油脂類型對(duì)肉雞不同組織甘油醛-3-磷酸脫氫酶基因相對(duì)表達(dá)量的影響

陶亞飛 陳 青 徐媛媛 石秀文 陳 文 黃艷群(河南農(nóng)業(yè)大學(xué)飼料營(yíng)養(yǎng)河南省工程實(shí)驗(yàn)室，河南農(nóng)業(yè)大學(xué)國(guó)家家養(yǎng)動(dòng)物種質(zhì)資源平臺(tái)，鄭州450002)

油脂類型對(duì)肉雞不同組織甘油醛-3-磷酸脫氫酶基因相對(duì)表達(dá)量的影響

陶亞飛 陳 青 徐媛媛 石秀文 陳 文 黃艷群*
(河南農(nóng)業(yè)大學(xué)飼料營(yíng)養(yǎng)河南省工程實(shí)驗(yàn)室，河南農(nóng)業(yè)大學(xué)國(guó)家家養(yǎng)動(dòng)物種質(zhì)資源平臺(tái)，鄭州450002)

本試驗(yàn)旨在探究油脂類型對(duì)肉雞不同組織甘油醛-3-磷酸脫氫酶(GAPDH)基因相對(duì)表達(dá)量的影響。試驗(yàn)選用240只1日齡的科寶肉雞母雛，隨機(jī)分為8個(gè)組(4個(gè)單一油脂組，分別在飼糧中添加5.00%亞麻油、玉米油、芝麻油和豬油；4個(gè)混合油脂組，分別在飼糧中添加2.50%豬油+2.50%玉米油、2.50%豬油+2.50%芝麻油、2.50%亞麻油+2.50%玉米油和2.50%亞麻油+2.50%芝麻油)，每組6個(gè)重復(fù)，每個(gè)重復(fù)5只雞。試驗(yàn)期42 d。結(jié)果表明：1)組織及油脂類型與組織的交互作用對(duì)42日齡肉雞組織GAPDH基因的相對(duì)表達(dá)量影響顯著(P<0.05)，油脂類型對(duì)42日齡肉雞組織GAPDH基因的相對(duì)表達(dá)量的影響不顯著(P>0.05)。42日齡肉雞胸肌GAPDH基因的相對(duì)表達(dá)量顯著高于肝臟和腹脂(P<0.05)，是肝臟的37.50～89.50倍，是腹脂的129.54～190.64倍，而42日齡肉雞的GAPDH基因的相對(duì)表達(dá)量在肝臟與腹脂之間差異不顯著(P>0.05)；玉米油組胸肌中GAPDH基因的相對(duì)表達(dá)量顯著高于豬油組(P<0.05)。2)21日齡肉雞肝臟GAPDH基因的相對(duì)表達(dá)量顯著或極顯著高于42日齡(P<0.05或P<0.01)。3)油脂組合及油脂組合與日齡的交互作用對(duì)肉雞肝臟GAPDH基因相對(duì)表達(dá)量的影響均不顯著(P>0.05)，但日齡對(duì)肉雞肝臟GAPDH基因相對(duì)表達(dá)量的影響顯著(P<0.05)。由此可見(jiàn)，油脂類型對(duì)肉雞GAPDH基因的相對(duì)表達(dá)量的影響呈現(xiàn)組織間的差異，玉米油可提高胸肌GAPDH基因的相對(duì)表達(dá)量。42日齡肉雞胸肌GAPDH基因的相對(duì)表達(dá)量顯著高于肝臟和腹脂，21日齡肉雞肝臟GAPDH基因的相對(duì)表達(dá)量顯著高于42日齡。

肉雞；油脂；GAPDH；基因表達(dá)

