Molecular cloning and tissue expression of chicken AdipoR1 and AdipoR2 complementary deoxyribonucleic acids

AdipoR1 and AdipoR2 belong to a novel class of transmembrane receptors that mediate the effects of adiponectin. We have cloned the chicken AdipoR1 and AdipoR2 complementary deoxyribonucleic acids (cDNA) and determined their expression in various tissues. We also investigated the effect of feed deprivation on the expression of AdipoR1 or AdipoR2 mRNA in the chicken diencephalon, liver, anterior pituitary gland, and adipose tissue. The chicken AdipoR1 and AdipoR2 cDNA sequences were 76-83% identical to the respective mammalian sequences. A hydrophobicity analysis of the deduced amino acid sequences of chicken AdipoR1/AdipoR2 revealed seven distinct hydrophobic regions representing seven transmembrane domains. By RT-PCR, we detected AdipoR1 and AdipoR2 mRNA in adipose tissue, liver, anterior pituitary gland, diencephalon, skeletal muscle, kidney, spleen, ovary, and blood. AdipoR1 or AdipoR2 mRNA expression in various tissues was quantified by real-time quantitative PCR, and AdipoR1 mRNA expression was the highest in skeletal muscle, adipose tissue and diencephalon, followed by kidney, ovary, liver, anterior pituitary gland, and spleen. AdipoR2 mRNA expression was the highest in adipose tissue followed by skeletal muscle, liver, ovary, diencephalon, anterior pituitary gland, kidney, and spleen. We also found that a 48 h feed deprivation significantly decreased AdipoR1 mRNA quantity in the chicken pituitary gland, while AdipoR2 mRNA quantity was significantly increased in adipose tissue (P<0.05). We conclude that the AdipoR1 and AdipoR2 genes are ubiquitously expressed in chicken tissues and that their expression is altered by feed deprivation in the anterior pituitary gland and adipose tissue.     

IGFBP-3基因在两个品种鸡组织中的表达及其与生长性状的相关性分析

为研究IGFBP-3基因表达与鸡生长性状的相关性,以生长速度差异较大的花山麻鸡和清远麻鸡两个黄羽肉鸡品种为研究素材,用实时荧光定量PCR法检测胚胎期和出雏后两个品种鸡胸肌和肝脏中IGFBP-3基因的表达规律,并将其与体重、胸肌重和肝脏重进行相关性分析。结果表明,在9胚龄时两个品种鸡胸肌和肝脏中均可检测到IGFBP-3基因表达,同一胚龄或日龄品种内组织间比较,胚胎期两个品种鸡胸肌IGFBP-3 mRNA表达量均高于肝脏,出雏后肝脏IGFBP-3 mRNA表达量迅速上升,胸肌IGFBP-3 mRNA表达量下降,两个品种鸡肝脏IGFBP-3 mRNA表达量均极显著高于胸肌（P<0.01）;组织中IGFBP-3 mRNA表达量与组织重量和体重相关研究表明,两个品种鸡肝脏IGFBP-3 mRNA表达量与其胸肌重、肝脏重和体重呈显著或极显著正相关（P<0.05;P<0.01）,胸肌IGFBP-3 mRNA表达量与其胸肌重、肝脏重和体重均呈极显著负相关（P<0.01）。研究结果揭示,IGFBP-3 mRNA在鸡中的表达具有品种、年龄和组织特异性,出雏前肝脏并不是鸡产生IGFBP-3的主要器官,出雏后鸡IGFBP-3可能主要由肝脏合成,肝脏中IGFBP-3 mRNA水平差异是导致两个品种鸡出雏后体重和胸肌重差异的重要因素。     

树鼩Tssk6基因cDNA克隆、组织表达谱及生物信息学分析

本研究旨在克隆出树鼩Tssk6基因cDNA的全长序列,阐明其组织表达谱及基本生物信息学特征。以GenBank中收录的树鼩Tssk6基因mRNA预测序列为参考设计特异性引物,对树鼩Tssk6基因的cDNA全长序列进行克隆,用实时荧光定量PCR检测该基因在树鼩不同组织中的表达情况,并对cDNA序列的CDS区核苷酸序列与蛋白质结构进行了生物信息学分析。结果显示,树鼩Tssk6基因cDNA的CDS区序列长度为822 bp,编码273个氨基酸;Tssk6基因仅表达于树鼩睾丸组织;对树鼩和其他8种哺乳动物Tssk6基因cDNA的CDS区核苷酸序列构建的系统进化树显示,树鼩与人、黑猩猩、恒河猴3种灵长类动物亲缘关系较近,与小鼠、大鼠、金仓鼠3种啮齿类动物亲缘关系较远。本研究为进一步研究树鼩Tssk6基因的功能奠定了基础。     

cDNA Cloning,Tissue Expression Profiles and Bioinformatical Analysis of Tssk6 Gene in Tree Shrews

