兰州大尾羊解偶联蛋白3(UCP3)基因荧光定量PCR检测方法的研究

试验通过提取兰州大尾羊不同组织中总RNA,反转录获得总cDNA样品,建立了用SYBR荧光定量PCR法检测兰州大尾羊解偶联蛋白(UCP3)基因mRNA在不同组织中的相对表达量的检测方法,并进行批内、批间重复性检验。结果表明:UCP3和GADPH基因标准曲线回归方程分别为CtUCP3=-3.2343x+41.6198和CtGADPH=-3.2851x+38.0344,相关系数(r2)分别为0.9958和0.9986,扩增效率分别为103%和97%;批内、批间重复性测定的变异系数分别为小于1.4%和10%。     

UCP3基因在巴马猪和藏猪脂肪组织中的表达和甲基化分析

为探究解偶联蛋白3(uncoupling protein 3,UCP3)基因在巴马猪和藏猪皮下脂肪组织中的表达和甲基化水平,试验采用实时荧光定量PCR技术检测UCP3基因在巴马猪和藏猪皮下脂肪组织中的mRNA表达水平;针对猪UCP3基因启动子区域(-3 580～+920 bp),利用在线软件MethPrimer对该区域进行CpG岛预测,并采用亚硫酸氢盐测序法(bisulfite sequencing PCR,BSP)检测其甲基化水平,探究UCP3基因甲基化水平在巴马猪和藏猪中的差异。结果显示,巴马猪皮下脂肪组织UCP3基因表达量显著高于藏猪(P0.05);在UCP3基因启动子区预测到3个CpG甲基化岛,分别是CpG island1(-3 171～-2 928 bp)、CpG island2(-154～-2 bp)和CpG island3(+648～+806 bp),其中CpG island1和CpG island3的甲基化水平在巴马猪和藏猪中差异较小,而藏猪CpG island2的甲基化水平(42.61%)高于巴马猪(24.49%)。本研究绘制了2个猪种CpG island2甲基化水平的黑白点图,其中CpG位点为4、8、9、10、11、12、15,藏猪甲基化频率分别比巴马猪高28.26%、17.39%、26.09%、26.09%、26.09%、23.91%和34.78%。在CpG island2处预测到3个转录因子结合位点(SP2、PPARγ和EGR1)。结果表明,巴马猪和藏猪皮下脂肪组织中UCP3基因mRNA水平的表达差异可能是由于CpG island2的甲基化水平不同所导致,藏猪DNA甲基化水平在一定程度上阻碍了转录因子与启动子调控区域的结合,从而抑制了UCP3基因的表达。     

内源性逆转录病毒在五指山小型猪体内存在状况的分子微生物学调查内源性逆转录病毒在五指山小型猪体内存在状况的分子微生物学调查 内源性逆转录病毒在五指山小型猪体内存在状况的分子微生物学调查

根据猪内源性逆转录病毒（PERV）的核心蛋白（gag）基因、多聚酶（pol）基因、囊膜（env）基因和β-肌动素（β-actin）基因核酸序列设计并筛选特异性引物6对,利用PCR方法对五指山小型猪PERVgag、pol和env基因存在情况进行系统检测;利用半定量RT-PCR方法对五指山小型猪PERVgag、pol和env基因在心、肝、脾、肺、肾、脑、肌肉7个组织中的表达情况进行系统检测与分析。结果显示,在202个被检样品中,gag、pol基因和envB基因亚型均存在,envA基因亚型仅3个样品未发现,envC基因亚型共检出34个,检出率为16.8%。所测试的7个组织均表达gag、pol和env基因序列,但envC仅在1头个体的组织中表达。各组织gag mRNA丰度以肾脏为最高,其次为肺、心、肝、脾,脑和肌肉组织的丰度最低。各组织pol mRNA丰度以肾脏为最高,其次为脾、肺、肝、心,肌肉和脑组织的丰度最低。各组织envA、envB mRNA丰度信号强度分布与gag、pol信号类似,亦以肾脏为最高,脑和肌肉组织的丰度最低。各组织envC mRNA的信号强度明显低于envA、envB。以上结果表明,在五指山小型猪中存在PERV,且在各组织中的表达量有差异。     

