摘要：
[目的]鹅豁眼性状呈隐性伴性遗传,其遗传基础有待揭示.基因FRAS-related extracellular matrix 1(FREM1)编码区的一些隐性突变导致了人类及模型小鼠上眼睑部分或完全缺失.本试验以鹅豁眼性状资源群为主要材料,通过对鹅基因FREM1的克隆、表达及基因多态性分析,验证FREM1是影响鹅上眼睑性状候选基因的假设,为深入研究眼睑性状的分子遗传机制奠定基础.[方法]采集鹅豁眼性状F2资源群中成年纯种豁眼鹅母鹅(3只)、四川白鹅(6只,雌雄各半)、♂豁眼×♀四川白鹅F1代公鹅(3只)的眼睑和肾组织,提取其总RNA,以鹅FREM1(XM_013193557)全长转录序列为参考设计引物,利用反转录RT-PCR克隆鹅FREM1基因,对其进行生物信息学分析,进而,采用实时荧光定量PCR法研究鹅眼睑和肾组织FREM1基因的表达特性.采集成年纯种豁眼鹅公鹅、四川白鹅公鹅和♂豁眼×♀四川白鹅正反交F1代公鹅各30只的血液,提取全血DNA,以鹅FREM1 (Anser cygnoides domesticus breed Zhedong scaffold203_32,NCBI)序列设计引物,利用直接测序法检测FREM1基因变异位点在不同鹅群体中的分布情况.[结果]①经测序和拼接,获得鹅FREM1基因cDNA序列7 305bp,该序列包含一个完整的CDS(Coding Sequences)区,编码2 184个氨基酸.与四川白鹅和浙东白鹅相比,在豁眼鹅FREM1基因CDS序列上发现第4 515bp:T＞C是错义突变,导致第1 505aa:Val＞Ala变化,位于FREM1蛋白的CSPG重复结构域中第1 0个CSPG上.利用在线工具SIFT预测该氨基酸替换对蛋白功能的影响较小.通过I-Mutant △△G和MUPro程序分析,p.1505V＞A位点氨基酸替换大幅度降低了FREM1蛋白的稳定性.②FREM1基因在四川白鹅公、母鹅的眼睑和肾脏2个组织中均有表达,但肾脏表达水平远远高于眼睑,更为重要的是,公鹅FREM1基因的组织表达水平正好接近母鹅的2倍.Z“W(正常眼睑)和ZhW(上眼睑部分缺失)基因型鹅FREM1基因相对表达量无差异(P＞ 0.05),虽然ZHZh(正常眼睑)基因型鹅的FREM1基因相对表达量是Z“W和ZhW基因型鹅的2倍多,但这可能是性别不同导致的差异.③豁眼鹅群体中基因型HH、Hh和hh的频率分别是0、0和1.0,等位基因H和h的频率分别是0和1.0,杂合度为0;四川白鹅群体中基因型HH、Hh和hh的频率分别是1.0、0和0,等位基因H和h的频率分别是1.0和0,杂合度为0;F1代群体中基因型HH、Hh和hh的频率分别是0、1.0和0,等位基因H和h的频率分别是0.5和0.5,杂合度为1.0.[结论]基因FREM1是决定鹅上眼睑性状的H基因座,该基因编码区1个纯合型错义突变导致了FREM1蛋白第10个CSPG结构域的变化,从而影响了FREM1蛋白的稳定性,基因FREM1的c.4514T＞C突变可作为鹅豁眼性状重要的分子标记.%[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 FREM 1 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 FREM 1 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 ofFREM1 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 FREM 1 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 △△G 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 ZHW(Normal eyelids)and ZhW(Upper eyelids colomobas) genotypes (P＞0.05).Although the relative expression of FREM1 gene of ZHZh(Normal eyelids) genotype was more than 2 times than that of ZHW and ZhW 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 FREMlgene caused an Valine to Alanine change (p.Vall505Ala) at a highly conserved amino acid residue in FREM1's tenth CSPG domain,which affected the stability of FREM 1 protein.Therefore,mutation c.4514T＞ C of gene FREM1 can be used as an important molecular marker.
