猪CAPN1基因mRNA表达特性分析

为了进一步揭示CAPN1的功能,研究以野猪、大白猪及野家杂交猪为研究对象,利用相对定量RT-PCR方法研究其编码基因的表达特性。结果表明:CAPN1基因在组织中普遍表达,但表达量存在差异,并且野猪和大白猪具有不同的表达模式;在大白猪肌肉组织中表达的发育性变化为波浪型;在360日龄大白猪和野家杂交猪肌肉组织中的表达量存在显著差异。     

滩羊肌肉组织钙蛋白酶系统基因表达及其与嫩度的相关分析

为了研究日粮不同蛋白水平下钙蛋白系统基因在滩羊肌肉组织的表达差异,采用实时荧光定量PCR(Quantitative Real-time PCR,qPCR)方法,根据GenBank已发表钙蛋白酶1(calpain 1)基因CAPN1、钙蛋白酶2(calpain 2)基因CAPN2和钙蛋白酶抑制蛋白(calpastatin)基因CAST的序列设计特异性引物,以滩羊肌肉组织总RNA为模板,分析了CAPN1、CAPN2和CAST基因在滩羊背最长肌的基因表达水平。结果发现CAPN1和CAPN2基因在高蛋白组中上调表达,qPCR扩增中CAST基因没有明显的扩增曲线,CAST基因的表达没有检测到。基因表达水平与肌肉剪切力值相关性分析表明,CAPN1、CAPN2与剪切力值均显著负相关(P0.05),相关系数分别为-0.864和-0.896。     

钙蛋白酶基因在鸡不同组织和品种中的表达差异

为了探讨钙蛋白酶(calpain,CAPN)基因在鸡不同组织和品种中的表达差异,本研究采用半定量RT-PCR方法研究了CAPN基因在鸡不同组织和品种中的表达差异。结果表明:CAPN基因表达于优质鸡的10个不同组织中,肝脏和胸肌中的表达量最高并显著高于其他组织(P<0.05);优质鸡胸肌组织中CAPN基因的表达量显著高于艾维茵肉鸡和宝万斯尼拉蛋鸡品种(P<0.05),优质鸡品种之间以及肉鸡与蛋鸡品种间差异不显著(P>0.05)。结果证明钙蛋白酶是一种在鸡体内普遍存在的蛋白,CAPN基因在鸡不同组织和品种中的表达存在显著差异。     

饲喂生物发酵饲料对延边黄牛背最长肌FABP4、CAPN1基因表达的影响

试验旨在研究育肥后期饲粮中添加生物发酵饲料对延边黄牛背最长肌组织中FABP4、CAPN1基因表达量的影响。选择12头平均体重(545.55±27.00)kg的体健无病延边黄牛阉牛,随机分成试验组和对照组,每组6头。试验组每头牛饲喂生物发酵饲料2 kg/d,对照组不饲喂生物发酵饲料,试验组和对照组的基础日粮营养水平基本相同。预饲期10 d,正式期90 d。试验结束后,分别从试验组与对照组各随机选取3头牛,在第14肋骨处采取背最长肌一小块,采用荧光定量聚合酶链式反应(RT-PCR)技术检测样品中FABP4、CAPN1基因表达量。结果表明:试验组中FABP4 mRNA的表达量是对照组的2.27倍,FABP4基因mRNA的表达量极显著高于对照组(P<0.01)。CAPN1 mRNA的表达量试验组高于对照组,差异显著(P<0.05)。综上所述,在育肥后期饲粮中添加生物发酵饲料可显著提高FABP4、CAPN1基因在延边黄牛背最长肌中的表达量。     

