Molecular cloning and gene expression analysis of the leptin receptor in the Chinese mitten crab Eriocheir sinensis

Abstract

Background: Leptin is an adipocyte-derived hormone with multiple functions that regulates energy homeostasis and reproductive functions. Increased knowledge of leptin receptor function will enhance our understanding of the physiological roles of leptin in animals.

Methodology/principal findings: In the present study, a full-length leptin receptor (lepr) cDNA, consisting of 1,353 nucleotides, was cloned from Chinese mitten crab (Eriocheir sinensis) using rapid amplification of cDNA ends (RACE) following the identification of a single expressed sequence tag (EST) clone in a cDNA library. The lepr cDNA consisted of a 22-nucleotide 5'-untranslated region (5' UTR), a 402-nucleotide open reading frame (ORF) and a 929-nucleotide 3' UTR. Multiple sequence alignments revealed that Chinese mitten crab lepr shared a conserved vacuolar protein sorting 55 (Vps55) domain with other species. Chinese mitten crab lepr expression was determined in various tissues and at three different reproductive stages using quantitative real-time RT-PCR. Lepr expression was highest in the intestine, thoracic ganglia, gonad, and accessory gonad, moderate in hepatopancreas and cranial ganglia, and low in muscle, gill, heart, haemocytes, and stomach. Furthermore, lepr expression was significantly higher in the intestine, gonad and thoracic ganglia in immature crabs relative to precocious and mature crabs. In contrast, lepr expression was significantly lower in the hepatopancreas of immature crabs relative to mature crabs.

Conclusions/significance: We are the first to identify the lepr gene and to determine its gene expression patterns in various tissues and at three different reproductive stages in Chinese mitten crab. Taken together, our results suggest that lepr may be involved in the nutritional regulation of metabolism and reproduction in Chinese mitten crabs.

Figure 1. Nucleotide sequence and deduced amino acid sequence of Chinese mitten crab lepr.

Nucleotides (upper) are numbered beginning at the 5′ end. Amino acids (lower), shown using one-letter abbreviations, are numbered beginning at the initiating methionine. The signal peptide is underlined, and the mature peptide is enclosed in the black box. The three conserved cysteine residues in the deduced amino acid sequence are shown in grey boxes. The stop codon is marked by an asterisk. The polyadenylation signal (AATAAA) is enclosed in the black ellipse. Arrows indicate the positions of primers; black boxes indicate the coding region; white lines indicate 5′ and 3′ UTRs; curly bracket indicate position of the EST clone. The E. sinensis lepr sequence was submitted to GenBank under accession number GU443952.

Figure 2. Genomic organization and nucleotide sequence of Chinese mitten crab lepr.

Lepr intron sequences are shown in bold and italics. Intron dinucleotide acceptor and donor sites for RNA splicing are underlined. The polyadenylation signal is shown in bold.

Figure 3. ClustalX alignment of lepr from E. sinensis and 25 other organisms.

The vacuolar protein sorting 55 (Vps55) domain in lepr starts at Leu7 and ends at Asp127.

Figure 4. Phylogenetic tree of lepr amino acid sequences in four vertebrates and 22 invertebrates using the neighbour-joining method.

The numbers in the phylogram nodes indicate percent bootstrap support for the phylogeny. The bar at the bottom indicates 5% amino acid divergence in sequences.

Figure 5. Tissue distribution of the lepr transcript measured by SYBR Green RT-PCR.

Quantitative analysis of lepr gene expression relative to β-actin expression in different mitten crab tissues. Lepr gene expression was analyzed in the following tissues: In – intestine, TG – thoracic ganglia, Gn – gonad, AG – accessory gonad, Hp – hepatopancreas, CG – cranial ganglia, Ms – muscle, Gi – gill, Hr – heart, Hm – haemocytes and St – stomach. Expression data for each tissue were analyzed from three individual crabs. Vertical bars represent the mean ± S.E. (n = 3). Significant differences relative to controls are indicated with an asterisk (P<0.05) or with two asterisks (P<0.01).

Figure 6. Lepr mRNA expression levels in six tissues from normal crabs (in the rapid developmental stage), precocious crabs and normal mature crabs.

Values are shown as mean ± S.E. (n = 3). Significant differences relative to intestine are indicated with one asterisk (P<0.05) or with two asterisks (P<0.01).