The purpose of this study was to clone the full-length cDNA of tree shrew's Tssk6 gene, to elucidate its tissue expression profiles and fundamental bioinformatical characteristics. In the present study, the full-length cDNA of tree shrew's Tssk6 gene was cloned with specific primers on the basis of predicted mRNA sequence of tree shrew's Tssk6 gene in GenBank, the mRNA expression of Tssk6 gene in various tree shrew's tissues was investigated via Real-time PCR. In addition, the bioinformatics on nucleotide sequence of CDS region of Tssk6 cDNA in tree shrew as well as putative protein structure had been analyzed. The results showed that:The CDS region of tree shrew's Tssk6 cDNA contained 822 bp encoding 273 amino acids; Tssk6 gene specifically expressed in tree shrew's testis; Based on the molecular phylogenetic tree constructed with CDS of Tssk6 cDNA among tree shrew and other eight mammalian species, it was inferred that tree shrew was genetically close to primates like human, chimpanzee and rhesus monkey, while it was genetically far to rodents like mouse, rat and golden hamster. This study would lay a foundation for the further study of Tssk6 gene function in tree shrew.     

白来航鸡半乳糖凝集素-1基因克隆、生物信息学及组织表达特性分析

【目的】克隆白来航鸡半乳糖凝集素-1(galectin-1,Gal-1)基因,对其编码蛋白进行生物信息学分析,并检测其在不同组织中的表达情况,为进一步阐明其抗病毒功能提供科学依据。【方法】以鸡脾脏cDNA为模板,通过PCR扩增鸡Gal-1基因完整CDS区序列,并进行相似性比对及系统进化树构建;运用生物信息学软件对其编码蛋白的理化性质、亲/疏水性、跨膜区、信号肽、修饰结构、保守结构域及高级结构进行预测。利用实时荧光定量PCR检测Gal-1基因在白来航鸡心脏、肝脏、脾脏、肺脏、肾脏、脑、腺胃、肌胃、十二指肠、空肠、盲肠、直肠、胸肌和腿肌组织中的表达情况。【结果】白来航鸡Gal-1基因CDS区序列长度为408 bp,编码135个氨基酸。相似性比对结果表明,白来航鸡Gal-1基因核苷酸序列与火鸡、绿头鸭和珍珠鸟的相似性分别为97.1%、88.4%和82.2%;系统进化树结果表明,白来航鸡与火鸡亲缘关系最近。Gal-1蛋白分子质量为15.06 ku,理论等电点为6.57,不稳定系数为36.05,脂肪系数为74.30,平均亲水指数为-0.259。Gal-1蛋白无信号肽,不存在跨膜区;存在2个明显的亲水区,其编码蛋白较稳定,为亲水性蛋白。二级结构预测显示,Gal-1蛋白以无规则卷曲(45.93%)和延伸链(41.48%)为主,三级结构预测结果与二级结构一致。实时荧光定量PCR结果显示,Gal-1基因mRNA在白来航鸡组织中广泛表达,在肺脏中表达量最高,在脑中表达最低。【结论】本研究成功克隆了白来航鸡Gal-1基因CDS区序列,Gal-1基因在白来航鸡心脏、肝脏等14种组织中广泛表达,结果可为鸡Gal-1蛋白功能的深入研究提供参考。     