鸭IL-1β基因实时荧光定量PCR方法的建立及初步应用

为了建立鸭白细胞介素-1β(IL-1β)基因的实时荧光定量PCR检测方法,并检测鸭机体器官/组织中IL-1β基因转录水平,试验根据GenBank上鸭IL-1β基因序列设计特异性引物,以鸭IL-1β基因克隆质粒为模板,SYBR GreenⅠ为荧光染料,建立鸭IL-1β基因实时荧光定量PCR检测方法,并用该方法检测鸭机体器官/组织中IL-1β基因的转录水平。结果表明:建立的鸭IL-1β基因实时荧光定量PCR方法的熔解曲线为特异性单峰,标准曲线方程为y=-3. 329x+39. 731,扩增效率为99. 7%,相关系数为1,批内重复变异系数小于1. 00%,批间重复变异系数小于2. 00%;实时荧光定量PCR方法的检测敏感性是普通PCR方法检测敏感性的1 000倍;以该方法进行检测发现,鸭的法氏囊、肺脏、肝脏、肌肉、脑、脾脏、肾脏、十二指肠、心脏和胸腺中IL-1β基因mRNA含量分别为428. 45,973. 43,1 394. 19,568. 22,551. 54,839. 91,2 586. 22,1 161. 92,459. 17,3 276. 16 copies/μL,其中胸腺中含量最高,而法氏囊中含量最低。说明试验建立的鸭IL-1β基因实时荧光定量PCR检测方法具有重复性好和敏感性高等特点,可用于鸭机体器官/组织中IL-1β基因转录水平的检测分析。     

内源性逆转录病毒在五指山小型猪体内存在状况的分子微生物学调查

根据猪内源性逆转录病毒(PERV)的核心蛋白(gag)基因、多聚酶(pol)基因、囊膜(env)基因和β-肌动素(β-actin)基因核酸序列设计并筛选特异性引物6对,利用PCR方法对五指山小型猪PERV gag、pol和env基因存在情况进行系统检测;利用半定量RT-PCR方法对五指山小型猪PERV gag、pol和env基因在心、肝、脾、肺、肾、脑、肌肉7个组织中的表达情况进行系统检测与分析.结果显示,在202个被检样品中,gag、pol基因和envB基因亚型均存在,envA基因亚型仅3个样品未发现,envC基因亚型共检出34个,检出率为16.8%.所测试的7个组织均表达gag、pol和env基因序列,但envC仅在1头个体的组织中表达.各组织gagmuRNA丰度以肾脏为最高,其次为肺、心、肝、脾,脑和肌肉组织的丰度最低.各组织pol mRNA丰度以肾脏为最高,其次为脾、肺、肝、心,肌肉和脑组织的丰度最低.各组织envA、envBmRNA丰度信号强度分布与gag、pol信号类似,亦以肾脏为最高,脑和肌肉组织的丰度最低.各组织envC mRNA的信号强度明显低于envA、envB.以上结果表明,在五指山小型猪中存在PERV,且在各组织中的表达量有差异.     

兰州大尾羊H-FABP基因荧光定量PCR检测方法的研究

通过对兰州大尾羊尾部、大网膜、肝脏、肾周脂肪组织中的总RNA提取,反转录获得总cDNA样品,结合PCR技术,建立了SYBR荧光定量PCR法检测兰州大尾羊心脏型脂肪酸结合蛋白(H-FABP)基因mRNA在不同组织中相对表达量的试验方法,并进行批内、批间重复性检验。结果表明,H-FABP和18S基因标准曲线回归方程分别为CtH-FABP=-3.24375x+38.34230和Ct18S=-3.33137x+33.4573,相关系数(r2)分别为0.9964和0.9992,扩增效率分别为103%和99%;批内、批间重复性测定的变异系数分别为小于1.8%和10%;说明该方法灵敏度高、特异性强、准确可靠、重复性好,是实时荧光定量PCR检测兰州大尾羊心脏型脂肪酸结合蛋白基因mRNA表达量的有效方法。     

