于金成，于寧，趙輝，李喆

（遼寧省農(nóng)業(yè)科學(xué)院草牧業(yè)研究所，沈陽 110161）

鵝豁眼性狀H基因座候選基因FREM1的驗證分析

于金成，于寧，趙輝，李喆

（遼寧省農(nóng)業(yè)科學(xué)院草牧業(yè)研究所，沈陽 110161）

【目的】鵝豁眼性狀呈隱性伴性遺傳，其遺傳基礎(chǔ)有待揭示?；騀RAS-related extracellular matrix 1（FREM1）編碼區(qū)的一些隱性突變導(dǎo)致了人類及模型小鼠上眼瞼部分或完全缺失。本試驗以鵝豁眼性狀資源群為主要材料，通過對鵝基因FREM1的克隆、表達及基因多態(tài)性分析，驗證FREM1是影響鵝上眼瞼性狀候選基因的假設(shè)，為深入研究眼瞼性狀的分子遺傳機制奠定基礎(chǔ)?！痉椒ā坎杉Z豁眼性狀F2資源群中成年純種豁眼鵝母鵝（3只）、四川白鵝（6只，雌雄各半）、♂豁眼×♀四川白鵝F1代公鵝（3只）的眼瞼和腎組織，提取其總RNA，以鵝FREM1（XM_013193557）全長轉(zhuǎn)錄序列為參考設(shè)計引物，利用反轉(zhuǎn)錄RT-PCR克隆鵝FREM1基因，對其進行生物信息學(xué)分析，進而，采用實時熒光定量PCR法研究鵝眼瞼和腎組織FREM1基因的表達特性。采集成年純種豁眼鵝公鵝、四川白鵝公鵝和♂豁眼×♀四川白鵝正反交F1代公鵝各30只的血液，提取全血DNA，以鵝FREM1（Anser cygnoides domesticus breed Zhedong scaffold203_32，NCBI）序列設(shè)計引物，利用直接測序法檢測FREM1基因變異位點在不同鵝群體中的分布情況。【結(jié)果】①經(jīng)測序和拼接，獲得鵝FREM1基因cDNA序列7 305bp，該序列包含一個完整的CDS(Coding Sequences）區(qū)，編碼2 184個氨基酸。與四川白鵝和浙東白鵝相比，在豁眼鵝FREM1基因CDS序列上發(fā)現(xiàn)第4 515bp:T＞C是錯義突變，導(dǎo)致第1 505aa:Val＞Ala變化，位于FREM1蛋白的CSPG重復(fù)結(jié)構(gòu)域中第10個CSPG上。利用在線工具SIFT預(yù)測該氨基酸替換對蛋白功能的影響較小。通過I-Mutant ΔΔG和MUPro程序分析，p.1505V＞A位點氨基酸替換大幅度降低了FREM1蛋白的穩(wěn)定性。②FREM1基因在四川白鵝公、母鵝的眼瞼和腎臟2個組織中均有表達，但腎臟表達水平遠遠高于眼瞼，更為重要的是，公鵝FREM1基因的組織表達水平正好接近母鵝的2倍。ZHW（正常眼瞼）和ZhW（上眼瞼部分缺失）基因型鵝FREM1基因相對表達量無差異（P＞0.05），雖然ZHZh（正常眼瞼）基因型鵝的FREM1基因相對表達量是ZHW和ZhW基因型鵝的2倍多，但這可能是性別不同導(dǎo)致的差異。③豁眼鵝群體中基因型HH、Hh和hh的頻率分別是0、0和1.0，等位基因H和h的頻率分別是0和1.0，雜合度為0；四川白鵝群體中基因型HH、Hh和hh的頻率分別是1.0、0和0，等位基因H和h的頻率分別是1.0和0，雜合度為0；F1代群體中基因型HH、Hh和hh的頻率分別是0、1.0和0，等位基因H和h的頻率分別是0.5和0.5，雜合度為1.0?！窘Y(jié)論】基因FREM1是決定鵝上眼瞼性狀的H基因座，該基因編碼區(qū)1個純合型錯義突變導(dǎo)致了FREM1蛋白第10個CSPG結(jié)構(gòu)域的變化，從而影響了FREM1蛋白的穩(wěn)定性，基因FREM1的c.4514T＞C突變可作為鵝豁眼性狀重要的分子標(biāo)記。

