水牛骨形态发生蛋白1基因的克隆及其在不同组织中的表达研究

本研究克隆了水牛骨形态发生蛋白1(bone morphogenetic protein-1,BMP1)基因序列,并运用生物信息学方法对其核苷酸序列的保守性和氨基酸的理化性质、蛋白质结构进行了系统分析,此外还运用实时荧光定量PCR技术对BMP1基因在水牛不同组织中的表达差异进行了检测。结果表明,应用RT-PCR技术克隆得到水牛BMP1基因的cDNA序列长度为3 195 bp,其中包含完整的2 967 bp的开放读码框(ORF),编码988个氨基酸。经序列相似性分析显示,水牛BMP1基因与牛、猪、马、人和小鼠相应氨基酸序列相似性分别为99%、96%、96%、96%和95%,具有很强的保守性。结合系统进化树分析结果推测,BMP1基因在不同物种及进化的过程中具有高度的保守性。对水牛BMP1蛋白的结构域预测结果发现,其存在1个信号肽区、1个前肽区、1个金属蛋白酶区、5个CUB区和2个EGF-like功能区。定量表达分析结果显示,BMP1基因在水牛心脏组织中相对表达量最高,睾丸、卵巢和生殖嵴等性腺器官表达量次之,骨头等其他组织表达量较低,肝脏表达量最低。     

水牛转录抑制因子CTCF基因克隆分析及不同组织中的表达研究

本研究克隆了水牛转录抑制因子CTCF基因序列,并运用生物信息学方法对其核苷酸序列的保守性和氨基酸的理化性质、蛋白质结构进行了系统分析,此外还对CTCF基因在水牛不同组织中的表达差异进行了检测。结果表明,应用RT-PCR技术克隆获得了长2239bp水牛CTCF基因序列,其中编码区全长2184bp,编码727个氨基酸,理论蛋白质分子质量82.7ku,等电点为6.57。多重序列比较分析显示,水牛CTCF核苷酸序列与牛、猪、马、人和小鼠相应序列的相似性分别为99%、96%、96%、94%和92%,结合系统进化树分析结果推测,CTCF基因在不同物种及进化的过程中具有高度的保守性。对水牛CTCF蛋白的二级和三级结构分析结果发现,其存在连续11个锌指C2H2结构,预测其为重要的DNA结合蛋白。定量表达分析结果显示,CTCF在水牛肝脏组织中相对表达量最高,大脑、肌肉和肾脏次之,卵巢和皮肤表达量较低。     

沼泽型水牛ALK3基因克隆分析与表达模式研究

试验旨在对沼泽型水牛ALK3基因进行克隆、生物信息学分析,并对其在水牛组织中的表达规律进行系统研究.根据GenBank中已公布的牛ALK3基因序列设计特异性引物,应用RT-PCR方法扩增、克隆获得目的基因片段;应用生物信息学方法分析和预测了水牛ALK3的遗传进化及蛋白质的理化性质、二级和三级结构;并应用QRT-PCR技术对ALK3基因在水牛组织中的表达进行了差异分析.结果表明,水牛ALK3基因编码区全长1 599 bp,共编码532个氨基酸.多重序列比较分析显示,水牛ALK3核苷酸序列与牛、绵羊、猪、马、人和小鼠相应序列的同源性分别为98%、96%、95%、93%、94%和91%.系统进化树分析显示,ALK3基因在不同物种以及进化的过程中具有高度保守性.对ALK3蛋白质的分析表明该蛋白呈弱碱性,有信号肽,细胞亚定位于胞膜上,存在丝氨酸/苏氨酸激酶和GS等结构.定量分析结果显示,ALK3在水牛生殖脊、心脏、肝脏、颗粒细胞、肺脏、卵丘细胞、肾脏、下丘脑、垂体、大脑等15种组织或细胞中有不同程度的表达,其中卵巢中表达量最高,垂体、肺脏和睾丸次之,卵丘细胞表达量最低.本研究成功克隆了沼泽型水牛ALK3基因,并研究了其在不同水牛组织细胞中的表达规律,为阐明其在水牛繁殖过程中的功能及转基因载体构建中的应用研究奠定了理论基础.     

