紹興鴨生長激素基因多態性與生長性能關聯性分析

紹興鴨生長激素基因多態性與生長性能關聯性分析

李麗1,2, 徐琪1, 陳陽1, 黃學濤3, 李柳萌4, 陶仲連5, 陳國宏1*

( 1.揚州大學動物科學與技術學院,江蘇 揚州225009;2.福建省農業科學院畜牧獸醫研究所,福州350013;

3.浙江省農業科學院畜牧獸醫研究所,杭州310021;4.諸暨國家級紹興鴨保種場,浙江 紹興311800;

5.縉云縣畜牧獸醫局,浙江 縉云321400)

摘要以紹興鴨為材料,選擇與生長發育密切相關的候選生長激素(growth hormone,GH)基因,通過直接測序方法掃描其單核苷酸多態性(single nucleotide polymorphism,SNP)位點,并分析其與生長性能的關聯性.結果表明:在GH基因的2 514 A>G和2 691 C>T處檢測到2個SNP位點,且2個基因座均處于Hardy-Weinberg平衡狀態。最小二乘法分析表明,GH基因(2 691 C>T)TT基因型個體的6周齡體質量、8周齡體質量、12周齡龍骨長、53周齡胸寬顯著高于CT基因型個體(P<0.05);TT基因型53周齡胸肌質量、腹脂質量、腹脂率顯著高于CT和CC基因型個體(P<0.05)。研究為在本品種選育中監測紹興鴨公鴨的生長發育和充分利用淘汰種公鴨肉用價值提供了一定的參考材料,同時從分子水平上豐富了紹興鴨遺傳信息。

關鍵詞紹興鴨; 生長激素基因; 單核苷酸多態性; 生長性能

中圖分類號S 834.89文獻標志碼A

Correlation between the polymorphism of growth hormone gene with the growth performance in Shaoxing ducks. Journal of ZhejiangUniversity(Agric. & LifeSci.), 2015,41(3):365-370

Li Li1,2, Xu Qi1, Chen Yang1, Huang Xuetao3, Li Liumeng4, Tao Zhonglian5, Chen Guohong1*(1.CollegeofAnimalScienceandTechnology,YangzhouUniversity,Yangzhou225009,Jiangsu,China; 2.InstituteofAnimalHusbandryandVeterinaryResearch,FujianAcademyofAgriculturalSciences,Fuzhou350013,China; 3.InstituteofAnimalHusbandryandVeterinaryResearch,ZhejiangAcademyofAgriculturalSciences,Hangzhou310021,China; 4.ShaoxingDuckNationalConservationFieldofZhuJi,Shaoxing311800,Zhejiang,China; 5.JinyunBureauofAnimalScienceandVeterinaryMedicine,Jinyun321400,Zhejiang,China)

SummaryAs excellent germplasm resources in China, Shaoxing duck is famous for its high egg production all over the world. Laying traits, rather than the growth and development traits, are the main breeding criteria in the duck breeding at present. We usually use egg laying traits as the main breeding goal, but ignore the growth and development traits, especially in drake breeding. Defective growth and development may lead to the loss of duck breeding value and the low fertilization rate, which will finally reduce the economic benefit of drakes. Thus, drakes’ growth traits must be emphasized in breeding of laying ducks. We must pay more attention to the selection of the drakes’ growth traits.

This study analyzed the correlation between the polymorphism of growth hormone (GH) gene with the growth performance and slaughter in Shaoxing ducks, which provided some reference material for monitoring the growth and development of Shaoxing ducks breeding.

As the candidate genes,GHgene is closely related to the growth and development. The SNP loci ofGHwere scanned by direct sequencing method. The body size was measured at the age of 12 weeks, and the slaughter indexes were measured at the age of 53 weeks. All ducks were sampled randomly according to the group, and the relevance of Shaoxing duck growth performance and slaughter performance were analyzed. The genomic DNA was extracted by phenol chloroform method from blood. All data were sampled randomly according to the group. According to the publishedGHgene sequence in GenBank, the primers were designed to amplify the sequences of intron 2, exon 4 and exon 5. The SNP loci ofGHwere scanned by direct sequencing method, and the sequences were compared by using DNAStar to look for the polymorphic loci. The genotype and allele frequencies were calculated and tested by Hardy-Weinberg balance. The correlation between the genotypes and traits was calculated by using statistical software JMP 4.0 (SAS Institute, 2002) to estimate the least square means of traits.

The results showed two loci:GH2 514 A>G,GH2 691 C>T. Theχ2 test revealed that all loci were in the balance of Hardy-Weinberg (P>0.05 ). ForGH(2 514 A>G & 2 691 C>T), 6-week body mass, 8-week body mass, 12-week keel bone length, 53-week chest width of the individuals with genotype TT were higher than the genotype CT. The breast muscle mass, abdominal fat mass and abdominal fat percentage of individuals with genotype TT were significantly higher than those with genotype CT and CC (P<0.05).

