山羊BMPR-IB基因编码蛋白结构及功能的生物信息学分析

试验旨在利用生物信息学方法分析山羊骨形态发生蛋白受体IB（bone morphogenetic protein receptor IB,BMPR-IB）基因编码蛋白的结构及功能。本研究通过在线软件预测和分析山羊BMPR-IB基因编码蛋白的一级结构、二级结构、亚细胞定位、三级结构、重要的功能基序及其分类,通过最大似然法对不同物种BMPR-IB基因编码蛋白序列构建系统发育树,进行系统发育分析。结果发现,山羊BMPR-IB基因共编码502个氨基酸,其编码蛋白属于不稳定的亲水性蛋白质。二级结构以β折叠为主,比例为63.5%,主要存在于细胞核中,在线粒体、细胞质、囊泡分泌系统和质膜中也有少量分布。该蛋白主要发挥嘌呤和嘧啶、监管、运输和结合、信号转导等功能。在127-149氨基酸位置存在1个跨膜结构域;在174-203、204-494氨基酸位置存在2个高度保守的基序,分别为GS结构域和蛋白激酶结构域。三级结构由sheet片段结构富集域和helix螺旋结构富集域组成。不同物种BMPR-IB基因系统发育分析结果与动物学分类结果一致,提示BMPR-IB基因与繁殖力性状存在联系。通过不同的在线预测软件分析了山羊BMPR-IB基因编码蛋白的结构及功能,为深入研究BMPR-IB基因提供理论指导。     

水貂AANAT基因克隆及生物信息学分析

【目的】试验旨在对美洲水貂(Neovison vison)褪黑素(N-acetyl-5-methoxytryptamine, MT)合成的限速酶5-羟色胺-N-乙酰基转移酶(aralkylamine N-acetyltransferase, AANAT)基因进行克隆和生物信息学分析,为探究AANAT基因在水貂中的生物学功能提供参考。【方法】对水貂尾静脉采血后提取DNA,利用同源重组的方法克隆AANAT基因并分析其编码区(CDS)序列,推导成氨基酸序列后进行相似性比对及系统进化树构建,并对AANAT蛋白进行生物信息学分析。【结果】水貂AANAT基因序列长约1 631 bp, CDS区长504 bp,可编码167个氨基酸。水貂AANAT氨基酸序列与雪貂相似性最高(98.2%),系统进化树分析也显示其与雪貂亲缘关系最近。生物信息学分析结果表明,水貂AANAT蛋白为疏水性蛋白,无跨膜结构域和信号肽,主要分布于细胞质中,有5个抗原结合位点为非分泌型蛋白,该蛋白含有多个磷酸化、糖基化等翻译后修饰位点。AANAT蛋白含有1个保守的N-酰基转移酶超家族结构域,该家族与哺乳动物的昼夜节律密切相关。AAN...     

水貂酪氨酸酶基因外显子1的鉴定及序列变异分析

根据GenBank中公布的雪貂(登录号:EF405957)、大熊猫(登录号:XM_002930310)、家犬(登录号:CFU42219)及猫(登录号:AY959314)的酪氨酸酶(tyrosinase, TYR)基因DNA序列保守区域,设计1对特异性引物,利用PCR及克隆技术测定了短毛黑水貂、红眼白水貂和米黄色水貂的TYR基因外显子1部分序列,并对该序列进行了BLAST鉴定及变异位点分析,基于该基因编码氨基酸序列构建了16个物种的系统进化树。结果表明,测定的3种毛色水貂TYR基因序列长度均为796 bp,包括795 bp的外显子1和1 bp 5'UTR,共检测到5个单一突变位点:g.A34G、g.T138A、g.T248C、g.A545G和g.C765A,包含4个错义突变:g.A34G(p.Ser12Gly)、g.T248C(p.Val83Ala)、g.A545G(p.Tyr182Cys)和g.C765A(p.His255Gln),1个移码突变分别出现在短毛黑水貂和红眼白水貂个体间:g.T138A(p.Cys46*),获得TYR基因GenBank登录号分别为:短毛黑水貂(KJ198847)、红眼白水貂(KJ658343)和米黄色水貂(KJ152769)。水貂与雪貂、大熊猫、家犬、猫、家兔、猪、羊驼、山羊、绵羊、牛、人、猕猴、黑猩猩、原鸡和日本鹌鹑的TYR基因核苷酸序列同源性为72.8%～98.6%,相应氨基酸序列同源性为75.0%～98.1%。TYR基因分子进化树显示水貂与雪貂遗传关系最近,分化时间约为1000万年前,且与原鸡和日本鹌鹑亲缘关系最远。该结果为进一步开展TYR基因多态性与水貂毛色表型的相关性研究奠定了生物信息学基础。     

