紹興鴨生長激素基因多態(tài)性與生長性能關(guān)聯(lián)性分析

李麗1,2, 徐琪1, 陳陽1, 黃學(xué)濤3, 李柳萌4, 陶仲連5, 陳國宏1*

( 1.揚(yáng)州大學(xué)動物科學(xué)與技術(shù)學(xué)院,江蘇 揚(yáng)州225009;2.福建省農(nóng)業(yè)科學(xué)院畜牧獸醫(yī)研究所,福州350013;

3.浙江省農(nóng)業(yè)科學(xué)院畜牧獸醫(yī)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(yī)局,浙江 縉云321400)

摘要以紹興鴨為材料,選擇與生長發(fā)育密切相關(guān)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(tài)性(single nucleotide polymorphism,SNP)位點,并分析其與生長性能的關(guān)聯(lián)性.結(jié)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(tài)。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(zhì)量、8周齡體質(zhì)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(zhì)量、腹脂質(zhì)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(jiān)測紹興鴨公鴨的生長發(fā)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(guān)鍵詞紹興鴨; 生長激素基因; 單核苷酸多態(tài)性; 生長性能

中圖分類號S 834.89文獻(xiàn)標(biāo)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(chǎn)蛋多、體質(zhì)量小、飼料報酬高、抗病力強(qiáng)等特點,是世界優(yōu)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(jìn)行深入研究,進(jìn)一步了解鴨體內(nèi)脂肪分布和生長發(fā)育的差異,為合理保護(hù)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(diào)節(jié)素。GH基因?qū)游矬w的影響主要表現(xiàn)為顯著提高生長速度,促進(jìn)肌肉生長,降低脂肪含量,進(jìn)而影響機(jī)體生長發(fā)育速度。GH基因由5或6個外顯子和4或5個內(nèi)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(fā)現(xiàn),GH基因存在豐富的多態(tài)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(shù),對GH基因進(jìn)行單核苷酸多態(tài)性(single nucleotide polymorphism,SNP)檢測,對不同外顯子、內(nèi)含子的遺傳變異進(jìn)行分析,并對其多態(tài)性與生長性能進(jìn)行關(guān)聯(lián)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(biāo)記,旨在從分子水平上分析紹興鴨GH基因單核苷酸多態(tài)性及其與生長性能的遺傳調(diào)控規(guī)律。

1材料與方法

1.1試驗材料及各生長性能指標(biāo)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(yǎng)方法相同,記錄其生長指標(biāo),12周齡時參照《畜禽遺傳資源調(diào)查手冊》[9]規(guī)定的方法測定試驗鴨的體質(zhì)量和體尺指標(biāo),53周齡時測定屠宰指標(biāo)。所有數(shù)據(jù)資料均按群組隨機(jī)取樣,同一指標(biāo)現(xiàn)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(jìn)行提取,0.6%瓊脂糖檢測,-20 ℃保存?zhèn)溆谩?/p>

1.2引物設(shè)計和聚合酶鏈反應(yīng)擴(kuò)增

根據(jù)GenBank公布的GH基因(登錄號:AB158760.2)序列設(shè)計引物,擴(kuò)增其內(nèi)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(shù)服務(wù)有限公司合成,用滅菌超純水溶解。

聚合酶鏈反應(yīng)(polymerase chain reaction,PCR)反應(yīng)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(bǔ)足體積至20.0 μL。PCR反應(yīng)條件:95 ℃ 2 min;隨后30個循環(huán),94 ℃ 30 s,56 ℃ 30 s,65 ℃ 1 min;最后65 ℃延伸10 min,10 ℃終止反應(yīng)。PCR產(chǎn)物經(jīng)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(shù)據(jù)統(tǒng)計分析

計算基因型和等位基因頻率,并進(jìn)行Hardy-Weinberg平衡性檢驗;運用DNAStar對序列進(jìn)行比對,尋找其多態(tài)性位點;采用如下固定模型進(jìn)行統(tǒng)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(yīng);E為隨機(jī)誤差。運用統(tǒng)計軟件JMP 4.0(SAS Institute,2002)分別計算基因型與性狀的相關(guān)性,估計性狀的最小二乘均值。

