for artificial breeding.
Table 3 The genes involved in procreation regulation.
Acknowledgments Classification of genes
Number
Prolactin Thyroid hormone Endothelial growth factor Aromatase Estrone Estradiol Growth hormone Growth hormone releasing hormone Gonadotropin releasing hormone Estrogen Androgen Somatostatin Preprosomatostatin Kisspeptin Prolactin-releasing peptide receptor Follicle-stimulating hormone Neurokinin B and neuropeptide Catecholamine Dopamine Tachykinin Insulin-like growth factor 5-Hydroxytryptamine Activin Inhibin Bombesin Cholecystokinin Galanin Ghrelin Testosterone Ghrelin
13 11 89 1 1 8 24 6 12 24 8 9 1 4 6 1 81 1 14 9 18 25 20 7 1 4 6 1 3 1
This work was supported by the National High Technology Research and Development Program of China (863 Program, 2012AA10A413), the Fundamental Research Funds for the Central Public Research Institutes (2012G30) and the National Marine Public Welfare Research Project (201205020).
Fig. 7. The expression of GnRH, NKB, FSH, GtH, Kiss, and PrRPR during gonad mature stage and gonad degradation stage. N1: The pituitary gland and hypothalamus of gonad maturation. N2: The pituitary gland and hypothalamus of gonad degenerate.
52
B. Wang et al. / Gene Reports 4 (2016) 45–52
References Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M., Sherlock, G., 2000. Gene Ontology: tool for the unification of biology [J]. Nat. Genet. 25, 25–29. Bao, Y.Y., Wang, Y., Wu, W.J., Zhao, D., Xue, J., Zhang, B.Q., Shen, Z.C., Zhang, C.X., 2012. De novo intestine-specific transcriptome of the brown planthopper Nilaparvata lugens revealed potential functions in digestion, detoxification and immune response. Genomics 99 (4), 256–264. Bobe, J., Goetz, F.W., 2000. A tumor necrosis factor decoy receptor homologue is upregulated in the brook trout (Salvelinus fontinalis) ovary at the completion of ovulation [J]. Biol. Reprod. 62 (2), 420–426. Bobe, J., Goetz, F.W., 2001. Molecular cloning and expression of a TNF receptor and two TNF ligands in the fish ovary [J]. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 129 (2/3), 475–481. Borsa, P., Blanquer, A., Berrebi, P., 1997. Genetic structure of the flounders Platichthys flesus and P. stellatus at different geographic scales [J]. Mar. Biol. 129, 233–246. Campana, S.E., Neilson, J.D., 1982. Daily growth increments in otoliths of starry flounder (Platichthys stellatus) and the influence of some environmental variables in their production. Can. J. Fish. Aquat. Sci. 39 (7), 937–942. Gao, Y., Zhang, H., Gao, Q., Wang, L., Zhang, F., Siva, V.S., Zhou, Z., Song, L., Zhang, S., 2013. Transcriptome analysis of artificial hybrid pufferfish Jiyan-1 and its parental species: implications for pufferfish heterosis. PLoS One 8 (3), e58453. Gen, K., Yamaguchi, S., Okuzawa, K., Kumakura, N., Tanaka, H., Kagawa, H., 2003. Physiological roles of FSH and LH in red seabream, Pagrus major [J]. Fish Physiol. Biochem. 28 (1), 77–80. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B.W., Nusbaum, C., Lindblad-Toh, K., Friedman, N., Regev, A., 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome [J]. Nat. Biotechnol. 29, 644–652. Hampton, M., Melvin, R.G., Kendall, A.H., Kirkpatrick, B., Peterson, N., Andrews, M.T., 2011. Deep sequencing the transcriptome reveals seasonal adaptive mechanisms in a hibernating mammal. PLoS One 6 (10), 1–13, e27021. Hua, W.P., Zhang, Y., Song, J., Zhao, L.J., Wang, Z.Z., 2011. Denovo transcriptome sequencing in Salvia miltiorrhiza to identify genes involved in the biosynthesis of active ingredients. Genomics 98 (4), 272–279. Kanehisa, M., Araki, M., Goto, S., Hattori, M., Hirakawa, M., Itoh, M., Katayama, T., Kawashima, S., Okuda, S., Tokimatsu, T., Yamanishi, Y., 2008. KEGG for linking genomes to life and the environment [J]. Nucleic Acids Res. 36, D480–D484. Lee, S.M., Lee, J.H., Kim, K.D., 2003. Effect of dietary essential fatty acids on growth, body composition and blood chemistry of juvenile starry flounder (Platichthys stellatus). Aquaculture 225 (1), 269–281. Levavi, Sivan B., Bogerd, J., Mananos, E.L., Gomez, Z., Lareyre, J.J., 2009. Perspectives on fish gonadotropins and their receptors [J]. Gen. Comp. Endocrinol. 165 (3), 412–437. Lin, H.R., 2011. Fish Physiology. Zhongshan University Press. Liu, S.L., Wang, Bo, Liu, Z.H., Xie, L.P., 2009a. Analysis and evaluation of the nutrition of Platichthys stellatus. Prog. Fish. Sci. 30 (6), 18–30.