甘油醛-3-磷酸脫氫酶(glyceraldehyde-3-phosphate dehydrogenase，GAPDH)是動(dòng)物能量代謝途徑的關(guān)鍵代謝酶，由4個(gè)分子質(zhì)量為30～40 ku的亞基組成，分子質(zhì)量為146 ku[1]。最初對(duì)GAPDH的研究主要集中在其在糖酵解過(guò)程中的作用，GAPDH作為糖酵解過(guò)程中的關(guān)鍵酶之一[2-4]，在細(xì)胞膜、細(xì)胞質(zhì)、細(xì)胞核上均有分布。隨著對(duì)GAPDH研究的深入，發(fā)現(xiàn)該酶還具有一些與糖酵解功能無(wú)關(guān)的生物活性，例如，在生物膜上，GAPDH參與膜運(yùn)動(dòng)、促進(jìn)膜融合等[5]；在細(xì)胞質(zhì)中，GAPDH有保護(hù)細(xì)胞、催化微管聚合等功能[6]；在細(xì)胞核內(nèi)，GAPDH參與tRNA出核、DNA修復(fù)[7]；在一些神經(jīng)性疾病上，GAPDH參與細(xì)胞凋亡以及與年齡相關(guān)的神經(jīng)性疾病[8]；研究表明，GAPDH還具有磷酸轉(zhuǎn)移酶/激酶的活性[9]。GAPDH基因具有高度保守的種屬序列并且廣泛存在于眾多生物體中，幾乎在所有組織中都高水平表達(dá)。在分子生物學(xué)研究方法上，GAPDH基因常被作為管家基因，廣泛用作RNA水平和蛋白質(zhì)水平下基因表達(dá)研究的標(biāo)準(zhǔn)化內(nèi)參。

玉米油和芝麻油含有較高的亞油酸和油酸，是n-6脂肪酸的代表[10-11]；亞麻籽油的n-6/n-3多不飽和脂肪酸(PUFA)的比值(約為0.34∶1.00)遠(yuǎn)遠(yuǎn)低于其他油脂，是n-3脂肪酸的代表[12-13]；豬油是富含單不飽和脂肪酸的動(dòng)物油，其油酸含量高達(dá)48.70%。已有研究表明，在肉雞飼糧中適量添加油脂可以滿足肉雞快速生長(zhǎng)對(duì)能量的需要，改善飼糧口感和提高飼糧利用率[14]。油脂類型會(huì)影響動(dòng)物脂類代謝及脂肪酸合成酶(FAS)mRNA的表達(dá)[15]。但尚未見(jiàn)油脂類型對(duì)肉雞GAPDH基因表達(dá)效應(yīng)的系統(tǒng)報(bào)道。本文主要研究單一油脂及其組合對(duì)不同日齡肉雞在不同組織中GAPDH基因表達(dá)量的影響。

1 材料與方法

1.1 試驗(yàn)動(dòng)物及試驗(yàn)設(shè)計(jì)

本試驗(yàn)選用240只1日齡科寶肉雞母雛，隨機(jī)分為8組，每組6個(gè)重復(fù)，每個(gè)重復(fù)5只雞。試驗(yàn)雞采用籠養(yǎng)，每籠5只雞，自由采食和飲水，免疫程序按常規(guī)進(jìn)行。飼糧參照NRC(1994)肉雞的營(yíng)養(yǎng)需要配制，分為1～3周齡和4～6周齡2個(gè)階段。試驗(yàn)期42 d。4個(gè)單一油脂組分別在飼糧中添加5%的亞麻油、玉米油、芝麻油和豬油，其飼糧組成及營(yíng)養(yǎng)水平見(jiàn)表1；4個(gè)混合油脂組包含2個(gè)豬油混合組(飼糧中添加2.5%豬油+2.5%玉米油、2.5%豬油+2.5%芝麻油)和2個(gè)植物油混合組(飼糧中添加2.5%亞麻油+2.5%玉米油、2.5%亞麻油+2.5%芝麻油)，其飼糧組成及營(yíng)養(yǎng)水平見(jiàn)表2。

表1 單一油脂組飼糧組成及營(yíng)養(yǎng)水平(風(fēng)干基礎(chǔ))

續(xù)表1項(xiàng)目Items1～3周齡1to3weeksofage植物油組Vegetableoilgroup豬油組Lardoilgroup4～6周齡4to6weeksofage植物油組Vegetableoilgroup豬油組Lardoilgroup賴氨酸Lys1．151．561．021．05蛋氨酸Met0．500．500．400．41

1)植物油組飼糧的油脂分別為亞麻油、玉米油和芝麻油。Oils in diets of vegetable oil groups were linseed oil, corn oil and sesame oil, respectively.

