关键词: FREM1基因, 豁眼性状, cDNA克隆, 豁眼鹅, 基因表达

Abstract: 【Objective】Recessive Z-link inheritance of the Huoyan trait has been confirmed, but its genetic basis still has not been defined. Recessive mutations in the coding region of FRAS-related extracellular matrix1 (FREM1) gene have been shown to cause partial or whole loss of upper eyelid in human and model mice. In this study, cloning, expression and polymorphism analysis of the goose FREM1 gene were carried out using goose resource populations with Huoyan trait as main materials, to verify the hypothesis that FREM1 is a candidate gene affecting eyelid traits of goose, which will lay a foundation for further study of molecular genetic mechanism of eyelid traits.【Method】The coding region of goose FREM1 gene was amplified and cloned from total RNA of goose upper eyelids and kidney tissues, which were collected from the Huoyan goose (3 females), Sichuan white goose(3males and 3females), F₁ population of male Huoyan goose mated with female Sichuan White goose(3males), byRT-PCR according to the full-length transcript of FREM1 (XM_013193557), and the bioinformation analysis was performed with it. Then, the expression of FREM1 gene in goose eyelid and kidney tissue was studied by real-time fluorescence quantitative PCR. The whole blood DNA was extracted from 30 adult male Huoyan geese, 30 male Sichuan white geese, and 30 male geese of F₁ population of Huoyan goose mated reciprocally with female Sichuan White goose, and the distribution of FREM1 gene mutations in different goose populations was detected by direct sequencing according to the genomic sequences of FREM1 (Anser cygnoides domesticus breed Zhedong scaffold203).【Result】①The 7 305bp cDNA sequences of FREM1 gene was obtained by sequencing and splicing, which contained a complete CDS region encoding 2 184 amino acids. Compared with the nucleotide sequences of the Sichuan White goose and Zhedong White goose, the c.4 515T＞ C missense mutation was found in CDS of FREM1 gene of Huoyan goose, which causes an Valine to Alanine change (p.Val1505Ala) at a highly conserved amino acid residue in FREM1’s tenth CSPG domain. Although, the p.1 505Val＞Ala change was predicted to be “Tolerated” by SIFT, it was subsequently predicted to lead to decreased protein stability by the I-Mutant ΔΔG and MUPro program. ②FREM1 gene was expressed in the tissues of eyelids and kidneys of Sichuan White goose, but the expression level in kidney was much higher than eyelid, more importantly, the expression level of the male is nearly twice as that of the female. There was no significant difference in the relative expression of FREM1 gene between Z^HW(Normal eyelids)and Z^hW(Upper eyelids colomobas) genotypes (P＞0.05). Although the relative expression of FREM1 gene of Z^HZ^h(Normal eyelids) genotype was more than 2 times than that of Z^HW and Z^hW genotype, it could be caused by differences in gender. ③In Huoyan Goose populations, the frequencies of HH, Hh and hh genotypes were 0, 0 and 1.0, the frequencies of alleles H and h were 0 and 1.0, the heterozygosity was 0; The frequencies of HH, Hh and hh were 1.0, 0 and 0 in Sichuan white goose population, the frequencies of the alleles H and h were 1.0 and 0, the heterozygosity was 0 in Sichuan White Goose populations; The frequencies of HH, Hh and hh were 0, 1.0 and 0, the frequencies of the alleles H and h were 0.5 and 0.5, the heterozygosity was 1.0 in F₁ generation population of Huoyan Goose mated reciprocally with female Sichuan White Goose.【Conclusion】The FREM1 gene is the H locus which determines the eyelid traits of goose. A homozygous missense mutation in the coding region of the FREM1gene caused an Valine to Alanine change (p.Val1505Ala) at a highly conserved amino acid residue in FREM1’s tenth CSPG domain, which affected the stability of FREM1 protein. Therefore, mutation c.4514T＞ C of gene FREM1can be used as an important molecular marker.

Key words: FREM1 gene, trait of upper eyelids colomobas, cloning of cDNA, Huoyan Goose, gene expression