FREM1基因；豁眼性狀；cDNA克?。换硌垸Z；基因表達

0 引言

【研究意義】鵝豁眼性狀呈伴性遺傳，其形成主要受H和M兩個基因座影響，其中H基因座位于Z染色體上，該基因座與眼瞼發(fā)育有密切的關(guān)聯(lián)[1]。從分子生物學(xué)角度研究鵝豁眼性狀的遺傳基礎(chǔ)，闡釋眼瞼缺失表型形成的原因，對豁眼鵝遺傳資源保護以及在肉鵝配套系中的利用方面具有重要的經(jīng)濟意義。此外，眼瞼發(fā)育異常也出現(xiàn)于兩類罕見的人類常染色體隱性遺傳疾病Manitoba-oculo-tricho-anal（MOTA）綜合征和Bifid nose and anorectal and renal anomalies（BNAR）綜合征[2-4]。通過現(xiàn)代分子生物學(xué)手段，人們找到了一些與眼瞼缺陷病癥相關(guān)的染色體區(qū)域、關(guān)鍵通路和基因[5-10]，但該病癥的遺傳機制尚未完全明了。因此，開展豁眼性狀候選基因研究，既可闡明該性狀的分子遺傳機制，又為人類眼瞼異常病癥研究提供參考。【前人研究進展】ALAZAMI等[11]通過連鎖分析，將MOTA綜合征關(guān)聯(lián)基因精細定位于人類染色體9p22.3，該區(qū)域FRAS-related extracellular matrix1（FREM1）基因與上眼瞼發(fā)育相關(guān)[7]?；騀REM1編碼基底膜的細胞外基質(zhì)組份，與Fras1、Frem2共同組成胞外基質(zhì)（extracellular matrix, ECM）蛋白, 從而調(diào)節(jié)胚胎發(fā)育過程中表皮基底膜和底層真皮之間的粘合；FREM1蛋白擁有典型的12個硫酸軟骨素蛋白多糖（CSPG）重復(fù)結(jié)構(gòu)域、氧化鈣β（Cax-β）結(jié)構(gòu)域和C型植物凝集素（C-type lectin）結(jié)構(gòu)域，這些結(jié)構(gòu)域在ECM蛋白家族都很保守，發(fā)揮著重要的生物學(xué)作用[12]。通過研究模型動物和人類MOTA綜合征家族的基因FREM1序列信息，發(fā)現(xiàn)該基因的移碼、外顯子刪除和錯義突變均導(dǎo)致上眼瞼殘缺表型的出現(xiàn)[13-18]?！颈狙芯壳腥朦c】檢索GenBank和Ensemble等數(shù)據(jù)庫，發(fā)現(xiàn)雞、鴨、火雞和斑胸草雀等禽類的FREM1基因均位于Z染色體上，而且像人類一樣，F(xiàn)REM1 與CER1、ZDHHC21基因一起連鎖[7]，表明FREM1基因在各物種中保守性強，發(fā)揮的生物學(xué)作用比較相似。雖然鵝基因組的物理圖譜尚未公布，但禽類基因組在很大程度上具有保守性和一致性[19-20]，且禽類Z染色體上的基因排序非常相似[21-22]，尤其雞、鴨和鵝Z染色體上的基因及其位置順序高度一致[23]。據(jù)此，假設(shè)基因FREM1是決定鵝上眼瞼性狀的H基因座，則與正常眼瞼鵝相比，豁眼鵝基因FREM1編碼區(qū)序列存在純合型變異，使得鵝豁眼性狀資源群中帶有該變異信息的FREM1基因呈現(xiàn)Z染色體連鎖模式。【擬解決的關(guān)鍵問題】本實驗以鵝豁眼性狀資源群為主要材料，克隆豁眼鵝和四川白鵝的FREM1基因，利用生物信息學(xué)手段分析該基因的結(jié)構(gòu)和變異信息，結(jié)合其mRNA在不同基因型鵝組織中的表達情況，確證基因FREM1是豁眼形成的原因之一，為全面揭示眼瞼性狀的分子遺傳機制奠定基礎(chǔ)。