2)預(yù)混料為每千克飼糧提供 Premix provide the following per kg of diets: VA 0.45 mg，VD30.005 mg，VE 10.00 mg，VK 0.50 mg，VB11.80 mg，VB23.60 mg，VB63.50 mg，VB120.002 5 mg，煙酸 niacin 35.00 mg，葉酸 folic acid 0.55 mg，生物素 biotin 0.20 mg，泛酸 pantothenic 10.00 mg，Zn (ZnSO4·7H2O) 40.00 mg，Mn (MnSO4·5H2O) 60.00 mg，F(xiàn)e (FeSO4·7H2O) 80.00 mg，Cu (CuSO4·5H2O) 10.00 mg，I (KI) 0.35 mg，Se (Na2SeO3·5H2O) 0.15 mg。表2同The same as Table 2。

3)營(yíng)養(yǎng)水平根據(jù)NRC(1994)肉雞的營(yíng)養(yǎng)需要計(jì)算得來(lái)。表2同。Nutrient levels were calculated in accordance with broilers nutrition needs of NRC (1994). The same as Table 2.

表2 混合油脂組飼糧組成及營(yíng)養(yǎng)水平(風(fēng)干基礎(chǔ))

植物油混合組飼糧的油脂分別為2.50%玉米油+2.50%亞麻油、2.50%亞麻油+2.50%芝麻油；豬油混合組飼糧的油脂分別為2.50%芝麻油+2.50%豬油、2.50%玉米油+2.50%豬油。

Oils in diets of mixed vegetable oil groups were 2.50% corn oil and 2.50% linseed oil, 2.50% linseed oil and 2.50% sesame oil, respectively; oils in diets of mixed lard oil groups were 2.50% sesame oil and 2.50% lard oil, 2.50% corn oil and 2.50% lard oil, respectively.

1.2 樣品采集

分別于21、42日齡時(shí)，從每組各重復(fù)隨機(jī)挑選1只肉雞屠宰，在無(wú)菌條件下取肝臟、胸肌和腹脂。所取樣品均用焦炭酸二乙酯(DEPC)水清洗后，放入液氮速凍，-80 ℃保存。

1.3 總RNA提取和反轉(zhuǎn)錄

1.4 引物設(shè)計(jì)

根據(jù)紅色原雞的GAPDH基因序列(GenBank登錄號(hào)：NM_204305.1)，用Oligo 6.0軟件設(shè)計(jì)熒光定量PCR所用的檢測(cè)引物，并由上海生物工程技術(shù)服務(wù)有限公司合成。PCR擴(kuò)增片段長(zhǎng)度為98 bp。熒光定量PCR引物見(jiàn)表3。

表3 熒光定量PCR引物

F:上游引物 forward primer；R:下游引物 reverse primer；P:探針 probe。

1.5 熒光定量PCR反應(yīng)條件

參照張?chǎng)┑萚16]文獻(xiàn)，運(yùn)用探針?lè)晒舛縋CR進(jìn)行基因的定量表達(dá)分析：反應(yīng)體系為25.0 μL，包括1.0 μL cDNA(0.1 μL總RNA)、0.5 μL上游引物(12.5 μmol/L)、0.5 μL下游引物(12.5 μmol/L)、2.5 μL TaqManTM探針、0.5 μL RoxⅡ、3.5 μL鎂離子(Mg2+，25 mmol/L)、12.5 μL 2×Mix(包含1.5 mmol/L Mg2+)、4.0 μL超純水；熒光定量PCR反應(yīng)條件為95 ℃預(yù)變性2 min；40個(gè)循環(huán)的95 ℃變性15 s；60 ℃退火1 min。每個(gè)樣品設(shè)置3個(gè)重復(fù)。純化的PCR產(chǎn)物采用微量分光光度計(jì)(NanoDrop 2000 UV spectrophotometer)測(cè)定濃度后，進(jìn)行梯度稀釋(10-4、10-5、10-6、10-7、10-8、10-9和10-10)，用于建立每板的標(biāo)準(zhǔn)曲線。每板均設(shè)陰性對(duì)照。參照Bustin[17]報(bào)道方法，本研究采用微量定量?jī)x測(cè)定RNA的濃度，反應(yīng)中添加等量的RNA和cDNA進(jìn)行每個(gè)樣品的均一化處理。

1.6 數(shù)據(jù)統(tǒng)計(jì)和分析

首先，把每個(gè)樣本的GAPDH拷貝數(shù)轉(zhuǎn)換成相對(duì)表達(dá)量。GAPDH基因的相對(duì)表達(dá)量=樣本拷貝數(shù)/42日齡平均拷貝數(shù)最低組(5.00%芝麻油組在腹脂組織的表達(dá)值)平均值。試驗(yàn)數(shù)據(jù)用SAS 8.1統(tǒng)計(jì)軟件中的ANOVA(GLM)過(guò)程進(jìn)行單變量?jī)梢蛩胤讲罘治?檢測(cè)油脂類型與組織、油脂類型與日齡、油脂組合與日齡對(duì)肉雞GAPDH基因的相對(duì)表達(dá)量的效應(yīng)。以P<0.05為差異顯著，P<0.01為差異極顯著。