It is suggested thatGHgene may be the major gene which directly control the abdominal fat and breast muscle related traits, or may be tightly linked with the major genes indirectly.

Key wordsShaoxing duck; growth hormone gene; single nucleotide polymorphism; growth trait

紹興鴨具有產蛋多、體質量小、飼料報酬高、抗病力強等特點,是世界優秀水禽品種之一[1]。但長期以來,因紹興鴨體型較小,對其肉用性能的研究較少,所以對紹興鴨公鴨的遺傳特征和生長性能進行深入研究,進一步了解鴨體內脂肪分布和生長發育的差異,為合理保護和利用我國地方鴨品種遺傳資源,具有重要的理論和實踐意義。

生長激素(growth hormone,GH)是一種具有廣泛生理功能的生長調節素。GH基因對動物體的影響主要表現為顯著提高生長速度,促進肌肉生長,降低脂肪含量,進而影響機體生長發育速度。GH基因由5或6個外顯子和4或5個內含子組成,cDNA長度一般為800～1 200 bp,信號肽和成熟肽分別為16～27個和186～191個氨基酸[2-4]。在對畜禽的研究中發現,GH基因存在豐富的多態性[5-8]。與哺乳動物相比,禽類GH基因的研究起步較晚。本試驗以紹興鴨公鴨為材料,運用DNA直接測序技術,對GH基因進行單核苷酸多態性(single nucleotide polymorphism,SNP)檢測,對不同外顯子、內含子的遺傳變異進行分析,并對其多態性與生長性能進行關聯分析,以期找到遺傳變異位點,尋找紹興鴨生長性能的主效基因和分子遺傳標記,旨在從分子水平上分析紹興鴨GH基因單核苷酸多態性及其與生長性能的遺傳調控規律。

1材料與方法

1.1試驗材料及各生長性能指標測定

紹興鴨195只,均為公鴨,來自浙江省諸暨市國家級紹興鴨保種場同一鴨舍、同一批次,飼料與飼養方法相同,記錄其生長指標,12周齡時參照《畜禽遺傳資源調查手冊》[9]規定的方法測定試驗鴨的體質量和體尺指標,53周齡時測定屠宰指標。所有數據資料均按群組隨機取樣,同一指標現場專人測定。采用酚-三氯甲烷法對全部個體血液基因組DNA進行提取,0.6%瓊脂糖檢測,-20 ℃保存備用。

1.2引物設計和聚合酶鏈反應擴增

根據GenBank公布的GH基因(登錄號:AB158760.2)序列設計引物,擴增其內含子2、外顯子4、外顯子5序列(表1)。引物由上海翼和生物工程技術服務有限公司合成,用滅菌超純水溶解。

聚合酶鏈反應(polymerase chain reaction,PCR)反應體系:緩沖液(10×,含20 mol/L Mg2+)2.0 μL,10 μmol/L脫氧核苷酸(dNTPs)2.0 μL,10 μmol/L上、下游引物各2.0 μL,模板DNA 50 ng,1 UTaqDNA聚合酶0.2 μL,加超純水補足體積至20.0 μL。PCR反應條件:95 ℃ 2 min;隨后30個循環,94 ℃ 30 s,56 ℃ 30 s,65 ℃ 1 min;最后65 ℃延伸10 min,10 ℃終止反應。PCR產物經1%瓊脂糖凝膠電泳回收。

表1　紹興鴨 GH基因引物信息

1.3數據統計分析

計算基因型和等位基因頻率,并進行Hardy-Weinberg平衡性檢驗;運用DNAStar對序列進行比對,尋找其多態性位點;采用如下固定模型進行統計分析:Y=μ+G+E。其中：Y為個體的表型記錄;μ為群體均值;G為該位點的基因型效應;E為隨機誤差。運用統計軟件JMP 4.0(SAS Institute,2002)分別計算基因型與性狀的相關性,估計性狀的最小二乘均值。

2結果與分析

2.1GH基因多態位點篩查

(1～4):PCR產物;M:DL1500標志物。 (1-4): PCR product; M: DL1500 marker. 圖1　GH基因內含子2凝膠電泳檢測結果 Fig.1　Gel electrophoresis results of GH gene intron 2

經DNA提取后,分別對GH基因內含子2、外顯子4、外顯子5進行PCR擴增,結果見圖1～3。各片段的擴增產物大小與預期的產物大小一致,且擁有較好的特異性。將PCR產物直接測序結果與GenBank中GH基因序列比對后發現,僅在GH基因內含子2發現2處突變:2 514 bp處A>G突變和2 691 bp處C>T突變(圖4、圖5)。