2結(jié)果與分析

2.1GH基因多態(tài)位點篩查

(1～4):PCR產(chǎn)物;M:DL1500標(biāo)志物。 (1-4): PCR product; M: DL1500 marker. 圖1　GH基因內(nèi)含子2凝膠電泳檢測結(jié)果 Fig.1　Gel electrophoresis results of GH gene intron 2

經(jīng)DNA提取后,分別對GH基因內(nèi)含子2、外顯子4、外顯子5進(jìn)行PCR擴(kuò)增,結(jié)果見圖1～3。各片段的擴(kuò)增產(chǎn)物大小與預(yù)期的產(chǎn)物大小一致,且擁有較好的特異性。將PCR產(chǎn)物直接測序結(jié)果與GenBank中GH基因序列比對后發(fā)現(xiàn),僅在GH基因內(nèi)含子2發(fā)現(xiàn)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(chǎn)物;M:DL600標(biāo)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(jié)果 Fig.2　Gel electrophoresis results of GH gene exon 4

(1～4):PCR產(chǎn)物;M:DL600標(biāo)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(jié)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(nèi)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(jìn)行統(tǒng)計,發(fā)現(xiàn)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(nèi)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(yōu)勢基因型,等位基因G為優(yōu)勢等位基因,χ2檢驗結(jié)果表明,該基因座處于Hardy-Weinberg平衡狀態(tài);2 691 C>T位點TT純合子為群體中的優(yōu)勢基因型,等位基因T為優(yōu)勢等位基因,χ2檢驗結(jié)果表明,該基因座處于Hardy-Weinberg平衡狀態(tài)。

2.3GH基因內(nèi)含子2多態(tài)性及其與生長發(fā)育性能和屠體性能的關(guān)聯(lián)性分析

連鎖不平衡分析結(jié)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(tài),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(jìn)行了關(guān)聯(lián)性分析。采用最小二乘均值法對GH基因多態(tài)性與其生長發(fā)育性能和屠體性能進(jìn)行關(guān)聯(lián)性分析的結(jié)果見表3和表4。

表2　紹興鴨 GH基因內(nèi)含子2的基因型頻率、等位基因頻率

表3　 GH基因內(nèi)含子多態(tài)位點(2 691 C>T)對鴨生長發(fā)育性能的影響

續(xù)表3　 GH基因內(nèi)含子多態(tài)位點(2 691 C>T)對鴨生長發(fā)育性能的影響

同行數(shù)據(jù)后的不同小寫字母表示在P<0.05水平差異有統(tǒng)計學(xué)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(nèi)含子多態(tài)位點(2 691 C>T)對鴨屠宰性能的影響

同行數(shù)據(jù)后的不同小寫字母表示在P<0.05水平差異有統(tǒng)計學(xué)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(yīng)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(zhì)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(zhì)量、8周齡體質(zhì)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(zhì)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(zhì)量、腹脂質(zhì)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(jìn)行綜合分析,發(fā)現(xiàn)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(yōu)勢標(biāo)記基因型。

3討論

關(guān)于GH基因多態(tài)性與生長性能的關(guān)系已有相關(guān)研究[10-13],對山羊、秦川牛、豁眼鵝、籽鵝進(jìn)行多態(tài)性檢測,均發(fā)現(xiàn)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(nèi)含子2的遺傳多樣性進(jìn)行分析發(fā)現(xiàn),不同基因型個體的生長性能如6周齡體質(zhì)量、胸寬、龍骨長以及胸肌質(zhì)量、腹脂質(zhì)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(jìn)行分析發(fā)現(xiàn)其內(nèi)含子2存在多態(tài)性。顏炳學(xué)等[10]在雞GH基因中發(fā)現(xiàn)了2個多態(tài)位點,研究結(jié)果表明這些多態(tài)位點與腹脂和胸肉等屠體性狀顯著相關(guān),與本研究分析結(jié)果一致,由此推測GH基因也許是直接控制腹脂和胸肌相關(guān)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(jié)果需在其后代或家鴨品種中驗證,同時還應(yīng)篩查紹興鴨GH基因編碼區(qū)和其他內(nèi)含子區(qū)的多態(tài)性是否對生長性能及抗病性也存在顯著效應(yīng),最終獲得真實可靠的分子遺傳標(biāo)記,以期用于紹興鴨公鴨群體生長性能標(biāo)記輔助選擇的育種實踐。

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