草原红牛CAPN1基因mRNA表达特性分析

为了进一步揭示CAPN1的功能,本研究采用实时荧光定量PCR技术对草原红牛初生牛与成年牛心脏、肝脏、脾脏、肺脏、肾脏、瘤胃、十二指肠、背最长肌8种组织中的CAPN1mRNA表达水平进行了分析。结果表明：CAPN1基因在组织中普遍表达,但表达量存在差异。且成年牛的CAPN1基因在肺脏和瘤胃组织中的表达显著高于初生牛（P〈0.05）,成年牛在肝脏、脾脏、肾脏和背最长肌组织中CAPN1基因的表达水平极显著高于初生牛（P〈0.01）。     

抗病毒基因MxA在鸡体内的表达

大量提取人抗病毒基因MxA的重组质粒pEGFP-C1-MxA,肌肉多点注射未成年广西土鸡,RT-PCR 免疫组化方法检测MxA基因在鸡体内各组织的分布表达.采用RT-PCR方法于注射后5 d、10 d在呼吸道上皮、脾脏、肌肉组织中均可检出MxA基因的转录;采用免疫组化方法检测到GFP和MxA基因均能在肌肉组织中得到表达,且注射5 d后的表达量高于注射10 d后.结果表明重组质粒肌肉多点注射可介导MxA在鸡体内多种组织中转录、表达,为进一步探讨MxA的生物学活性以及禽类抗病毒免疫制荆的研发奠定了基础.     

抗病毒基NMxA在鸡体内的表达

大量提取人抗病毒基因MxA的重组质粒pEGFP-C1-MxA，肌肉多点注射未成年广西土鸡，RT-PCR及免疫组化方法检测MxA基因在鸡体内各组织的分布表达。采用RT-PCR方法于注射后5d、10d在呼吸道上皮、脾脏、肌肉组织中均可检出MxA基因的转录；采用免疫组化方法检测到GFP和MxA基因均能在肌肉组织中得到表达，且注射5d后的表达量高于注射10d后。结果表明重组质粒肌肉多点注射可介导MxA在鸡体内多种组织中转录、表达，为进一步探讨MxA的生物学活性以及禽类抗病毒免疫制剂的研发奠定了基础。     

散养黄羽肉鸡CAPN1、H-FABP基因表达的发育性变化及其肌内脂肪含量的研究

为探讨在散养条件下黄羽肉鸡CAPN1、H-FABP基因与其生长发育和肌内脂肪(IMF)含量的关系,试验以130羽3周龄体重接近,健康商品代黄羽肉鸡母鸡为试验材料,随机分为笼养组(对照组)和散养组(试验组),采集在不同周龄(3、4、6、8、10、13 w)的胸肌、腿肌和腹脂组织样品共360份,采用实时荧光定量PCR法对不同生长阶段组织中CAPN1和H-FABP基因mRNA表达量进行检测,同时采用索氏抽提法测定胸肌和腿肌的IMF含量。结果表明:黄羽肉鸡的CAPN1、H-FABP基因在胸肌、腿肌和腹脂中均有不同程度的表达。随着周龄的增加,散养肉鸡CAPN1基因表达的整体变化趋势为增加—减少—增加—减少,且3～6 w其在胸肌和腹脂中的表达量均表现为笼养显著高于散养(P<0.05)。H-FABP基因的整体表达趋势随着周龄的增加而降低,在胸肌和腿肌中变化趋势相同,且均表现为散养显著高于笼养(P<0.05),在腹脂中表现为笼养高于散养(P>0.05)。与散养鸡相比,笼养鸡生长速度较快,腹脂率、IMF含量较高。本试验结果可为散养黄羽肉鸡CAPN1和H-FABP基因分子选育提供一定参考。     

五指山猪不同组织中Myf5与MyoD1基因的表达研究

 MRFs家族成员包括Myf5、MyoD1、Myf4和Mfy6,利用Real time PCR技术,检测Myf5、MyoD1基因在从出生到体成熟（30 d、210 d、360 d）五指山猪背部肌肉组织中的表达变化趋势,以及Myf5、MyoD1基因在体成熟五指山猪心脏、肝脏、肺脏、脾脏、肾脏、肌、胃和小肠以上组织中的mRNA表达水平。结果表明：Myf5和MyoD1基因在出生后五指山猪背部肌肉组织中的mRNA表达水平与五指山猪的生长年龄成正比（P<0.05）;Myf5及MyoD1基因在成年五指山猪以上8种组织中均有表达,其中在肌肉组织中相对表达量最高。     