猪圆环病毒3型TaqMan-MGB荧光定量PCR方法的建立及应用

为建立猪圆环病毒3型(PCV3)荧光定量PCR检测方法,本研究根据GenBank中PCV3基因序列设计特异性引物和探针,经过反应体系和条件优化,建立了特异性检测PCV3的TaqMan-MGB荧光定量PCR方法。该检测方法在4.78×10~1拷贝/μL～4.78×10~9拷贝/μL质粒标准品范围内均有良好的线性关系;该方法特异性试验结果显示,其与多种常见猪病病毒均无交叉反应,特异性良好;本研究建立的方法敏感性是常规PCR方法的100倍,敏感性较高;批内批间重复性试验变异系数均小于2.3%,重复性良好。对临床样品的检测结果显示,该方法对PCV3的检出率高于常规PCR方法,并且PCV3阳性样品多存在混合感染情况。该方法的建立为PCV3的实验室诊断及流行病学调查提供了快速、准确的检测手段。     

猪UCP3基因的分离与多态分析

解偶联蛋白3是一种线粒体内膜上的转运蛋白,可以使氧化过程与ADP磷酸化过程解偶联。本研究分离了猪UCP3基因的外显子2~3和外显子6。经过序列分析,发现猪UCP3基因共有3处框移突变和18个SNP位点。在猪UCP3片段中分别包含一个SmaⅠ和SduⅠ酶切位点。UCP3片段SmaⅠ位点在大白×梅山F2家系中经PCR-RFLP分析,表现为AA、AB和BB 3种基因型,A基因频率为0.57,B基因频率为0.43。因此,可以将UCP3基因作为猪分子遗传育种的候选基因。     

猪血清淀粉样蛋白3分子SYBR Green Ⅰ实时荧光定量PCR检测方法的建立及初步应用

分别根据猪血清淀粉样蛋白3(serum amyloid A3,SAA3)基因序列以及猪三磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因全长序列设计特异性扩增引物,进行PCR扩增,并将PCR扩增片段连接至相应载体上构建重组质粒。两个重组质粒经测序鉴定和纯化后,倍比稀释作为标准曲线样品,用于实时荧光定量PCR中SAA3、GAPDH标准曲线的制备,并进行反应的灵敏性、特异性和重复性检测。结果显示,标准曲线线性关系R2均在0.98以上;特异性检测显示该引物可以特异性检测到猪SAA3、GAPDH扩增曲线;组内和组间变异系数均小于5%,说明本研究成功建立猪SAA3的荧光定量PCR检测方法。运用建立的荧光定量RT-PCR对正常以及感染猪繁殖与呼吸综合征病毒的肺泡巨噬细胞和猪组织进行检测,可检测到猪SAA3的表达,正常组与接毒组之间显示出明显的表达差异。本研究初步建立了检测猪SAA3基因的SYBR Green荧光定量RT-PCR的方法,为后续对猪传染性疾病与猪SAA3之间相互关系的研究提供了一种特异、灵敏的检测方法。     

UCP3基因在巴马猪和藏猪脂肪组织中的表达和甲基化分析

为探究解偶联蛋白3（uncoupling protein 3,UCP3）基因在巴马猪和藏猪皮下脂肪组织中的表达和甲基化水平,试验采用实时荧光定量PCR技术检测UCP3基因在巴马猪和藏猪皮下脂肪组织中的mRNA表达水平;针对猪UCP3基因启动子区域（-3 580~+920 bp）,利用在线软件MethPrimer对该区域进行CpG岛预测,并采用亚硫酸氢盐测序法（bisulfite sequencing PCR,BSP）检测其甲基化水平,探究UCP3基因甲基化水平在巴马猪和藏猪中的差异。结果显示,巴马猪皮下脂肪组织UCP3基因表达量显著高于藏猪（P<0.05）;在UCP3基因启动子区预测到3个CpG甲基化岛,分别是CpG island1（-3 171~-2 928 bp）、CpG island2（-154~-2 bp）和CpG island3（+648~+806 bp）,其中CpG island1和CpG island3的甲基化水平在巴马猪和藏猪中差异较小,而藏猪CpG island2的甲基化水平（42.61%）高于巴马猪（24.49%）。本研究绘制了2个猪种CpG island2甲基化水平的黑白点图,其中CpG位点为4、8、9、10、11、12、15,藏猪甲基化频率分别比巴马猪高28.26%、17.39%、26.09%、26.09%、26.09%、23.91%和34.78%。在CpG island2处预测到3个转录因子结合位点（SP2、PPARγ和EGR1）。结果表明,巴马猪和藏猪皮下脂肪组织中UCP3基因mRNA水平的表达差异可能是由于CpG island2的甲基化水平不同所导致,藏猪DNA甲基化水平在一定程度上阻碍了转录因子与启动子调控区域的结合,从而抑制了UCP3基因的表达。     