2 結(jié)果與分析

2.1 單一油脂對(duì)42日齡肉雞不同組織中GAPDH基因的相對(duì)表達(dá)量的影響

由表4可知，42日齡時(shí)，組織以及油脂類型與組織的交互作用顯著影響了肉雞不同組織中GAPDH基因的相對(duì)表達(dá)量(P<0.05)，油脂類型對(duì)肉雞GAPDH基因的相對(duì)表達(dá)量的影響不顯著(P>0.05)。肉雞不同組織中GAPDH基因的相對(duì)表達(dá)量表現(xiàn)為胸肌>肝臟>腹脂。在不同單一油脂組中，胸肌GAPDH基因的相對(duì)表達(dá)量均顯著高于肝臟和腹脂(P<0.05)，是肝臟的37.50～89.50倍，是腹脂的129.54～190.64倍，而GAPDH基因的相對(duì)表達(dá)量在肝臟和腹脂之間差異不顯著(P>0.05)。在胸肌中，玉米油組GAPDH基因的相對(duì)表達(dá)量顯著高于豬油組(P<0.05)，而在肝臟和腹脂中，不同油脂類型組中GAPDH基因的相對(duì)表達(dá)量均無(wú)顯著差異(P>0.05)。以上結(jié)果表明，肉雞GAPDH基因的相對(duì)表達(dá)量具有明顯的時(shí)空表達(dá)特性，受肉雞不同組織的顯著影響。

表4 單一油脂對(duì)42日齡肉雞不同組織中GAPDH基因的相對(duì)表達(dá)量影響

同行數(shù)據(jù)(不同組織之間)和同列數(shù)據(jù)(不同油脂類型之間)肩標(biāo)不同小寫字母表示差異顯著(P<0.05)，肩標(biāo)相同小寫字母表示差異不顯著(P>0.05)。

Values with different small letter superscripts in the same row (among different tissues) and column (among different oil types) mean significant difference (P<0.05), while with the same small letter superscripts mean no significant difference (P>0.05).

2.2 單一油脂類型對(duì)不同日齡肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量的影響

由表5可知，日齡顯著影響了肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量(P<0.05)，油脂類型以及油脂類型與日齡的交互作用對(duì)肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量的影響不顯著(P>0.05)。亞麻油組、玉米油組、芝麻油組和豬油組21日齡肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量均顯著或極顯著高于42日齡(P<0.05或P<0.01)，是42日齡肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量的2.46～4.15倍。但21和42日齡肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量在不同油脂類型之間均無(wú)顯著差異(P>0.05)。以上結(jié)果表明，肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量受肉雞日齡的顯著影響。

表5 單一油脂對(duì)不同日齡肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量的影響

同行數(shù)據(jù)肩標(biāo)*表示差異顯著(P<0.05)，肩標(biāo)**表示差異極顯著(P<0.01)。同列數(shù)據(jù)肩標(biāo)無(wú)字母表示差異不顯著(P>0.05)。下表同。

In the same row, values with * superscripts mean significant difference (P<0.05), with ** superscripts mean extremely significant difference (P<0.01). In the same column, values with no letter superscripts mean no significant difference (P>0.05). The same as below.

2.3 混合油脂對(duì)不同日齡肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量的影響

由表6和表7可知，油脂組合以及日齡與油脂組合的交互作用對(duì)肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量影響不顯著(P>0.05)，但日齡仍顯著影響了肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量(P<0.05)。與單一油脂一樣，21日齡肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量均顯著或極顯著高于42日齡(P<0.05或P<0.01)，是肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量的2.46～3.57倍。

表6 亞麻油混合油脂對(duì)肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量的影響

表7 豬油混合油脂對(duì)肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量的影響

3 討 論

本研究發(fā)現(xiàn)，肉雞GAPDH基因的相對(duì)表達(dá)量在不同組織間有顯著的差異，胸肌是其優(yōu)勢(shì)表達(dá)部位。在肉雞肝臟中，GAPDH基因的相對(duì)表達(dá)量也隨日齡呈現(xiàn)顯著變化，表明肉雞GAPDH基因具有明顯的時(shí)空表達(dá)特性。Barber等[18]研究表明，人GAPDH基因在組織間的表達(dá)也呈現(xiàn)顯著差異，在骨骼肌中的相對(duì)表達(dá)量最高，而在乳腺中的相對(duì)表達(dá)量最低，兩者相差15倍。