水貂PRLR基因的克隆及生物信息学分析

本研究旨在从水貂卵巢组织中克隆催乳素受体(prolactin receptor,PRLR)基因编码区全长序列,并进行生物学信息分析,为研究其结构和功能奠定基础。根据GenBank中登录的犬PRLR基因的mRNA预测序列(登录号:XM_025436332.1)设计1对引物,采集性成熟的健康水貂卵巢组织样本,提取RNA并反转录合成cDNA,以cDNA为模板PCR扩增PRLR基因,将其插入pEASY-Blunt Simple Vector中,筛选阳性克隆测序后进行生物信息学分析。结果显示,水貂PRLR基因编码区序列全长为1 878bp,编码了625个氨基酸,水貂PRLR基因核苷酸序列与海獭同源性最高,为97.7%。系统进化树分析也显示其和海獭亲缘关系最近。对水貂卵巢PRLR基因编码蛋白进行生物信息学分析表明,PRLR蛋白含有1个信号肽,1个细胞外区域,1个跨膜区和1个膜内区;水貂PRLR蛋白为单次跨膜蛋白,其N端的D1部分含有2对保守的二硫键(Cys36—Cys46和Cys75—Cys86),D2部分含有保守的WS-基序(WS-E-WS)。水貂PRLR蛋白具有和其他PRLR蛋白相似的膜外和膜内结构。本试验结果为进一步研究和揭示水貂PRLR蛋白的结构及功能奠定了基础。     

水貂酪氨酸酶(TYR)基因克隆、SNPs筛查及其皮肤组织mRNA差异表达分析

旨在克隆和分析水貂酪氨酸酶(tyrosinase,TYR)基因编码区,揭示该基因编码区SNPs及其皮肤组织mRNA差异表达规律与水貂毛色表型的关系.本研究采集7月龄雄性金州黑水貂、名威银蓝水貂和红眼白水貂共计301个样本的血液,利用聚合酶链式反应(PCR)方法对水貂TYR基因5个外显子进行分段克隆,并拼接获得其完整编码...     

水貂阿留申病毒结构蛋白与非结构蛋白的研究进展

水貂阿留申病毒(Aleutian mink disease virus,ADV)是一种主要侵染水貂的自主复制型细小病毒,是一种在水貂中广泛存在的重要病原体。病毒粒子的蛋白分为结构蛋白(VP1、VP2)和非结构蛋白(NS1、NS2)两类。VP1蛋白对病毒粒子产生感染性有重要作用;VP2蛋白是主要免疫功能区,能刺激机体产生中和抗体;NS1和NS2主要参与病毒的复制和基因的表达调节。文中对近年来国内外学者关于水貂阿留申病毒结构蛋白和非结构蛋白的研究情况进行归纳和总结。     

牛GDF8基因编码蛋白结构和功能预测

旨在探究GDF8基因（Growth differentiation factor 8）编码蛋白的结构和功能,对后期研究其生理机制提供理论依据。利用生物信息学技术对牛GDF8蛋白二级和三级结构进行了预测,分析了保守结构域、信号肽剪切位点、理化性质、亲疏水性、亚细胞定位和蛋白网络通路。结果显示,牛GDF8蛋白与人和小鼠具有相似的保守结构域,不稳定指数85.52,属于不稳定蛋白,其亲水性氨基酸明显多于疏水性氨基酸,为亲水性蛋白;牛GDF8蛋白在内质网分布的概率44.4%,在线粒体分布的概率33.3%,二级结构以无规则卷曲为主,折叠缠绕后形成三级结构;牛GDF8蛋白PPI网络图有11个节点,其KEGG通路主要与骨骼肌纤维发育、活化素受体信号通路、成肌细胞融合等相关。综上,推测GDF8蛋白存在于内质网和线粒体中,通过与亲水性蛋白结合调节机体生物学功能。     

