Molecular cloning, sequence analysis, and cadmium stress-rated expression changes of BTG1 in freshwater pearl mussel (Hyriopsis schlegelii)

Abstract

The B cells translocation gene 1 (BTG1) is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among verteates. Here, for the first time we cloned the full-length cDNA sequence of Hyriopsis schlegelii (Hs-BTG1), an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of cDNA ends (RACE) together with splicing the EST sequence from a haemocyte cDNA liary, we found that Hs-BTG1 contains a 525 bp open reading frame (ORF) encoding a 174 amino-acid polypeptide, a 306 bp 5' untranslated region (5' UTR), and a 571 bp 3' UTR with a Poly(A) tail as well as a transcription termination signal (AATAAA). Homologue searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the inverteate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas (2.03-fold), mantle (2.07-fold), kidney (2.2-fold) and haemocyte (2.5-fold) were enhanced by cadmium (Cd²⁺) stress, suggesting that Hs-BTG1 may have played a significant role in H. schlegelii adaptation to adverse environmental conditions.

Keywords: BTG1; Cadmium stress; Gene cloning; Hyriopsis schlegelii; mRNA expression.

Figure 1

cDNA and deduced amino acid sequence of BTG1 in Hyriopsis schlegelii Nucleotide and deduced amino acid sequence were numbered on the left. The BTG/TOB function domain was shaded and the two highly conserved domains (BTG Boxes A and B) were indicated with a rounded rectangle. The stop codon TGA and the polyadenylation signal AATAAA were tagged with an asterisk and underline, respectively. The lower image depicts the BlastP result for Hs-BTG1.

Figure 2

Multiple alignment of BTG1 between Hyriopsis schlegelii with other species (A) Alignment of the deduced amino acid sequence of H. schlegelii BTG1 with the corresponding sequences from other species; (B) the tertiary (3D) structures of Homo sapiens BTG1 (a), Crassostrea gigas BTG1 (b), and H. schlegelii BTG1 (c) modeled in SWISS-MODEL. The highly conserved A Box and B Box regions are marked in (A) and a schematic representation of the α-helical (yellow) and β-sheet (purple) structural elements are present in (B). a. H. sapiens: NP_001722.1 BTG1, b. C. gigas: EKC27510.1 BTG1, c. H. schlegelii BTG1: AGT79958.1; C. gigas: EKC27510.1; Danio rerio: NP_956314.1; Xenopus laevis: NP_001080825; Gallus gallus: CAA45507.1; Mus musculus: NP_031595.1; H. sapiens: NP_001722.1. “…” indicates the same amino acid; “---” indicates the default at the corresponding sites.

Figure 3

Phylogenetic tree of BTG1 from Hyriopsis schlegelii and other animals Mus musculus BTG1: NP_031595.1; Homo sapiens BTG1: NP_001722.1; Gallus gallus BTG1: NP_990681.1; Danio rerio BTG1: NP_956314.1; Oryzias latipes BTG1: XP_004083105.1; Takifugu rubripes BTG1: XP_003972839; Xenopus laevis BTG1: NP_001080825; G. gallus BTG2: XP_418053; M. musculus BTG2: NP_031595.1; H. sapiens BTG2: NP_006754.1; Coptotermes formosanus BTG1: AGM32529; Apis florae BTG1: XP_003697190; Lycosa singoriensis BTG1: ABX75488; Lottia gigantea BTG1: ESO88805; H. schlegelii BTG1: AGT79958.1; C. gigas BTG1: EKC27510.1. The number in the branch is the bootstrap value.

Figure 4

Expression level of Hs-BTG1 mRNA in different tissues

Figure 5

Fold inductions of Hs-BTG1 mRNA levels in hepatopancreas (a), kidney (b), haemocytes (c) and mantle (d) after exposure to waterborne Cd exposure (5 mg/L) Data represent group mean fold induction±SD (n=3 in each time). Asterisks indicate mean fold induction, which are significantly (P<0.05) different from control values (0.005 mg/L).