水牛脑源性神经营养因子基因克隆、序列分析及其在不同组织中的表达研究

本研究克隆了水牛脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因序列,并运用生物信息学方法对序列的同源性、生物进化树、蛋白的理化性质及二、三级结构等进行了分析预测,同时利用QRT-PCR方法研究了BDNF mRNA在胎儿水牛及成年水牛不同组织中的表达情况。结果表明,应用RT-PCR技术克隆获得了长800bp水牛BDNF基因序列,其中编码区全长753bp,编码250个氨基酸。多重序列比对分析显示,水牛BDNF核苷酸序列与黄牛、野猪、犬、人、马、小鼠同源性分别为99%、94%、93%、90%、90%和89%;生物进化树分析显示,BDNF基因在不同物种进化过程中具有较高的保守性;BDNF蛋白理论分子质量28 173.36u,等电点9.12;蛋白二级结构由多个α-螺旋、β-折叠、T-转角及无规则卷曲组成,三级结构由多个α-螺旋、3对反向平行的β-折叠结构等构成活性中心(即NGF功能结构域)。QRT-PCR结果显示,BDNF mRNA在胎儿水牛及成年水牛的心脏、肺脏、肾脏、大脑、肌肉、卵巢、睾丸组织中都有表达,且成年水牛的表达量高于胎儿水牛。     

水牛Keap1基因的克隆、序列分析及组织表达研究

本研究克隆了水牛Keap1基因的全长编码区,并对其序列进行了生物信息学分析,同时探索了其在水牛各组织中的表达差异。根据GenBank中公布的牛Keap1基因的序列信息,设计特异性引物并扩增出水牛Keap1的目的片段,利用生物信息学分析方法,对测序所得水牛Keap1基因序列、预测蛋白质序列进行了分析,并采用实时荧光定量PCR技术对Keap1基因mRNA在水牛各组织中的表达进行了研究。结果表明,水牛Keap1基因编码区全长1 875 bp,预测编码624个氨基酸;多重分析结果显示,水牛与牛、绵羊、野猪和人Keap1基因的同源性分别为99%、96%、92%和90%;进化树分析表明Keap1在物种间具有较高保守性,不同物种间Keap1序列的差异符合物种间的进化性。对Keap1蛋白质二级结构预测发现,其包含24个α-螺旋、40个β-螺旋、38个T转角和27个无规则卷曲。实时荧光定量PCR结果显示,Keap1基因在水牛的心脏、肝脏、脾脏、肺脏、肾脏、卵巢和肌肉组织中均有表达,但心脏中表达量最高,肝脏、脾脏中表达量较低。本研究成功克隆了水牛Keap1基因,并进行了相关生物信息学分析及其mRNA在水牛各组织的表达情况研究,为阐明Keap1-Nrf2-ARE信号通路,提高水牛胚胎体外培养的抗氧化能力奠定基础。     

Cloning and Sequence Analysis of Buffalo Keap1 Gene and Investigation of Its Expression Pattern in Different Tissues

In this study,the CDS sequence of buffalo Keap1 gene was cloned and analyzed,then its expression pattern in different tissues was also investigated.A pair of primers of buffalo Keap1 gene was designed based on the nucleotide sequence of Bos taurus Keap1 gene from GenBank,and then the buffalo Keap1 gene was amplified.Using the bioinformation techniques,the gene sequence and the protein structure were analyzed.The expression level of Keap1 gene in different tissues were detected with Real-time quantitative PCR.The results showed that the length of buffalo Keap1 gene coding sequence was 1 875 bp and encoded 624 amino acids.The multiple sequence alignment results showed that buffalo Keap1 gene shared 99%,96%,92% and 90% of similar nucleotide sequence with that of Bos taurus,Ovis aries,Sus scrofa and Homo sapiens,respectively.And the phyogenetic tree also showed the conservatism between several different species.The second structure of buffalo Keap1 protein was predicted as 24 alpha regions,40 beta regions,38 turn regions and 27 coil regions.In addition,the results of Real-time quantitative PCR showed that Keap1 mRNA exists in all seven tissues,but the most abundant expression was in heart and the minimal expression was in liver and spleen.The results provided an foundation for further study of Keap1-Nrf2-ARE signal pathway,for enhancing the ability of antioxidant of buffalo embryo in vitro culture.     