大白猪和山西黑猪三种肌肉JAZF1基因的发育性表达研究

本文旨在探究JAZF1基因mRNA在大白猪和山西黑猪肌肉组织中的发育性表达规律,为研究猪肌内脂肪的形成机理提供有力的理论参考。采用实时荧光定量PCR技术检测大白猪和山西黑猪在25d、70d和150d的背最长肌、腰大肌和股二头肌三种肌肉组织中JAZF1基因mRNA表达量。结果表明:随着日龄的增长,JAZF1基因在三种肌肉中呈现逐渐下降的表达趋势。大白猪25d腰大肌和股二头肌、70d股二头肌中的表达量显著高于山西黑猪(P<0.05)。三种肌肉中的表达量比较发现,JAZF1基因在两个品种腰大肌中表达量最高,其次是股二头肌,背最长肌表达量最低。结果表明JAZF1基因在肌肉发育和肌内脂肪沉积过程中起着很重要的作用。本研究结果可为探索肌肉发育及肌内脂肪沉积相关研究提供理论参考和研究基础。     

Study on association of single nucleotide polymorphism of CAPN1 gene with muscle fibre and carcass traits in quality chicken populations

This study was aimed at investigating the effect of the calpain 1 (CAPN1) gene on carcass and meat quality traits in eight meat-type chicken populations, including five pure lines (developed from Chinese local breeds) and three cross-breeds. Primer pairs for the Coding Sequence (CDS) region in CAPN1 were designed from the chicken genomic sequence database. Polymorphisms were detected by polymerase chain reaction (PCR)-Single Strand Conformation Polymorphism (SSCP) and DNA sequencing. Three single nucleotide polymorphisms (SNP; C2546T, G3535A and C7198A) were detected among individuals in each population. The associations of their haplotypes (H1 = CGA, H2 = CGC, H3 = CAA, H4 = CAC, H5 = TGA and H7 = TAA) with chicken breast muscle fibre and carcass traits were analysed. Results showed that the haplotypes were associated with live weight (LW), carcass weight (CW), breast muscle weight (BMW) and leg muscle weight (LMW) (p < 0.05), and were also related to eviscerated percentage (%EP) and breast muscle fibre density (p < 0.01). H1H3 haplotype was dominant for LW, CW and BMW; H1H5 haplotype was dominant for EP; H3H4 haplotype was dominant for LMW and H1H1 haplotype was dominant for BFD. It was concluded that the CAPN1 gene may be a major gene affecting meat quality traits of chicken or it is linked with the major gene. H1H3, H1H5 and H3H4 were the most advantageous haplotypes for carcass traits whereas H1H1 was the positive haplotype for breast muscle fibre trait.     

The normalisation of CAPN gene expression in M. pectoralis superficialis in broiler lines differing in growth rate and their relationship to breast muscle tenderness

1. The aim of this study was to assess mRNA abundance of calpain 1 (CAPN1) and calpain 3 (CAPN3) in breast muscle of 80 fast-growing (FG) and slow-growing broilers (SG) and relate gene expression in relation to growth and Warner Bratzler (WB) shear force of breast muscle.

2. The expression of CAPN1 and CAPN3 genes was higher in the FG compared to the SG line, but significant results were obtained only for CAPN1. The CAPN1 mRNA level was strongly dependent on line and gender interaction.

3. Lower values of shear force were observed in the FG line, where a higher level of calpain expression was shown.

4. A new panel of housekeeping genes (RPL4 and SDHA) for normalisation of gene expression in muscle tissues could be used in other studies of gene expression in chicken.