Effect of heat exposure on uncoupling protein-3 mRNA abundance in porcine skeletal muscle

Exposure to cold increases abundance of mRNA for uncoupling protein-3 (UCP3) in skeletal muscle, whereas the influence of exposure to heat is unknown. Thus, we conducted a study to investigate the influence of heat exposure on UCP3 mRNA abundance in porcine skeletal muscle. Three pigs aged 110 to 120 d, with an average BW of 75 kg, from each of eight litters were used. Each littermate was assigned to one of three treatment groups; one group was reared at 32 degrees C and fed ad libitum (32AL) for 4 wk, whereas the other two groups were maintained at 23 degrees C for the same period, and either pair-fed the intake of their 32AL littermates (23PF), or fed ad libitum (23AL). The RNase protection assay revealed that UCP3 mRNA abundance in longissimus dorsi and rhomboideus muscles was higher (P < 0.05) in the 32AL group than the 23PF group. The 23AL group also had significantly higher UCP3 mRNA abundance than the 23PF group in these muscles. Plasma total 3,5,3'-triiodothyronine concentration of the 32AL group was lower (P < 0.05) than that of the 23PF group, whereas mRNA abundance of thyroid hormone receptor (TR) isoforms, TRalpha1 and TRalpha2, in these muscles was not affected, suggesting that the 32AL group was in a relatively hypo-thyroid state. Because thyroid hormone up-regulates UCP3 expression, these results indicate that factors other than thyroid hormone may play a role in regulating UCP3 mRNA abundance in skeletal muscle of heat-exposed pigs.     

Study on Differences of UCP3 Gene Expression of Reciprocal Crosses F1 with Luchuan Pig and Duroc Pig

In order to breed high quality pig strain and improve the pork quality,the intramuscular fat content and the differences of UCP3 gene expression were studied in Luchuan pigs with Duroc reciprocal cross F1 and its parents Luchaun pigs.8 head of Luchuan pigs,Luchuan pigs with Duroc reciprocal cross F1 generation were respectively selected to analyze the correlation of UCP3 gene expression with intramuscular fat and eye muscle area.Expression of UCP3 mRNA in muscles of different pig breeds were measured by Real-time RT-PCR,and the eye muscle area and intramuscular fat content were measured too.The results indicated that the eye muscle area,intramuscular fat and the UCP3 gene expression were significantly different in the three groups of pigs (P<0.05).The crossbreds inherited the big body size of Duroc,and the high intramuscular fat content of Luchuan pig.The extremely significantly negative correlations were observed between UCP3 gene expression and the pig eye muscle area,the extremely significantly positive correlations were observed between UCP3 gene expression and intramuscular fat content.UCP3 gene should be the main candidate gene for fat trait research in pigs.     

陆川猪与杜洛克猪正反交F1代UCP3基因表达差异研究

为培育优质猪品系,提高猪肉品质,本试验探索了陆川猪与杜洛克猪正反交F1代及其亲本陆川猪肌内脂肪含量和UCP3基因表达差异。试验分别选取陆川猪、陆川猪与杜洛克猪正反交F1代各8头,采用实时荧光定量RT-PCR方法测定肌肉中UCP3基因mRNA在不同品种猪中的表达量,并测定眼肌面积和肌内脂肪含量,以分析UCP3基因的表达量与脂肪和眼肌面积的相关性。结果显示,3组猪在眼肌面积、肌内脂肪含量和UCP3基因mRNA表达量上均差异显著(P<0.05),且正反交F1代均遗传了杜洛克猪体型大、陆川猪肌内脂肪含量高的优势,UCP3基因的表达量与猪眼肌面积呈极显著负相关,与肌内脂肪含量呈极显著正相关。初步推断UCP3基因可作为影响猪脂肪性状的主效基因。     

Effects of dietary lysine levels on apparent nutrient digestibility and cationic amino acid transporter mRNA abundance in the small intestine of finishing pigs,Sus scrofa