Lowe等[19]研究報(bào)道，老齡(37月齡)大鼠的GAPDH基因和蛋白質(zhì)水平在骨骼肌的快肌中均顯著低于幼齡(9月齡)大鼠，而在慢肌之間則無(wú)顯著差異，顯示快肌的糖酵解能力隨著年齡的增長(zhǎng)而下降。Slagboom等[20]研究發(fā)現(xiàn)，36月齡雌性大鼠脾臟GAPDH基因的相對(duì)表達(dá)量顯著高于24月齡，而在肝臟和腦部GAPDH基因的相對(duì)表達(dá)量沒(méi)有出現(xiàn)伴隨年齡的明顯變化。Mozdziak等[21]研究發(fā)現(xiàn)，7日齡時(shí)雞胸肌中GAPDH基因的相對(duì)表達(dá)量顯著高于其他不足7日齡的小雞。本試驗(yàn)中21日齡肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量顯著或極顯著高于42日齡，顯示肉雞肝臟的糖酵解能力隨著肉雞日齡的增長(zhǎng)而下降。日齡對(duì)動(dòng)物GAPDH基因的相對(duì)表達(dá)量的影響因物種、組織或肌肉類型的不同而異。

Hanke等[22]研究發(fā)現(xiàn)，在野兔骨骼肌細(xì)胞培養(yǎng)過(guò)程中，低濃度的葡萄糖可以直接抑制GAPDH啟動(dòng)子的活性，從而降低其轉(zhuǎn)錄水平和酶活性。Mozdziak等[21]報(bào)道，3日齡肉雞胸肌GAPDH基因的相對(duì)表達(dá)量以飼糧飼喂組顯著高于禁飼組，表明營(yíng)養(yǎng)狀態(tài)可以改變GAPDH基因的轉(zhuǎn)錄水平。而本研究發(fā)現(xiàn)5%的玉米油上調(diào)了胸肌中GAPDH基因的相對(duì)表達(dá)量，而油脂類型和油脂組合對(duì)42日齡肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量無(wú)顯著影響，顯示油脂以機(jī)體組織特異性和種類特異性的方式影響GAPDH的轉(zhuǎn)錄水平。

近幾年來(lái)，越來(lái)越多的研究者發(fā)現(xiàn)GAPDH在作為內(nèi)參基因時(shí)表現(xiàn)出不穩(wěn)定的現(xiàn)象[23-24]，其表達(dá)量在轉(zhuǎn)錄或轉(zhuǎn)錄后受到其他因子調(diào)節(jié)，基因水平和蛋白質(zhì)水平會(huì)隨著不同刺激而變化[20]。因此在分子生物學(xué)研究中應(yīng)謹(jǐn)慎選擇GAPDH基因作為內(nèi)參基因。

4 結(jié) 論

① 油脂類型對(duì)肉雞GAPDH基因的相對(duì)表達(dá)量的影響呈現(xiàn)組織間的差異，玉米油可提高42日齡肉雞胸肌GAPDH基因的相對(duì)表達(dá)量。

② 42日齡肉雞胸肌GAPDH基因的相對(duì)表達(dá)量顯著高于肝臟和腹脂。

③ 21日齡肉雞肝臟中GAPDH基因的相對(duì)表達(dá)量顯著或極顯著高于42日齡。

致謝：

感謝河南農(nóng)業(yè)大學(xué)牧醫(yī)工程學(xué)院王志祥教授對(duì)該文稿所提出的寶貴意見(jiàn)。

[1] YAN B X,ZHAO R,WANG J P,et al.Effect of different dietary oil sources on the growth performance,blood characteristics,fatty acid profiles,and expression of lipogenic genes in the liver of broiler chickens[J].Czech Journal of Animal Science,2015,60(11):487-497.

[2] KUBO T,NAKAJIMA H,NAKATSUJI M,et al.Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death[J].Nitric Oxide,2016,53:13-21.

[3] TANG Z J,YUAN S Q,HU Y M,et al.Over-expression of GAPDH in human colorectal carcinoma as a preferred target of 3-bromopyruvate propyl ester[J].Journal of Bioenergetics and Biomembranes,2012,44(1):117-125.