水貂TYR基因T138A位点多态性及其与毛色性状的关联分析

试验旨在检测水貂酪氨酸酶（tyrosinase,TYR）基因T138A位点的多态性,并分析其与水貂毛色表型的相关性。提取5种被毛色型430只水貂血液基因组DNA,采用PCR-RFLP技术,对TYR基因T138A位点进行多态性检测,统计等位基因频率与基因型频率,通过卡方（χ²）独立性检验分析该位点多态性与水貂毛色性状的相关性。结果表明,T138A位点存在2个等位基因T和A,形成TT、TA和AA 3种基因型,AA基因型在吉林白水貂群体中为优势基因型（0.9069）,而TT基因型为金州黑水貂、珍珠水貂、咖啡水貂和银蓝水貂群体的优势基因型,其中在金州黑水貂群体中基因型频率最高（1.0000）。关联分析表明,TYR基因T138A位点的多态性与毛色性状呈极显著相关（P<0.0001）。表明TYR基因T138A位点可能是影响水貂毛色的主控位点或与调控白色被毛表型主控位点连锁的分子标记。     

黔北麻羊TYR基因多态性及生物信息学分析

为研究黔北麻羊毛色形成的遗传机制,本实验对黔北麻羊哺乳动物酪氨酸酶(TYR)基因外显子区域运用混合DNA池结合PCR产物直接测序方法进行多态性分析,在TYR基因外显子区域共筛选出5个SNPs,分别为Exon1-G617A、Exon1-G685T、Exon3-C1236T(Asn-Lys)、Exon5-C1578T(Gly-Glu)、Exon5-T1862C;进而利用生物信息学分析软件对PCR扩增所获序列进行m RNA二级结构及蛋白质二级结构和三级结构分析,发现Exon3-C1236T(Asn-Lys)、Exon5-C1578T(Gly-Glu)引起m RNA二级结构的改变,其中Exon1-G685T、Exon3-C1236T(Asn-Lys)、Exon5-T1862C突变导致其m RNA二级结构最小自由能增大,即二级结构稳定性降低,其错义突变位点Exon3-C1236T(Asn-Lys)、Exon5-C1578T(Gly-Glu)均引起蛋白质二级结构和三级结构的改变。     

中国黄牛酪氨酸关联蛋白1（TYRP1）基因多态性的研究

[目的]验证TYRP1基因的错义突变(g.1300C>T)与中国黄牛毛色性状是否存在相关性。[方法]采用PCR测序及生物信息学的方法,对991头中国黄牛、48头安格斯牛、104头婆罗门牛的TYRP1基因进行多态性分析。[结果]通过对中国黄牛的等位基因频率和基因型频率的分析,发现此变异与中国黄牛棕色毛色性状不相关。[结论]TYRP1基因的错义突变(g.1300C>T)与中国黄牛棕色表型不相关。该位点可能在一些品种中受到了选择,中国黄牛的棕色表型可能受其他基因调控。     

Age‐dependent baseline values of faecal cortisol metabolites in the American mink (Neovison vison) under semi‐natural housing conditions

The welfare of an animal is ensured if it is able to fully satisfy its essential species‐typical needs in all functional aspects of behaviour. In mink, stereotypies and apathy, internal and/or external injuries as well as increased susceptibility to disease have been known to occur as a result of chronic stress. The non‐invasive method of analysing faecal cortisol metabolites (FCM) allows conclusions to be drawn about the stress level in the respective housing system. The objective of this study is to find out how the cortisol metabolites content in the faecal changes with increasing age of the mink under semi‐natural housing conditions. Thus, 40 American mink (Neovison vison) were housed in two outdoor enclosures imitating natural conditions. Throughout the entire study (13th to 32nd week of life), faecal samples were collected to measure cortisol metabolites. No differences in FCM concentrations between the two outdoor enclosures were found. In the young mink lower, less fluctuating FCM levels were found than in older animals. After the first faecal collection in the 13th/14th week of life, the level of metabolites decreased slightly (p = 0.032; 17th/18th week). From the 22nd/23rd week onwards until the 30th/31st week, shortly before the animals were pelted, continuously increasing concentrations were then measured. Increasing FCM levels with advancing age of the animals are probably attributable to the onset of sexual maturity and/or the respective season. This has to be taken into account in future studies using this method for assessing welfare and when comparing different mink housing systems.     