1 材料與方法

試驗于2016年3—9月在遼寧省農(nóng)業(yè)科學(xué)院創(chuàng)新中心分子實驗室和遼寧省農(nóng)業(yè)科學(xué)院彰武隆江牧業(yè)種鵝場進行。

1.1 試驗動物和組織采集

從遼寧省農(nóng)業(yè)科學(xué)院彰武隆江牧業(yè)種鵝場的鵝豁眼性狀F2資源群中挑選出成年純種豁眼鵝母鵝3只、四川白鵝6只（雌雄各半）、♂豁眼×♀四川白鵝F1代公鵝3只，頸動脈放血處死后，取其上眼瞼和腎組織，立即投入液氮中，-80℃保存?zhèn)溆谩?/p>

挑選出成年純種豁眼鵝公鵝、四川白鵝公鵝和♂豁眼×♀四川白鵝正反交F1代公鵝各30只，翅靜脈采血2mL，肝素抗凝并置于-20℃保存?zhèn)溆谩?/p>

1.2 總RNA 的制備和反轉(zhuǎn)錄cDNA合成

鵝的上眼瞼和腎組織，在液氮中研磨成粉末狀，加入TRIzol裂解液裂解組織，用氯仿/異戊醇、異丙醇抽提，75%乙醇洗滌，適量無RNA 酶的水溶解。NanoDrop2000分光光度計測定總RNA的純度和濃度，凍存于-80℃。

采用PrimeScript? RT reagent Kit with gDNA Eraser進行cDNA反轉(zhuǎn)錄，實驗操作按產(chǎn)品說明書進行。將所得的cDNA置于-20℃保存。

1.3 FREM1基因cDNA克隆與測序

根據(jù)GenBank已公布的鵝FREM1基因序列（XM_013193557）設(shè)計8 對引物（表1和圖1），以合成的鵝cDNA為模板進行PCR擴增。PCR反應(yīng)體系（25 μL）：1.0μLcDNA、上下游引物各1.0 μL、2.5 μL dNTP Mix、0.25 μLKODFX、加ddH2O至25 μL。

反應(yīng)條件：95°C 5 min；94°C 1min，53°C 1min，72°C 1.5min，35個循環(huán)；4℃保存。將擴增產(chǎn)物在1.0%瓊脂糖凝膠中電泳，DNA磁珠法回收純化試劑盒將PCR產(chǎn)物純化，送北京華康同創(chuàng)生物技術(shù)有限公司測序。

1.4 鵝血紅細胞基因組DNA提取

采用天根生化科技有限公司提供的血液基因組DNA提取試劑盒提取鵝基因組DNA。用1%瓊脂糖檢測提取DNA的質(zhì)量。全波長紫外/可見光掃描分光光度計Nanovue檢測DNA的濃度和純度，并置于-20℃保存?zhèn)溆谩?/p>

圖1 RT-PCR擴增鵝基因FREM1片段所用引物示意圖Fig.1 Schematic diagram of the primers used for amplication of the gene FREM1 by RT-PCR

1.5 PCR擴增與突變位點檢測

根據(jù)GenBank已公布的鵝FREM1基因序列（Anser cygnoides domesticus breed Zhedong scaffold203_32, NCBI）設(shè)計引物（表1），以鵝總DNA為模板進行擴增。反應(yīng)體系為：2×Taq PCRMaster Mix10μL，模板DNA2.0μL，引物F11的上下游引物各2.0μL，加滅菌蒸餾水至25μL。反應(yīng)條件為：94℃預(yù)變性4min；94℃變性30s，60℃退火30s，72℃延伸2min，30個循環(huán)；72℃延伸10 min；4℃保存。1%的瓊脂糖凝膠電泳檢查PCR產(chǎn)物。PCR產(chǎn)物送北京華康同創(chuàng)生物技術(shù)有限公司進行直接測序。