巨型玫瑰冠鸡H-FABP、A-FABP基因表达的发育性变化及其肌内脂肪含量研究

为探讨心脏型脂肪酸结合蛋白（heart-type fatty acid-binding protein，H-FABP）和脂肪型脂肪酸结合蛋白（adipocyte fatty acid-binding protein，A-FABP）基因与巨型玫瑰冠鸡生长发育及肌内脂肪含量（IMF）的关系，研究H-FABP、A-FABP基因对巨型玫瑰冠鸡IMF含量和腹脂沉积的作用机制，本试验采集了巨型玫瑰冠鸡与良凤花鸡在不同周龄（2、4、6、8、10、12周龄）的腹脂、心肌、胸肌、腿肌组织样品共480份，采用实时荧光定量PCR对巨型玫瑰冠鸡与良凤花鸡不同生长阶段组织中H-FABP、A-FABP基因mRNA表达量进行了检测，并采用索氏浸提法测定了两种鸡12周龄的胸肌与腿肌的IMF含量。结果表明，巨型玫瑰冠鸡与良凤花鸡的H-FABP、A-FABP基因mRNA在腹脂、心肌、胸肌和腿肌组织中均有不同程度的表达，且两种鸡H-FABP基因mRNA在心肌组织中高度表达，在脂肪组织中表达量较低，在胸肌、腿肌组织中中度表达，而A-FABP基因相反，推测H-FABP基因主要在肌肉组织中表达，而A-FABP基因主要在脂肪组织中表达。良凤花鸡生长速度高于巨型玫瑰冠鸡；巨型玫瑰冠鸡的腹脂率均低于同性别的良凤花鸡，但胸肌、腿肌的IMF均高于同性别的良凤花鸡，表明巨型玫瑰冠鸡的肉品质优于良凤花鸡。本试验结果为巨型玫瑰冠鸡H-FABP、A-FABP基因分子选育奠定了基础。     

Expression and Distribution Difference Analysis of STC-1 Gene in Big and Small Body Shape Pigs

The heart,liver,spleen,lung,kidney,skull,skeletal muscle were collected from 40-day-old Bama Mini pig and Large White pig,stanniocalcin 1(STC-1) gene mRNA and STC-1 protein were detected with Real-time PCR and Western blotting methods, respectively. The Real-time PCR results showed that the expression level of STC-1 gene mRNA was higher in lung and kidney, but was the lowest in skeletal muscle of Bama Mini pig and Large White pig. Except for heart and skeletal muscle tissues, the expression level of STC-1 gene mRNA in other tissues of Bama Mini pig was significantly higher than Large White pig (P < 0.05).The Western blotting results showed that the STC-1 protein distribution were the highest in liver and spleen of Bama Mini pig and Large White pig, respectively, and the difference was significant (P < 0.05). The STC-1 protein expression in lung, liver, skeletal muscle and heart of Bama Mini pig were extremely significantly higher than Large White pig (P < 0.01),and the STC-1 protein expression in kidney and spleen of Bama Mini pig were extremely significantly lower than Large White pig (P < 0.01). STC-1 gene mRNA and protein were firstly detected in different tissues from big and small body shape pigs,this might have a relationship with various external environments stress and development.     

STC-1基因在大、小体型猪中表达与分布的差异分析

试验分别采集40日龄小体型猪（巴马猪）和大体型猪（大白猪）的心脏、肝脏、脾脏、肺脏、肾脏、头骨、骨骼肌组织,利用实时荧光定量PCR检测斯钙素-1（stanniocalcin 1,STC-1）基因mRNA在各个组织中的表达水平,并通过Western blotting检测STC-1蛋白在各个组织中的分布。实时荧光定量PCR检测结果表明,STC-1基因mRNA在巴马猪和大白猪肺脏、肾脏中相对表达水平较高,在骨骼肌中的表达水平最低;除心脏和骨骼肌外,巴马猪其余各组织中STC-1基因mRNA表达水平均显著高于大白猪（P < 0.05）。Western blotting检测结果表明,巴马猪肝脏中STC-1蛋白的表达量最高,而大白猪脾脏中STC-1蛋白表达量最高,两者差异显著（P < 0.05）;巴马猪肺脏、肝脏、骨骼肌及心脏组织中STC-1蛋白表达量均极显著高于大白猪（P < 0.01）;而巴马猪肾脏、脾脏中STC-1蛋白表达量极显著低于大白猪（P < 0.01）。本研究首次对大、小体型猪不同组织的STC-1基因mRNA表达水平及其STC-1蛋白分布进行检测,导致该基因表达与分布差异的原因可能与两种猪受外界环境应激及生长发育差异有关。     

