Cloning and expression profiling of the VLDLR gene associated with egg performance in duck (Anas platyrhynchos)

Abstract

Background: The very low density lipoprotein receptor gene (VLDLR), a member of the low density lipoprotein receptor (LDLR) gene family, plays a crucial role in the synthesis of yolk protein precursors in oviparous species. Differential splicing of this gene has been reported in human, rabbit and rat. In chicken, studies showed that the VLDLR protein on the oocyte surface mediates the uptake of yolk protein precursors into growing oocytes. However, information on the VLDLR gene in duck is still scarce.

Methods: Full-length duck VLDLR cDNA was obtained by comparative cloning and rapid amplification of cDNA ends (RACE). Tissue expression patterns were analysed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). Association between the different genotypes and egg performance traits was investigated with the general linear model (GLM) procedure of the SAS® software package.

Results: In duck, two VLDLR transcripts were identified, one transcript (variant-a) containing an O-linked sugar domain and the other (variant-b) not containing this sugar domain. These transcripts share ~70 to 90% identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences showed that duck VLDLR proteins were closely related with those of chicken and zebra finch. The two duck VLDLR transcripts are differentially expressed i.e. VLDLR-a is mainly expressed in muscle tissue and VLDLR-b in reproductive organs. We have localized the duck VLDLR gene on chromosome Z. An association analysis using two completely linked SNP sites (T/C at position 2025 bp of the ORF and G/A in intron 13) and records from two generations demonstrated that the duck VLDLR gene was significantly associated with egg production (P < 0.01), age of first egg (P < 0.01) and body weight of first egg (P < 0.05).

Conclusions: Duck and chicken VLDLR genes probably perform similar function in the development of growing oocytes and deposition of yolk lipoprotein. Therefore, VLDLR could be a candidate gene for duck egg performance and be used as a genetic marker to improve egg performance in ducks.

Figure 1

Nucleotide and deduced amino acid sequences of the cDNA encoding the duck VLDLR. Letters in bold character indicate the start codon (ATG); the cleaved signal sequence is boxed at the N-terminus; different ligand binding motifs within the ligand domain are underlined; YWXD repeats are indicated by a thick underline.

Figure 2

Nucleotide and deduced amino acid sequences of the cDNA encoding the duck VLDLR. An asterisk indicates the stop codon (TGA) and the polyadenylation signal sequence (AATAAA) is underlined and in bold character; YWXD repeats are indicated by a thick underline; serine and threonine residues that correspond to the O-linked sugar domain are indicated by double underline; the 23 amino-acid putative transmembrane domain is shaded; the FDNPVY sequence targeting the receptor to coated pits is marked by a wavy underline.

Figure 3

The phylogenetic tree of duck VLDLR amino acid sequences with other eleven animal species. The phylogenetic tree was constructed by the Neighbor-Joining (NJ) method of MEGA 4.0 using the deduced amino acid VLDLR sequence of duck and eleven other animal species; the number at the branches denotes the bootstrap majority consensus values on 1000 replicates; the branch lengths represent the relative genetic distance among these species.

Figure 4

Tissue expression profiles of the two splicing variants of duck VLDLR gene. Tissue samples are heart, liver, spleen, lung, kidney, muscle, brain, intestine, adipose tissue, pituitary gland, ovary and oviduct from adult female ducks; β-actin is used as control.

Figure 5

(A) AccI-PCR-RFLP and (B) RsaI-PCR- RFLP genotyping of duck VLDLR gene. TT (315 bp) and CC (191/124 bp) genotypes for the Acc1 site (A) and GG (168 bp) and AA (115/53 bp) genotypes for the Rsa1 site (B) were generated. The genotypes are shown at the top of each lane; M is marker1.