One hundred and twenty pigs were used to evaluate the effects of different dietary lysine levels on the growth performance, apparent nutrient digestibility, and abundance of cationic amino acid transporter messenger RNA (mRNA) in the small intestine of finishing pigs. Pigs received a low lysine diet (LL, 0.60% lysine), moderate lysine diet (ML, 0.80% lysine) or a high lysine diet (HL, 1.00% lysine) for 28 days. A digestion test was carried out during the third week. Although the apparent nutrient digestibility in pigs fed experimental diets were different (P < 0.05) and the highest when pigs were fed ML diet, diets did not change the growth performance. In the duodenum, mRNA abundance of PepT-1, as detected by real-time RT-PCR, was reduced in the LL diet (P < 0.05). A greater abundance of b^0,+AT and PepT-1 mRNA was associated with the ML diet (P < 0.05) in the jejunum and ileum, respectively. In the ileum, the HL diet had a lower abundance of CAT-1 mRNA compared with other diets. These results showed that the finishing pigs would gain better nutrient digestibility when the dietary lysine content was 0.80%, and dietary lysine levels influenced the expression of cationic amino acid transporter mRNA in the small intestine of finishing pigs.     

Betaine suppresses carnitine palmitoyltransferase I in skeletal muscle but not in liver of finishing pigs

An experiment was conducted to investigate the effect of dietary betaine supplementation on the enzyme activity and mRNA abundance for carnitine palmitoyltransferase I (CPT I) in liver and skeletal muscle of finishing pigs. Forty-eight crossbred barrows and gilts [Duroc × (Seghers × Seghers)] weighing about 55 kg were divided into two dietary treatments, each with three replicates of eight pigs (four barrows and four gilts) per replicate. Pigs were fed a corn–soybean meal basal diet supplemented with betaine at 0 or 1250 mg/kg feed for 42 days. At trial termination, two pigs (one barrow and one gilt) weighing about 90 kg were selected from each replicate (six pigs per dietary treatment) and slaughtered for analyses. The results showed that intramuscular fat content in the longissimus muscle of pigs fed betaine was 23.6% higher than that of controls (P < 0.05), whereas hepatic fat content was not affected with dietary betaine treatment. Muscle-type CPT I (M-CPT I) activity, but not liver-type CPT I (L-CPT I) activity was decreased by betaine supplementation. Furthermore, betaine supplementation reduced M-CPT I mRNA abundance by 14.6% (P < 0.05) but did not affect L-CPT I mRNA abundance. There was a positive correlation between enzyme activity and mRNA abundance for both L-CPT I and M-CPT I (r = 0.67 and r = 0.72 for L-CPT I and M-CPT I, respectively; P < 0.05). The study suggests that betaine may be involved in fat partitioning in pigs by reducing the activity and mRNA abundance of M-CPT I, with a resultant increase in intramuscular fat content.     

Triiodothyronine differentially regulates key metabolic factors in lean and obese cats

The effect of a 2-week administration of 75microg triiodothyronine (T3) on substrate oxidation, heat production, non-esterified fatty acids, and leptin was evaluated in eight lean (three females and five males) and eight obese (five females and three males) age-matched adult neutered cats. In addition, using real-time RT-PCR, expression of muscle and adipose tissue uncoupling proteins (UCP2 and UCP3), deiodinase 1 and 2 (D1; D2), and peroxisome proliferator-activated receptor (PPAR) alpha and gamma and peroxisome-proliferator-activator receptor-gamma co-activator 1alpha (PGC1) was examined. Compared to lean cats, obese cats had increased NEFA, leptin, UCP2, and D1mRNA in muscle and UCP3mRNA levels in fat, but lower heat production, and fat PPARs and PGC1. T3 administration increased thermogenesis and NEFA in lean and obese cats, and adipose tissue PPARgamma in lean cats. It also increased muscle D1 in lean and D2 in obese cats. The increase in muscle D2 was interpreted to be reflective of the reduced serum total T4 concentration following T3 suppression of the pituitary. No effect was seen on leptin, or UCP2 and 3. This shows that T3 regulates thermogenesis but not through changes in uncoupling protein expression. It also indicates that PPARs have an important role in the pathogenesis of obesity in cats.     