表1 各引物基本信息Table 1 Descriptions of the 11 primers used in this study

1.6 qRT-PCR檢測不同基因型鵝FREM1基因的表達水平

以梯度稀釋的含有目的片段的質(zhì)粒為標(biāo)準品，做標(biāo)準曲線，每個樣品設(shè)置3個重復(fù)，對FREM1和ACTB進行實時定量PCR擴增。根據(jù)所獲得的鵝FREM1基因序列設(shè)計定量PCR引物（表1）。反應(yīng)體系（20 μL）:模板cDNA 2.0 μL，2×Power SYBR? MasterMix 10.0 μL，上游引物0.5 μL，下游引物0.5 μL，加ddH2O至20 μL。反應(yīng)條件：95℃ 30s；95℃ 5s，60℃ 40s（收集熒光）, 45個循環(huán)；熔點曲線分析60—95℃。以鵝ACTB（XM_013174886）在肝臟組織中的表達作內(nèi)標(biāo)，通過Ct值與標(biāo)準曲線，計算的目標(biāo)基因的相對表達量和標(biāo)準差，用 Excel 作圖。

1.7 數(shù)據(jù)統(tǒng)計與分析

采用Primer 6.0和Beacon designer 7.8軟件設(shè)計PCR、RT-PCR和qRT-PCR引物。獲得的序列利用DNAMan軟件、MEGA4.0和NCBI的Blastn等軟件進行生物信息學(xué)分析。采用Excel和SPSS18.0軟件處理試驗數(shù)據(jù)、進行t檢驗、方差分析和顯著性檢驗。

2 結(jié)果

2.1 鵝FREM1cDNA 克隆及生物信息學(xué)分析

以豁眼鵝和四川白鵝腎臟組織 cDNA 為模板，利用引物 F1-F8 （表1）進行 PCR 擴增。通過拼接多次RT-PCR序列獲得了豁眼鵝和四川白鵝兩個品種鵝的FREM1cDNA序列7 305 bp，該序列包含一個完整的CDS區(qū)，編碼2 184個氨基酸。

圖2 基因FREM1突變導(dǎo)致鵝豁眼性狀示意圖。Fig. 2 Mutation in FREM1 is responsible for Huoyan trait of goose.

與正常眼瞼表型的四川白鵝和浙東白鵝[24]（XM_013193557.1）序列相比，在豁眼鵝FREM1基因CDS序列上發(fā)現(xiàn)10個單核苷酸突變，其中1個是錯義突變，為第4 514 P:T＞C（位于外顯子24上）（圖2-A），第1 505aa：纈氨酸（Val）＞丙氨酸（Ala）變化（圖2-B），位于FREM1蛋白的CSPG重復(fù)結(jié)構(gòu)域中第10個CSPG上（圖2-C）。利用在線工具SIFT（http://sift.jcv.org）預(yù)測氨基酸替代對蛋白功能的影響（預(yù)測閾值小于0.05則為有害）p.1505V＞A位點預(yù)測值為0.51，表明該氨基酸替換對蛋白功能的影響較小[25]。由于檢測到人類FREM1基因移碼、外顯子刪除和錯義突變等影響蛋白功能的變異情況，使得相應(yīng)病例不但有眼瞼缺失的表型，同時帶有異常的發(fā)際線，隱眼、鼻裂、腎發(fā)育不全等癥狀，甚至有些不到成年就夭折[13-18]，表明FREM1蛋白有重要的生物學(xué)功能，極其有害的突變會降低該等位基因攜帶者的適合度，因而，從另一個角度提示，豁眼鵝FREM1基因p.1505V＞A的突變雖然可能是導(dǎo)致豁眼表型的原因之一，但是其影響的程度是比較溫和的。

由于眾多致病性的錯義突變都會引起蛋白穩(wěn)定性的變化，利用在線工具分析p.1505V＞A位點氨基酸變化是否導(dǎo)致了FREM1蛋白穩(wěn)定性的降低[26-27]。通過I-Mutant ΔΔG程序（http://gpcr2.biocomp. unibo.it/cgi/predictors/I-Mutant3.0/I-Mutant3.0.cgi;