(1～4):PCR產物;M:DL600標志物。 (1-4): PCR product; M: DL600 marker. 圖2　GH基因外顯子4凝膠電泳檢測結果 Fig.2　Gel electrophoresis results of GH gene exon 4

(1～4):PCR產物;M:DL600標志物。 (1-4): PCR product; M: DL600 marker. 圖3　GH基因外顯子5凝膠電泳檢測結果 Fig.3　Gel electrophoresis results of GH gene exon 5

圖4　GH基因2 514 bp處突變位點測序峰圖 Fig.4　Partial chromatogram of GH gene mutation at 2 514 bp

圖5　GH基因2 691 bp處突變位點測序峰圖 Fig.5　Partial chromatogram of GH gene mutation at 2 691 bp

2.2GH基因內含子2的群體遺傳特性

對紹興鴨群體中GH突變位點基因型分布進行統計,發現存在TT基因型、CT基因型、CC基因型以及GG基因型、GA基因型和AA基因型個體。紹興鴨GH基因內含子2的基因型頻率和等位基因頻率見表2。2 514 A>G位點GG純合子為群體中的優勢基因型,等位基因G為優勢等位基因,χ2檢驗結果表明,該基因座處于Hardy-Weinberg平衡狀態;2 691 C>T位點TT純合子為群體中的優勢基因型,等位基因T為優勢等位基因,χ2檢驗結果表明,該基因座處于Hardy-Weinberg平衡狀態。

2.3GH基因內含子2多態性及其與生長發育性能和屠體性能的關聯性分析

連鎖不平衡分析結果表明,2 514 A>G和2 691 C>T位點之間的R2=1,處于完美連鎖不平衡狀態,一個位點的信息完全可以代表另外一個位點。因此,本研究只對2 691 C>T位點進行了關聯性分析。采用最小二乘均值法對GH基因多態性與其生長發育性能和屠體性能進行關聯性分析的結果見表3和表4。

表2　紹興鴨 GH基因內含子2的基因型頻率、等位基因頻率

表3　 GH基因內含子多態位點(2 691 C>T)對鴨生長發育性能的影響

續表3　 GH基因內含子多態位點(2 691 C>T)對鴨生長發育性能的影響

同行數據后的不同小寫字母表示在P<0.05水平差異有統計學意義.

Values within a row followed by different lowercase letters are significantly different at the 0.05 probability level.

表4　 GH基因內含子多態位點(2 691 C>T)對鴨屠宰性能的影響

同行數據后的不同小寫字母表示在P<0.05水平差異有統計學意義.

Values within a row followed by different lowercase letters are significantly different at the 0.05 probability level.

GH的基因型效應對生長和屠宰性能均有不同程度的影響。從生長性能(表3)可知,基因型對6周齡體質量、12周齡龍骨長和53周齡胸寬有顯著影響(P<0.05)；最小二乘均值分析表明,TT基因型個體6周齡體質量、8周齡體質量、12周齡龍骨長、53周齡胸寬顯著高于CT基因型個體(P<0.05)。從屠宰性能(表4)可知,基因型對53周齡腹脂質量以及53周齡腹脂率有顯著影響(P<0.05)；最小二乘均值分析表明,TT基因型個體胸肌質量、腹脂質量、腹脂率顯著高于CC和CT基因型個體(P<0.05)。本研究對2個性能進行綜合分析,發現GH基因TT型在2個性狀方面顯著大于CC和CT基因型,可視其為紹興鴨公鴨生長和屠宰性能的優勢標記基因型。

3討論

關于GH基因多態性與生長性能的關系已有相關研究[10-13],對山羊、秦川牛、豁眼鵝、籽鵝進行多態性檢測,均發現基因型對生長性能有極顯著(P<0.01)或顯著(P<0.05)影響。采用DNA測序法對GH基因內含子2的遺傳多樣性進行分析發現,不同基因型個體的生長性能如6周齡體質量、胸寬、龍骨長以及胸肌質量、腹脂質量和腹脂率等性狀間存在顯著差異。說明GH可能對紹興鴨公鴨胸肌附著的龍骨長及胸寬均有顯著影響,并且對腹脂及腹脂率有一定影響。本研究對GH基因的遺傳多樣性進行分析發現其內含子2存在多態性。顏炳學等[10]在雞GH基因中發現了2個多態位點,研究結果表明這些多態位點與腹脂和胸肉等屠體性狀顯著相關,與本研究分析結果一致,由此推測GH基因也許是直接控制腹脂和胸肌相關性狀的主效基因,又或者是間接與主效基因緊密連鎖的基因。

本研究結果需在其后代或家鴨品種中驗證,同時還應篩查紹興鴨GH基因編碼區和其他內含子區的多態性是否對生長性能及抗病性也存在顯著效應,最終獲得真實可靠的分子遺傳標記,以期用于紹興鴨公鴨群體生長性能標記輔助選擇的育種實踐。

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