[4] REIS M,ALVES C N,LAMEIRA J,et al.The catalytic mechanism of glyceraldehyde 3-phosphate dehydrogenase from Trypanosoma cruzi elucidated via the QM/MM approach[J].Physical Chemistry Chemical Physics,2013,15(11):3772-3785.

[5] GLASER P E,HAN X L,GROSS R W.Tubulin is the endogenous inhibitor of the glyceraldehyde 3-phosphate dehydrogenase isoform that catalyzes membrane fusion:implications for the coordinated regulation of glycolysis and membrane fusion[J].Proceedings of the National Academy of Sciences,2002,99(22):14104-14109.

[6] COLELL A,RICCI J E,TAIT S,et al.GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation[J].Cell,2007,129(5):983-997.

[7] SINGH R,GREEN M R.Sequence-specific binding of transfer RNA by glyceraldehyde-3-phosphate dehydrogenase[J].Science,1993,259(5093):365-368.

[8] BAUMGARNER B L,RILEY C P,SEPULVEDA M S,et al.Increased expression of GAPDH protein is not indicative of nitrosative stress or apoptosis in liver of starved rainbow trout (Oncorhynchusmykiss)[J].Fish Physiology and Biochemistry,2012,38(2):319-327.

[9] ENGEL M,SEIFERT M,THEISINGER B,et al.Glyceraldehyde-3-phosphate dehydrogenase and Nm23-H1/nucleoside diphosphate kinase A.Two old enzymes combine for the novel Nm23 protein phosphotransferase function[J].Journal of Biological Chemistry,1998,273(32):20058-20065.

[10] PATHAK N,RAI A K,KUMARI R,et al.Value addition in sesame:a perspective on bioactive components for enhancing utility and profitability[J].Pharmacognosy Reviews,2014,8(16):147-155.

[11] 盧建,王克華,曲亮,等.飼糧中不同水平芝麻油對(duì)蘇禽青殼蛋雞產(chǎn)蛋性能、蛋品質(zhì)、血清脂質(zhì)指標(biāo)和蛋黃膽固醇含量的影響[J].動(dòng)物營(yíng)養(yǎng)學(xué)報(bào),2013,25(10):2474-2480.

[12] LEE J Y,KANG S K,HEO Y J,et al.Influence of flaxseed oil on fecal microbiota,egg quality and fatty acid composition of egg yolks in laying hens[J].Current Microbiology,2016,72(3):259-266.

[13] BOURRE J M.Effect of increasing the ω-3 fatty acid in the diets of animals on the animal products consumed by humans[J].Médecine/Sciences,2005,21(8/9):773-779.

[14] 倪紅玉,魯菲,溫超,等.飼糧不同油脂來(lái)源對(duì)肉雞脂類代謝及相關(guān)基因表達(dá)的影響[J].動(dòng)物營(yíng)養(yǎng)學(xué)報(bào),2011,23(10):1677-1683.

[15] 岳穎,劉國(guó)華,鄭愛(ài)娟,等.生長(zhǎng)動(dòng)物脂肪代謝關(guān)鍵酶基因表達(dá)調(diào)控[J].動(dòng)物營(yíng)養(yǎng)學(xué)報(bào),2012,24(2):232-238.

[16] 張?chǎng)?魯衛(wèi)衛(wèi),王婷,等.油脂類型和水平對(duì)肉雞組織18S rRNA表達(dá)的影響[J].動(dòng)物營(yíng)養(yǎng)學(xué)報(bào),2014,26(12):3814-3820.

[17] BUSTIN S.Quantification of mRNA using real-time reverse transcription PCR (RT-PCR):trends and problems[J].Journal of Molecular Endocrinology,2002,29(1):23-39.

[18] BARBER R D,HARMER D W,COLEMAN R A,et al.GAPDH as a housekeeping gene:analysis of GAPDH mRNA expression in a panel of 72 human tissues[J].Physiological Genomics,2005,21(3):389-395.

[19] LOWE D A,DEGENS H,CHEN K D,et al.Glyceraldehyde-3-phosphate dehydrogenase varies with age in glycolytic muscles of rats[J].The Journals of Gerontology Series A：Biological Sciences and Medical Sciences,2000,55(3):B160-B164.