猪CRTC3基因表达的品种差异及forskolin对其表达的影响

本试验旨在探究糖脂代谢通路关键基因CRTC3在不同品种猪肌肉和脂肪组织中的表达情况,并通过forskolin处理猪皮下脂肪前体细胞,研究forskolin对脂肪前体细胞分化聚酯和CRTC3基因表达的影响,阐明猪CRTC3基因表达与脂肪沉积的关系。试验选取杜长大猪和莱芜猪各5头,检测肌肉、脂肪组织中CRTC3的mRNA和蛋白表达水平以及脂肪代谢相关基因的mRNA表达水平;选取2头3日龄的杜长大仔猪,分离猪皮下脂肪前体细胞,待完全融合后用MDI诱导培养基诱导4 d,然后用分化培养基继续诱导4 d,完成诱导分化。Forskolin组在诱导分化的第1天即加入forskolin,使其终浓度为10μmol/L,对照组则加入同浓度的二甲基亚砜(DMSO)进行诱导分化。结果表明:在莱芜猪的背最长肌和腰大肌中,CRTC3的蛋白表达水平高于杜长大猪;在莱芜猪的皮下和内脏脂肪组织中,CRTC3及脂肪沉积相关基因过氧化物酶体增殖剂激活受体γ(PPARγ)、脂肪酸结合蛋白4(FABP4)、CCAAT/增强子结合蛋白α(C/EBPα)、围脂滴蛋白(PLIN)和瘦素(LEP)的mRNA表达水平显著或极显著高于杜长大猪(P<0.05或P<0.01),而脂肪棕色化相关基因NF-E2相关因子1(NRF1)、过氧化物酶体增殖物激活受体-γ共激活因子-1α(PGC⁃1α)、PRDM16、解偶联蛋白2(UCP2)、解偶联蛋白3(UCP3)的mRNA表达水平则显著或极显著低于杜长大猪(P<0.05或P<0.01)。进一步的研究发现,猪皮下脂肪前体细胞分化后CRTC3和脂肪沉积相关基因的mRNA表达水平极显著提高(P<0.01),脂肪棕色化相关基因的mRNA表达水平也均极显著升高(P<0.01)。10μmol/L forsko⁃lin处理能抑制猪皮下脂肪前体细胞分化,极显著升高环磷腺苷效应元件结合蛋白(CREB)和脂肪棕色化相关基因的mRNA表达水平(P<0.01),促进CRTC3的进核,极显著降低CRTC3和脂肪沉积相关基因的mRNA表达水平(P<0.01)。上述研究结果表明,CRTC3基因与猪脂肪沉积密切相关,forskolin处理可以调控猪CRTC3及脂质代谢相关基因表达,调控猪皮下脂肪前体细胞分化聚酯。     

β‐hydroxy‐β‐methyl butyrate promotes leucine metabolism and improves muscle fibre composition in growing pigs

The aim of this study was to investigate the effects of excess leucine (Leu) vs. its metabolites α‐ketoisocaproate (KIC) and β‐hydroxy‐β‐methyl butyrate (HMB) on Leu metabolism, muscle fibre composition and muscle growth in growing pigs. Thirty‐two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed 1 of 4 diets for 45 days: basal diet, basal diet + 1.25% L‐Leu, basal diet + 1.25% KIC‐Ca, basal diet + 0.62% HMB‐Ca. Results indicated that relative to the basal diet and HMB groups, Leu and KIC groups exhibited increased Leu concentrations and decreased concentrations of isoleucine, valine and EAAs in selected muscle (p < 0.05) and had lower mRNA levels of MyHC I and higher expression of MyHC IIx/IIb (p < 0.05), and there was no significant difference between the basal and HMB‐supplemented groups. Moreover, the mRNA expression levels of AMPKα and UCP3 were higher but the myostatin mRNA levels were lower in the soleus muscle of the HMB group than those from other groups (p < 0.05). These findings demonstrated that doubling dietary Leu content exerted growth‐depressing effects in growing pigs; dietary KIC supplementation induced muscular branched‐chain amino acid imbalance and promoted muscle toward a more glycolytic phenotype; while dietary HMB supplementation promoted the generation of more oxidative muscle types and increased muscle growth specially in oxidative skeletal muscle, and these effects of HMB might be associated with the AMPKα‐Sirt1‐PGC‐1α axis and mitochondrial biogenesis.