水貂ASIP基因单倍型检测及其皮肤组织mRNA差异表达分析

试验旨在探究鼠灰色（agouti signaling protein,ASIP）基因单核苷酸多态性（single nucleotide polymorphisms,SNPs）形成的单倍型及皮肤组织差异表达mRNA对水貂被毛色素沉积的影响。通过PCR扩增、Sanger测序技术对金州黑水貂、红眼白水貂和名威银蓝水貂ASIP基因进行SNPs单倍型检测分析,利用实时荧光定量PCR技术检测3种毛色皮肤组织ASIP基因的表达量,分析单倍型及mRNA差异表达与毛色表型的相关性。结果表明,301个样本中共检测到10个SNPs,内含子2中4个SNPs （G18A、A159G、G235T、C1189T）共形成10种单倍型（Hap1~Hap10）,其中Hap1（GAGC）和Hap2（GAGT）是3种不同毛色水貂群体的共享单倍型;部分内含子3中6个SNPs （C252T、A290C、G298C、A340G、T343C、T379C）形成4种单倍型（Hap1~Hap4）,且Hap2（CCCGCC）是名威银蓝水貂群体的主体单倍型。5个位点（A290C、G298C、A340G、T343C、T379C）均处于完全连锁不平衡状态。实时荧光定量PCR检测显示,金州黑水貂和名威银蓝水貂ASIP基因mRNA表达量分别是红眼白水貂的1.25和0.95倍,三者间差异不显著（P>0.05）。研究结果初步提示,ASIP基因调控水貂不同毛色表型形成的分子机制可能存在差异。     

猫泛白细胞减少症病毒北京株NS1基因克隆与生物信息学分析

为分析猫泛白细胞减少症病毒（FPV）北京株（FPV-BJ 05株）NS1基因的分子特征及其编码蛋白的生物学功能,本研究对FPV-BJ 05株NS1基因进行PCR扩增、克隆及序列测定。应用生物信息学软件分析FPV-BJ 05株与GenBank上登录的11株FPV参考株NS1基因的同源性,并预测NS1蛋白理化性质、信号肽、跨膜结构、B细胞抗原表位、磷酸化位点、亚细胞定位及蛋白结构与功能、高级结构等。结果显示,NS1基因全长2 007 bp,编码668个氨基酸,且与其他FPV分离株NS1基因核苷酸序列同源性为98.8%~99.3%,氨基酸序列同源性为97.9%~98.8%。系统进化树分析结果显示,FPV-BJ 05株NS1蛋白与GenBank上登录的3株FPV参考株处于同一大分支,但由于氨基酸的突变导致其属于独立的一小分支。NS1蛋白既无信号肽也无跨膜结构域,属于非分泌型的疏水性蛋白。B细胞抗原表位预测结果显示,NS1蛋白柔韧性较好,抗原性及表面可及性较高,预测该蛋白含有13个优势抗原位点。修饰结构预测表明,NS1蛋白含有62个潜在磷酸化位点,27个O-糖基化位点和2个N-糖基化位点。亚细胞定位结果显示,NS1蛋白在细胞质和细胞核的概率较高,分别为69.6%和17.4%。NS1蛋白二级结构中α-螺旋、延伸链、无规则卷曲和β-转角分别占39.37%、15.42%、39.37%和5.84%。应用在线软件SWISS-Modle对NS1蛋白进行建模,预测其三级结构,结果发现NS1蛋白主要以α-螺旋为主。本试验结果将为北京地区FPV免疫诊断及核酸疫苗研究提供理论依据。     

Phylogenetic Analysis and Detection of Positively Selected Site on the Coding Regions of TYRP1 Gene

Tyrosinase-related protein 1 (TYRP1) is one of the tyrosinase family members, which was also the first successfully cloned pigment gene.The study aimed at exploring the selective pressure on TYRP1 gene in the process of biological evolution.The study used NCBI to download nucleotide and protein sequences of TYRP1 gene coding regions in different species, through the Clustal X to compare TYRP1 gene encoding protein sequences, using Pal2nal online tool to generate codon multiple sequence alignment, DAMBE software was used to determine the base substitution saturation for constructing phylogenetic trees, using maximum likelihood method and PAML software to analyse TYRP1 gene in different species phylogenetic relationships and selection pressure.The results showed as follows:TYRP1 gene evolution and evolutionary classification of mammals was most close, and the site specific model revealed that TYRP1 genes were mainly affected by purifying selection, and it had been subjected to positive selection in its adaptive evolution, the software found thirteen positive selection amino acid sites.The study analyzed the evolutionary pattern of the TYRP1 gene from the molecular evolutionary perspective, providing a new way for the study of the TYRP1 gene structure and function.     