[20] SLAGBOOM P E,DE LEEUW W J F,VIJG J.Messenger RNA levels and methlation patterns of GAPDH and β-actin genes in rat liver,spleen and brain in relation to aging[J].Mechanisms of Ageing and Development,1990,53(3):243-257.

[21] MOZDZIAK P E,DIBNER J J,MCCOY D W.Glyceraldehyde-3-phosphate dehydrogenase expression varies with age and nutrition status[J].Nutrition,2003,19(5):438-440.

[22] HANKE N,MEISSNER J D,SCHEIBE R J,et al.Metabolic transformation of rabbit skeletal muscle cells in primary culture in response to low glucose[J].Biochimica et Biophysica Acta (BBA)-Molecular Cell Research,2008,1783(5):813-825.

[23] TONG Z G,GAO Z H,WANG F,et al.Selection of reliable reference genes for gene expression studies in peach using real-time PCR[J].BMC Molecular Biology,2009,10:71.

[24] MOE T K,ZILIANG J,BARATHI A,et al.Differential expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH),β actin and hypoxanthine phosphoribosyltransferase (HPRT) in postnatal rabbit sclera[J].Current Eye Research,2001,23(1):44-50.

*Corresponding author, professor, E-mail: hyanqun@aliyun.com

(責(zé)任編輯 武海龍)

Effects of Oil Types on Glyceraldehyde-3-Phosphate Dehydrogenase Gene Relative Expression Level in Different Tissues of Broilers

TAO Yafei CHEN Qing XU Yuanyuan SHI Xiuwen CHEN Wen HUANG Yanqun*
(HenanAgriculturalUniversityNationalGermplasmResouecesPlatformforAnimals,FeedNutritionEngineeringLaboratoryofHenanProvince,HenanAgriculturalUniversity,Zhengzhou450002,China)

This experiment was conducted to study the effects of oil types on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene relative expression level in different tissues of broilers. A total of 240 one-day-old female Cobb broilers were selected and randomly assigned to 8 groups (four single oil groups were fed diets added with 5.00% linseed oil, corn oil, sesame oil and lard oil, respectively; four oil combination groups were fed diets added with 2.50% lard oil+2.50% corn oil, 2.50% lard oil+2.50% sesame oil, 2.50% linseed oil+2.50% corn oil and 2.50% linseed oil+2.50% sesame oil, respectively) with 6 replicates per group and 5 broilers per replicate. The experiment lasted for 42 days. The results showed as follows: 1) the relative expression level ofGAPDHgene in broiler tissues was significantly affected by tissues and the interaction between oil types and tissues (P<0.05), however, the effects of oil types were not significant (P>0.05). The relative expression level in pectorals ofGAPDHgene of 42-day-old broilers was significantly higher than that in liver and abdominal fat (P<0.05), the relative expression level ofGAPDHgene in liver was 37.50 to 89.50 and 129.54 to 190.64 times than that in liver and abdominal fat, respectively, however, the relative expression level ofGAPDHgene between liver and abdominal fat was not significant (P>0.05). The relative expression level ofGAPDHgene in corn oil group was significantly higher than that in sesame oil group in pectorals (P<0.05). 2) The relative expression level ofGAPDHgene in liver of 21-day-old broilers was significantly higher than that of 42-day-old broilers (P<0.05 orP<0.01). 3) The relative expression level ofGAPDHgene in liver of broilers was significantly affected by days of age (P<0.05), however, the effects of oil combination and the interaction between oil combination and days of age were not significant (P>0.05). In conclusion, the effect of oil types on the relative expression level ofGAPDHgene in broilers shows difference among different tissues, corn oil can improve the relative expression level ofGAPDHgene in pectorals. The relative expression level in pectorals ofGAPDHgene of 42-day-old broilers is significantly higher than that in liver and abdominal fat, and the relative expression level ofGAPDHgene in liver of 21-day-old broilers is significantly higher than that of 42-day-old broilers.[ChineseJournalofAnimalNutrition, 2017, 29(5)：1750-1758]

broilers; oil; GAPDH; gene expression

10.3969/j.issn.1006-267x.2017.05.035

2016-11-17

國(guó)家自然科學(xué)基金(31272434)

陶亞飛(1990—)，男，河南商丘人，碩士研究生，動(dòng)物遺傳育種與繁殖專業(yè)。E-mail: 15515517571@163.com

*通信作者：黃艷群，教授，碩士生導(dǎo)師，E-mail: hyanqun@aliyun.com

S831

A

1006-267X(2017)05-1750-09