鸡Smad4基因克隆、生物信息学及组织表达分析

试验旨在克隆鸡Smad4基因，并对其进行生物信息学和组织表达分析。以苏禽3号优质黄羽肉鸡为试验对象，使用RACE技术扩增并克隆了鸡Smad4基因全长序列，对其进行生物信息学分析，构建系统进化树，并利用实时荧光定量PCR检测了Smad4基因在鸡各组织中的表达情况。结果显示，鸡Smad4基因全长序列包含17 bp的5'UTR、1 842 bp的开放阅读框和466 bp的3'UTR，有11个外显子和10个内含子，mRNA序列全长2 325 bp，可编码613个氨基酸。系统进化树显示，鸡Smad4与其他鸟类聚为一类。生物信息学分析显示，Smad4蛋白分子质量为65.43 ku，理论等电点为10.04，为亲水蛋白；含有2个保守结构域：MH1和MH2；二级结构由α-螺旋（18.11%）、延伸链（17.29%）和无规则卷曲（64.60%）组成。组织表达分析表明，Smad4基因在鸡的心脏、肝脏、空肠、卵巢中均有较高表达，而在胸肌中不表达。本试验成功获得了鸡Smad4基因全长序列，并初步研究了其组织表达规律，为进一步研究Smad4基因在鸡繁殖活动中的分子机制提供了理论依据。     

鸡CSRP3基因编码区SNPs及其与肌纤维性状的相关分析

CSRP3基因是肌细胞分化所必需的正向调节因子,在肌肉生长发育过程中发挥重要作用。研究以220只63日龄(上市日龄)"花山麻鸡"为研究素材,应用PCR扩增与测序首次扫描了鸡CSRP3基因编码区SNPs,并将其与腓肠肌外侧头肌纤维平均面积、直径和密度等肌纤维性状进行关联分析。结果显示:在鸡CSRP3基因编码区共发现G180A、C240T、G246A、C252T、C546T、A633G 6个SNPs,均为同义突变;相关分析表明,A633G位点的多态性与花山麻鸡腓肠肌外侧头肌纤维平均面积存在极显著的基因型效应(P<0.01),与肌纤维平均直径、肌纤维平均密度存在显著的基因型效应(P<0.05)。结果表明,CSRP3基因可作为影响鸡骨骼肌生长发育的候选基因,可选择CSRP3基因编码区的633位点作为鸡骨骼肌肌纤维性状的分子标记辅助选择的候选标记。     

山地乌骨鸡不同组织中CAPN 1基因表达的发育性变化

本试验采用SYBR GreenⅠ荧光定量法,首次测定了四川山地乌骨鸡1d、14d、28d、42d、56d、70d、84d的胸肌、腿肌、肝脏、心、脑组织中钙蛋白酶1CAPN1基因的相对表达量。结果表明：CAPN1基因在鸡体内普遍存在,CAPN1基因在肌肉组织中的表达量与鸡肌肉组织的生长发育呈相似的变化规律。     

鸡HMIT基因的克隆与表达分析

旨在克隆鸡H+/肌醇转运蛋白（H+/myoinositol transporter，HMIT）基因的转录序列，并检测HMIT基因在鸡不同组织中的表达情况，以及外源胰岛素处理对HMIT基因相对表达量的影响。本研究以AA肉鸡为试验动物，采用RT-PCR技术克隆鸡HMIT基因全编码区序列，采用生物信息学技术分析其编码蛋白的生物学特性，利用荧光定量PCR检测HMIT基因在大脑、肝、腹脂、腿肌、心、肾、空肠和回肠等组织中的表达情况，并检测其在肾和大脑中进行胰岛素处理120和240 min后的表达情况。结果表明，以NCBI预测的鸡HMIT（XM_001232939.5）序列为模板设计引物，成功克隆出鸡HMIT基因（1 694 bp，GenBank登录号：MN708211）。克隆的鸡HMIT编码区全长1 380 bp，相比XM_001232939.5预测的编码序列少561 bp，预测编码含有459个氨基酸组成的蛋白。核苷酸和氨基酸序列比对发现，鸡HMIT在物种间高度同源，共线性分析显示，HMIT所在的1号染色体区域在物种间也是高度保守的。HMIT编码的蛋白质是一种不稳定的亲水性蛋白质，其二级结构和三级结构以无规则卷曲和α-螺旋为主。跨膜结构分析显示，鸡HMIT存在8个明显的跨膜结构域。亚细胞定位结果表明，鸡HMIT蛋白分布于质膜（52.3%）、内质网（21.7%）、液泡（17.4%）、线粒体（4.3%）和高尔基体（4.3%）。实时荧光定量PCR检测结果显示，HMIT基因在鸡大脑和肾表达量最高，且显著高于其他组织（P<0.05）。胰岛素处理240 min后，HMIT在肾和大脑中的表达量都显著低于PBS对照组（P<0.05）。本研究克隆获得了鸡HMIT的全编码区，生物信息学分析及组织表达特性研究为深入探索鸡HMIT基因的生理功能和调控机制奠定基础。     