Liu, S.L., Wang, B., Liu, Z.H., Xie, L.P., Jiang, M.J., 2009b. Nutrition analysis and evaluation of starry flounder. Prog. Fish. Sci. 30 (6), 18–24. Liu, Z.L., Shimada, M., Richards, J.S., 2008. The involvement of the toll-like receptor family in ovulation [J]. J. Assist. Reprod. Genet. 25 (6), 223–228. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCt method. Methods 25 (4), 402–408. Ma, A.J., Zhuang, Z.M., Li, Chen, Sun, X.L., Wang, X.A., J.L., Lei, 2006. The biologic characteristics of starry flounder (Platichthy stelltus Pallas). Mar. Fish. Res. 27 (5), 91–95. Margulies, M., Egholm, M., Aitman, W.E., Attiya, S., Bader, J.S., 2005. Genome sequencing in microfabricated highdensity picolitre reactors. Nature 437, 376–380. McKenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A., Garimella, K., Altshuler, D., Gabriel, S., Daly, M., 2010. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20 (9), 1 297–1 303. Minoru, K., Susumu, Goto, Kawashima, C.S., Yasushi, O., Masahiro, H., 2004. The KEGG resource for deciphering the genome [J]. Nucleic Acids Res. 32 (Database issue), D277–D280 (January 1). Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L., Wold, B., 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq [J]. Nat. Methods 5, 621–628. Park, J.Y., Kijma, A., 1991. Genetic variability and differentiation within and between the stone flounder (Kareius bicoloratus) and the starry flounder (Platichthys stellatus) [J]. Tohoku J. Agric. Res. 41 (3–4), 69–82. Qi, G.S., Li, D., Chen, S.Q., Zhuang, Z.M., Liu, C.L., Song, Z.C., Deng, Y.S., 2008. Morphological characteristics and internal structure of Platichthys stellatus Pallas. J. Fish. Sci. China 15 (1), 1–11. Radakovits, R., Jinkerson, R.E., Fuerstenberg, S.I., Tae, H., Settlage, R.E., Boore, J.L., Posewitz, M.C., 2012. Draft genome sequence and genetic transformation of the oleaginous alga: Nannochloropsis gaditana [J]. Nat. Commun. 21 (3), 686. Richards, J.S., Liu, Z.L., Shimada, M., 2008. Immune-like mechanisms in ovolution [J]. Trends Endocrinol. Metab. 19 (6), 191–196. Rocha, A., Gomez, A., Zanuy, S., Cerdá-Reverter, J.M., Carrillo, M., 2007. Molecular characterization of two sea bass gonadotropin receptors: cDNA cloning, expression analysis, and functional activity [J]. Mol. Cell. Endocrinol. 272 (1–2), 63–76. Tatusov, R.L., Fedorova, N.D., Jackson, J.D., Jacobs, A.R., Kiryutin, B., Koonin, E.V., Krylov, D.M., Mazumder, R., Mekhedov, S.L., Nikolskaya, A.N., Rao, B.S., Smirnov, S., Sverdlov, A.V., Vasudevan, S., Wolf, Y.I., Yin, J.J., Natale, D.A., 2003. The COG database: an updated version includes eukaryotes [J]. BMC Bioinf. 4, 41 (11). Wang, B., Wang, Z.L., Sun, P.X., Liu, Z.H., Wang, S.X., 2006. The development prospect of the breeding conditions of Platichthys stellatus. Fish. Modernization 5, 16–18. You, F., Wu, Z.H., Li, J., Wang, B., Xu, D.D., Ni, J., Yu, D.D., Zhu, X.P., Zhang, P.J., Xu, Y.L., 2007. RAPD analysis of genetic diversity in cultured stock of Platichthys stellatus. Adv. Mar. Sci. 25 (1), 73–78. Zheng, M.G., Tian, J.H., Yang, G.P., Zheng, L., Chen, G.G., Chen, J.L., Wang, B., 2013. Transcriptome sequencing, annotation and expression analysis of Nannochloropsis sp. at different growth phases. Gene 523 (2), 117–121.