     

鸡肉嫩度候选基因CAPN1多态性的检测研究

建立基于聚合酶链反应-连接酶检测反应的基因多态性并行检测系统,探讨鸡肉嫩度候选基因钙蛋白酶I（CAPN1）基因的多态性,并分析其在优良地方鸡种-清远麻鸡和快大型隐性白羽鸡中的分布差异。用PCR-LDR方法检测CAPN1 2546、CAPN1 3535和CAPN19950的基因型作单倍型分析,进行各组间基因型分布和等位基因频率x2检验。结果CAPN12546位点未检测到多态,其他两个位点的结果同直接测序分型结果一致,其基因型分布在不同品种中均存在差异,并且在CAPN1 3535位点上达到显著水平,两个位点的单倍型分布在不同品种间也存在较大差异。     

广灵驴CAPN1基因克隆、生物信息学分析及表达研究

本研究旨在对钙蛋白酶1（CAPN1）基因CDS区进行克隆及生物信息学分析，并鉴定其在广灵驴各组织中的表达量。应用生物信息学方法对广灵驴CAPN1基因CDS区进行序列分析，并对其编码蛋白的理化性质、亚细胞定位、翻译后修饰结构和蛋白结构进行预测；利用实时荧光定量PCR技术对CAPN1基因在心脏、肝脏、脾脏、肺脏、肾脏和背最长肌6种组织中的表达水平进行分析。结果显示，广灵驴CAPN1基因CDS区全长2 148 bp，可编码715个氨基酸，已提交至NCBI，登录号为：MN158194，其核酸序列与马、绵羊、牛、人、山羊、小鼠、猪的同源性分别为99.7%、92.3%、92.5%、92.0%、92.5%、85.9%和92.9%；CAPN1蛋白的分子质量为82.01 ku，理论等电点为5.59，平均疏水性为-0.374，不稳定系数为36.42，不存在跨膜区及信号肽；其编码蛋白的二级结构由无规则卷曲、α-螺旋、β-转角和延伸链组成；CAPN1基因在广灵驴的6种组织中均表达，其中在背最长肌中的表达量最丰富，其次是肝脏，在心脏中的表达最低。本研究成功克隆了广灵驴CAPN1基因CDS，并对其在不同组织中的表达量进行了分析，为进一步研究CAPN1基因在肌肉嫩度方面的表达调控功能及发展地方品种广灵驴肉制品产业提供了理论依据。     

Linkage mapping of four chicken calpain genes

Calpains are intracellular Ca²⁺‐dependent proteases and enzymes that contribute to growth and meat quality. In the present study, we identified polymorphisms in four calpain genes (CAPN1, CAPN2, CAPN3, and CAPN1.5) expressed ubiquitously in chicken using polymerase chain reaction‐restriction fragment length polymorphism, and mapped them using two backcross families (East Lansing (EL) and Kobe University (KU)). CAPN2 and CAPN1.5 mapped to two locations on chromosome 3 about 30 cM apart, while CAPN3 mapped to chromosome 5. CAPN1 was linked to a previously unlinked microsatellite marker LEI0140 to form a new linkage group called E66. CAPN2 and CAPN3 extend the amount of conserved synteny between chicken chromosome 3 and human chromosome 1, and between chicken chromosome 5 and human chromosome 15, respectively. Although CAPN2, CAPN3, and CAPN1.5 were found in the University of California Santa Cruz chicken genome browser gateway, CAPN1 and LEI0140 were not in specific genomic positions.     

鸡CAPN1基因3′-UTR多态性及其与肉质性状的关联分析

试验利用PCR-SSCP技术对寿光鸡CAPN1基因3′-UTR的多态性进行了检测,并进一步分析了其与肉质性状的相关性。结果显示,在寿光鸡群体中检测到AA、AB 2种基因型,基因型频率分别为0.43、0.57,AB为优势基因型;A、B等位基因频率分别为0.715、0.285,A为优势等位基因;群体中未检测到BB基因型个体。经χ2检验,群体处于Hardy-Weinberg非平衡状态(P0.05)。CAPN1基因3′-UTR的多态性对胸肌pHu、腿肌pHu、肌内脂肪含量和胸肌剪切力有显著性影响(P0.05);对腿肌剪切力、失水率、胸肌蒸煮损失和腿肌蒸煮损失无显著性影响(P0.05)。     