酪氨酸相关蛋白酶1基因编码区系统发育分析和正选择位点检测

酪氨酸相关蛋白酶1(tyrosinase-related protein 1,TYRP1)是酪氨酸酶家族成员之一,也是第一个成功克隆的色素基因。为了探究选择压力对TYRP1基因在生物进化过程中的影响。本研究利用NCBI下载不同物种TYRP1基因编码区核苷酸及其蛋白序列,通过Clustal X对TYRP1基因编码蛋白序列进行比对,利用Pal2nal在线工具生成密码子多序列比对,DAMBE软件判断该碱基替代饱和度适合构建系统树后,利用最大似然法和PAML软件分别分析不同物种TYRP1基因的亲缘关系和选择压力。结果表明,TYRP1基因进化与哺乳动物的进化分类最为接近,位点模型检测表明TYRP1基因在适应性进化中主要受负选择的约束,而且在进化过程中曾承受正选择作用,共检测到13个正选择位点。本研究从分子进化的角度分析了TYRP1基因的生物进化模式,为该基因结构和功能的研究提供了新的思路。     

Effects of different dietary protein levels and DL‐methionine supplementation on hair growth and pelt quality in mink (Neovision vision)

The effect of different dietary protein levels and DL‐methionine (Met) supplementation on hair growth and the resulting pelt quality in mink was studied. Four groups of male mink were fed with four isocaloric diets containing 32% (P32), 24% (P24), 16% (P16) or P24+Met (0.8%) crude protein of dry matter (DM) from September to December. Skin biopsies were taken at the pelting. Histological techniques and computer‐assisted light microscopy were used to determine the ratio of activity (ROA) of under hairs and guard hairs respectively. The results showed that when the dietary protein level reduced from 32% to 16%, body length, number and diameter of under hairs and guard hairs of minks declined, and pelt length and pelt weight of minks decreased significantly (p < 0.05). These parameters were similar between P32 and P24 with Met supplementation (p > 0.05). The hair follicle density of the winter coat was not influenced by the dietary protein levels and Met supplementation (p > 0.05). Low‐protein diets content led to a reduction of hair follicle developing to next phase. It was documented that 24% crude protein of DM with Met supplementation during growing‐furring period was sufficient for minks to express their genetic capacity to develop hair follicles and achieve the prime fur characteristics. Overall this study demonstrated that hair growth and hair properties in pelts are very dependent on the dietary protein and Met supply in the growing‐furring period of minks.     

Effects of dietary protein levels on digestibility of nutrients and growth rate in young female mink (Mustela vison)

This study investigated digestibilities of nutrients and feed efficiency in female mink at the different dietary protein levels during the mink growth period. Effects of dietary protein on growth performance of minks were also measured. Sixty 45‐day‐old healthy female minks were randomly assigned to 6 treatment groups with 10 animals in each group. Animals were fed diets varying in protein levels: 28% (Group I), 30% (Group II), 32% (Group III), 34% (Group IV), 36% (Group V) and 38% (Group VI), respectively. The digestibilities of key nutrients were determined on Day 14 after initiating the experiment and the last 3 days. From the beginning of the study, body weight and feed intake were weighed and recorded every other week in order to calculate the average daily bodyweight gain and the feed efficiency. The trial had demonstrated that nitrogen intake was greatly significantly different, which was affected by dietary protein levels (p < 0.001). Growth performance of minks was impaired when dietary protein level was at 28%. When dietary protein level was at 34%, minks had the best daily gains, feed efficiency, and digestibilities of some key nutrients.     

Cloning and Bioinformatic Analysis of L1 Gene of BPV from Guizhou Province

In order to study and analyze L1 gene of bovine papillomavirus（BPV）in Guizhou province,the L1 gene of BPV-GZ01 strain was amplified,cloned and sequenced using bioinformatic softwares and methods,and the secondary structure,tertiary structure,B-cell preponderant epitope,conserved domains analysis, transmembrane domain and signal peptide of L1 gene were predicted.The results showed that the length of L1 gene was 1 494 bp,encoding 497 amino acids.The L1 gene of BPV-GZ01 strain shared an amino acid identities of 98.6%,99.4%,98.4%,94.4% and 91.3%,and a nucleotide identities of 99.1%,99.8%,99.4%,87.6% and 82.8% with those of BPV2,BPV2-SW01,BPV2-AKS01,BPV13 and BPV1 strains,respectively.The results of phylogenetic tree analysis indicated that there was a close relationship between BPV-GZ01 and BPV2-SW01 strains.The prediction of secondary structure of L1 protein indicated that the random coil,extended strand and alphahelix took a higher percentage.The L1 protein was supposed contain 6 potential antigen epitopes.And no transmembrane domains and no signal peptide were found.The tertiary structure of L1 protein was curved spiral structure.These results provided a theoretical basis for immunologic diagnosis and further research of nucleic acid vaccine of BPV.