利用实时荧光定量RT-PCR检测鸡A-FABP和H-FABP基因的差异表达

依据鸡A-FABP和H-FABP基因及参照基因GAPDH序列设计引物,以鸡心肌、胸肌和腿肌、肝脏和腹脂总RNA逆转录合成的cDNA为模板,GAPDH基因为参照基因,应用SYBRGreenI实时荧光定量PCR检测如皋黄鸡和安卡红鸡A-FABP和H-FABP基因的差异表达。结果表明:A-FABP、H-FABP基因和参照基因GAPDH基因融解曲线为单峰,无杂峰及二聚体,其扩增效率分别为99.7%、99.8%和100.0%。如皋黄鸡H-FABP基因在心肌、胸肌和腿肌的差异表达量分别是安卡红鸡的0.0860、0.0680倍和0.0580倍(P0.05)。H-FABP基因mRNA表达量与肌内脂肪含量呈显著负相关。如皋黄鸡A-FABP基因在腹脂和肝脏的表达量分别是安卡红鸡的15.9640倍和10.9640倍(P0.05),而在心肌、胸肌和腿肌的表达量差异不显著。     

Characterisation of glucose transporter (GLUT) gene expression in broiler chickens

1. Glucose transporter (GLUT) proteins, one of which is the major insulin-responsive transporter GLUT4, play a crucial role in cellular glucose uptake and glucose homeostasis in mammals. The aim of this study was to identify the extent of mRNA expression of GLUT1, GLUT2, GLUT3 and GLUT8 in chickens intrinsically lacking GLUT4. 2. GLUT1 mRNA was detected in most tissues of 3-week-old broiler chickens, with the highest expression measured in brain and adipose tissue. GLUT2 was expressed only in the liver and kidney. GLUT3 was highly expressed in the brain. GLUT8 was expressed ubiquitously, with expression in kidney and adipose tissue relatively higher than that of other tissues. 3. Expression levels of GLUT isoforms 1, 3 and 8 in skeletal muscle tissue were very low compared to the other tissues tested. 4. [3H]Cytochalasin B binding assays on tissue from 3-week-old chickens showed that the number of cytochalasin B binding sites in skeletal muscle plasma membranes was higher than in liver plasma membranes. These results suggest that GLUT proteins and/or GLUT-like proteins that bind cytochalasin B are expressed in chicken skeletal muscles. 5. It is proposed that GLUT expression and glucose transport in chicken tissues are regulated in a manner different from that in mammals.     

猪ADAR2基因全长cDNA克隆、序列特征及表达模式分析

旨在克隆猪作用于RNA的腺苷脱氨酶2基因（ADAR2）全长cDNA序列,同时对该基因在猪不同组织中的表达规律进行探索。利用RACE （rapid-amplification of cDNA ends）对大白猪ADAR2基因mRNA全长序列进行克隆,并进行生物信息学分析;用荧光定量PCR方法检测35日龄大白猪心、肝、肺、肾、脾、脑、小肠、背最长肌和背部脂肪9种组织中ADAR2的表达水平。结果表明,猪ADAR2基因cDNA全长6 305 bp,共包含12个外显子,编码704个氨基酸,与人、黑猩猩、猕猴、长臂猿、黄牛、山羊和绵羊的CDS区核酸序列和氨基酸序列的一致性均在84%以上。该基因编码的蛋白含有2个双链RNA结合基序和一个脱氨酶结构域。猪ADAR2在检测的各组织中均表达,其中在肺中的表达量最高。综上所述,本研究成功克隆了猪ADAR2基因全长cDNA序列,并且发现其在猪体内广泛表达,为深入研究ADAR2的功能奠定了良好的基础。