CAPN3基因在草原红牛不同组织中的表达差异

钙激活中性蛋白酶（CAPN3）参与哺乳动物体内多种重要的生理生化过程,但是有关它的确切生理功能和作用机制研究较少。本研究采用实时荧光定量PCR技术检测CAPN3基因在草原红牛多种组织中的表达水平差异情况。试验结果表明,CAPN3基因在所检测草原红牛的心脏、肝脏、脾脏、肺脏、肾脏、十二指肠、瘤胃和背最长肌8种组织中均有表达,且在不同组织中的表达水平有所不同。草原红牛脾脏中的CAPN3基因表达量显著高于其他组织,十二指肠和瘤胃中的CAPN3基因表达量显著高于心脏、肝脏、肺脏、肾脏和背最长肌。本试验为肉牛肉质性状的改良提供了分子遗传学依据,并为深入研究草原红牛体内CAPN3基因的生物学作用及作用机制奠定了基础。     

Gene constitution of South-East Asian native chickens, commercial chickens and jungle fowl using polymorphisms of four calpain genes

The gene constitution of polymorphisms of the four calpain genes (µ‐calpain, m‐calpain, p94, and µ/m‐calpain) were analyzed in South‐East Asian native chickens, White Leghorn and Broiler commercial chickens, and Red and Green jungle fowl. Polymorphisms were detected at all loci in chickens and Red jungle fowl, but only for CAPN1 (µ‐calpain gene) in Green jungle fowl. CAPN2 and CAPN1.5 are linked on chicken chromosome 3, and the genotype for these loci were treated as haplotype. Some combinations of calpain loci were tested using principal component analysis, and the best combination (CAPN1, CAPN3, and CAPN1.5) was determined. The proportion of polymorphic loci (P_poly) and heterozygosity (H?) were 1.00 and 0.316–0.465 in domestic chickens and red jungle fowl, and 0.33 and 0.137 in Green jungle fowl, respectively. G_ST values suggested that the degree of subdivision among native chickens was relatively low except for Thailand, which was highest. Pair‐wise F_ST testing, dendrogram and principal component analysis from the results of calpain loci showed that the four South‐East Asian native and commercial chicken populations were close genetically.     

An 8bp indel in exon 1 of Ghrelin gene associated with chicken growth

Ghrelin, acts as the endogenous ligand for growth hormone secretagogues receptor (GHS-R), is a novel growth hormone (GH) releasing peptide with reported effects on food intake in chickens. In this study, an 8 bp indel polymorphism in exon 1 of the chicken Ghrelin (cGHRL) gene was genotyped in a F(2) designed full-sib population to analyze its associations with chicken growth and carcass traits. Later, mRNA level in the proventriculus was determined by real-time PCR to reveal the expression feature of cGHRL gene. Result showed that this 8 bp indel was significantly associated with body weight at the age of 28 days (BW28) and 56 days (BW56), eviscerated weight (EW) and leg muscle weight (LMW) (P<0.05), highly significantly associated with hatch weight (HW), BW14, 21, 35, 42, 49, 90 and body length (BL), dressed weight (DW), eviscerated weight with giblet (EWG), wing weight (WW), breast muscle weight (BMW) and head and neck weight (HNW) (P<0.01). Meanwhile, A allele (with 'CTAACCTG') was positive for chicken growth as individuals with AA genotype had the highest value of all traits. Analysis on cGhrelin mRNA level revealed that it differed significantly among individuals with three genotypes (P<0.05). Individuals with AB genotype had the highest mRNA level, whereas that of AA had the lowest one. It was concluded that this 8 bp indel of cGHRL gene was significantly associated with most body weight and body composition traits, and negative effect of endogenous Ghrelin on chicken growth were indicated by this study.