Tem.25℃, pH 7.0）分析，結(jié)果顯示，p.1505V＞A位點的自由能變化為-1.95Kcal·mol-1，表明該氨基酸替換大幅度降低了FREM1蛋白的穩(wěn)定性[26,28]。利用MUPro（http://mupro. proteomics.ics.uci.edu）在線工具分析也得到了同樣的結(jié)果[29]，其中，基于支持向量機（support vector machine，SVM）方法分析得分是-1，表明蛋白穩(wěn)定性降低；基于神經(jīng)網(wǎng)絡(luò)（neural network，NW）方法分析得分是-0.95，表明蛋白穩(wěn)定性降低。

2.2 鵝眼瞼和腎臟組織FREM1基因mRNA表達水平分析

人類MOTA綜合征表現(xiàn)為眼瞼部分缺失，異常的發(fā)際線，隱眼、鼻裂、腎發(fā)育不全等[3-4]，而基因FREM1也主要在人類和小鼠的表皮、眼部、腎臟和肺臟中表達[30]，檢測鵝基因FREM1在眼瞼和腎臟組織中的表達情況，以驗證該基因是否存在性別和不同表型之間的差別。

FREM1基因在四川白鵝公、母鵝的眼瞼和腎臟2個組織中均有表達，但腎臟組織表達水平遠遠高于眼瞼，更為重要的是，公鵝FREM1基因的組織表達正好是母鵝的2倍（圖3），表明鵝基因FREM1表達可能與性別關(guān)聯(lián)，即公鵝有2個FREM1基因，而母鵝只有1個。根據(jù)鵝FREM1基因變異和H基因座情況[1]，定義c.4514T＞C位點為H基因座重要的分子標(biāo)記，其中，定義野生型（T）為H基因座的H等位基因，突變型（C）為H基因座的h等位基因。

根據(jù)鵝豁眼性狀遺傳模式分析[1]，四川白鵝母鵝（正常眼瞼）、豁眼鵝母鵝（上眼瞼部分缺失）及其雜交F1代公鵝（正常眼瞼）H基因座的基因型分別為ZHW、ZhW和ZHZh。圖4顯示了FREM1基因在不同基因型鵝眼瞼組織中的相對表達情況。ZHW和ZhW基因型鵝的FREM1基因相對表達量無差異（P＞0.05）；ZHZh基因型鵝的FREM1基因相對表達量是ZHW和ZhW基因型鵝的2倍，這可能是不同性別鵝帶有FREM1基因的數(shù)量不同所致（圖3）。這些結(jié)果提示，F(xiàn)REM1基因兩個外顯子的錯義突變可能并未影響該基因的表達，證實了豁眼鵝FREM1蛋白氨基酸序列變化可能只是影響了該蛋白穩(wěn)定性的預(yù)測。

圖3 FREM1基因在公母鵝眼瞼和腎臟中的表達情況Fig. 3 Relative mRNA expression level of FREM1 gene in eyelid and kidney of male and female geese

圖4 FREM1基因在不同基因型鵝的上眼瞼組織中表達情況Fig. 4 Relative mRNA expression level of FREM1 gene in upper eyelid of different genotype of goose

2.3 鵝豁眼性狀資源群基因FREM1變異及基因型頻率情況

由于H座位呈伴性遺傳，檢測豁眼鵝、四川白鵝和其正反交F1群體中公鵝的基因型更具代表性。檢測結(jié)果顯示（表2），豁眼鵝H基因座上基因型HH、Hh和hh的頻率分別是0、0和1.0，等位基因H和h的頻率分別是0和1.0，雜合度為0；四川白鵝H基因座上基因型HH、Hh和hh的頻率分別是1.0、0和0，等位基因H和h的頻率分別是1.0和0，雜合度為0；F1代H基因座上基因型HH、Hh和hh的頻率分別是0、1.0和0，等位基因H和h的頻率分別是0.5和0.5，雜合度為1.0。結(jié)果表明，豁眼鵝群體是h等位基因純合型，四川白鵝群體是H等位基因純合型，這與鵝豁眼性狀遺傳模式分析的理論推理一致[1]。

表2 鵝豁眼性狀資源群H基因座變異情況Table 2 Current status of genetic variation at the H locus in F2resource population with Huoyan trait

3 討論

本試驗克隆了鵝FREM1基因的編碼區(qū)序列，通過與正常眼瞼鵝FREM1基因序列的比對分析，在豁眼鵝群體中發(fā)現(xiàn)純合型錯義突變c.4514T＞C（圖2-A），通過I-Mutant ΔΔG程序和MUPro在線工具分析，表明該突變位點對蛋白功能影響較小，可能只是導(dǎo)致了蛋白穩(wěn)定性的降低，這需要進一步的驗證。通過對FREM1基因在不同基因型鵝組織中的實時定量分析（圖3和圖4），表明鵝FREM1基因與Z染色體存在較強的關(guān)聯(lián)性，同時，結(jié)合鵝豁眼性狀資源群中基因FREM1變異分析（表2），筆者初步認為基因FREM1是鵝豁眼性狀H座位的候選基因，其編碼區(qū)c.4514T＞C突變可作為鵝豁眼性狀重要的分子標(biāo)記。

FREM1蛋白擁有典型的12個CSPG重復(fù)結(jié)構(gòu)域、Cax-β結(jié)構(gòu)域和C-type lectin結(jié)構(gòu)域，這些結(jié)構(gòu)域在ECM蛋白家族都很保守，發(fā)揮著重要的生物學(xué)作用[12]。眾多研究表明，帶有CSPG結(jié)構(gòu)域的蛋白家族與血小板衍生生長因子（platelet-derived growth factor）、成纖維細胞生長因子（fibroblast growth factor）、膠原蛋白V（collagen V）和膠原蛋白VI（collagen VI）等因子有互作效應(yīng)[31-33]，提示FREM1蛋白可能也有類似的作用[34]?；硌垸Z基因FREM1的氨基酸替換位于FREM1蛋白高度保守的CSPG重復(fù)結(jié)構(gòu)域中第10個CSPG上（圖2-C），通過在線工具預(yù)測，表明該位置的突變大大降低了蛋白的穩(wěn)定性。由于豁眼性狀是由H和M兩個基因座互作而形成[1]，推測豁眼鵝H基因座的FREM1氨基酸序列p.1505Val＞Ala變化可能影響了與之有互作效應(yīng)的因子，而該因子可能是M基因座[1]，這為開展豁眼性狀另一個常染色體上基因座研究指明了方向。

4 結(jié)論

基因FREM1是決定鵝上眼瞼性狀的H基因座，該基因編碼區(qū)1個純合型錯義突變導(dǎo)致了FREM1蛋白第10CSPG結(jié)構(gòu)域的變化，基因FREM1的c.4514T＞C突變可作為鵝豁眼性狀重要的分子標(biāo)記。

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（責(zé)任編輯 林鑒非）

Validation of Candidate Gene FREM1 of H Locus of Huoyan Trait in Goose

YU JinCheng, YU Ning, ZHAO Hui, LI Zhe
(Pratacultural & Animal Science Research Institute, Liaoning Academy of Agricultural Sciences, Shenyang 110161)

FREM1 gene; trait of upper eyelids colomobas; cloning of cDNA; Huoyan Goose; gene expression

2016-11-15；接受日期：2017-03-09

遼寧省博士啟動基金資助項目（20141164）、國家水禽產(chǎn)業(yè)技術(shù)體系專項資金（CARS-43-23）、遼寧省科技型中小企業(yè)技術(shù)創(chuàng)新基金（2013-1）

聯(lián)系方式：于金成，Tel：024-31029891；13898156386；E-mail：yujincheng_pi@126.com。通信作者趙輝，E-mail：zhaohui_sy@126.com。通信作者李喆，Tel：024-31029891；E-mail：acj423@163.com。

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), F1population 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 F1population 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 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 F1generation 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.