水牛缝隙连接蛋白43基因克隆及表达

本研究克隆了水牛缝隙连接蛋白43(connexin 43,Cx43)基因序列,并运用生物信息学方法对其核苷酸序列的保守性和氨基酸的理化性质、蛋白质结构进行了系统分析,对Cx43基因在水牛不同组织和不同发育阶段卵泡中的表达差异进行了检测。结果表明,应用RT-PCR技术克隆获得了水牛Cx43基因序列,其中编码区全长1152 bp,编码383个氨基酸,蛋白质理论分子质量43.13 ku,等电点为8.88。多重序列比对结果显示,水牛Cx43核苷酸序列与牛、羊、猪、马和人相应序列的同源性分别为99%、98%、94%、93%和92%,系统进化树分析结果推测,Cx43基因在不同物种进化过程中具有高度保守性;对水牛Cx43蛋白的二级和三级结构分析发现,其具有缝隙连接蛋白的特有结构。定量RT-PCR结果显示,Cx43在水牛卵巢组织中相对表达量最高,睾丸、肾脏、心脏和皮肤次之,肝脏和大脑表达量较低。免疫组化结果发现,Cx43蛋白表达随卵泡发育时期的不同而变化,Cx43蛋白随卵泡发育表达逐渐增强。     

Cloning,Bioinformatics and Expression Pattern Analysis of Buffalo MC1R Gene

The aim of this study was to clone and analyze the expression pattern of buffalo MC1R gene.A pair of specific primers was designed according bovine MC1R sequence (GenBank accession No.:JN123363.1),with genome DNA of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle as template,MC1R was amplified by PCR.Then relative expression level of MC1R gene of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle was analyzed using QRT-PCR,and protein expression was detected by Western blotting method.The results showed that the 954 bp coding region of buffalo MC1R gene was successfully cloned and sequenced,which code for 317 amino acids.The MC1R gene nucleotide sequences and amino acid sequences of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle were highly conserved.Five polymorphic sites were found between White swamp buffalo and swamp buffalo of MC1R gene,including 476 T→C,618 G→C,881 G→A,930 G→A and 931 A→G,which caused three nonsynonymous mutation sites of Phe159Ser,Glu310Ala and Asp294Ala.The QRT-PCR result showed that relative expressions of MC1R gene of Murrah buffalo,swamp buffalo and Yellow cattle were significant higher than that of White swamp buffalo (P<0.05).The Western blotting results revealed that MC1R protein expression level in swamp buffalo was higher than that of White swamp buffalo.In conclusion,there were amino acids mutation in White swamp buffalo MC1R gene,and MC1R gene relative expression of White swamp buffalo was lower than that of other buffalo,which was the main reason of lacking of melanin production in White swamp buffalo.     

水牛黑色素皮质素1受体基因克隆、生物信息学分析及表达模式研究

本试验旨在对水牛黑色素皮质素1受体(MC1R)基因进行克隆、生物信息学分析及表达模式研究。参考牛MC1R基因(GenBank登录号:JN123363.1)序列设计引物,以本地沼泽水牛、白沼泽水牛、摩拉水牛和黄牛基因组DNA为模板,应用PCR方法扩增克隆MC1R基因片段并进行测序分析。运用QRT-PCR方法检测摩拉水牛、沼泽水牛、白沼泽水牛和黄牛皮肤组织中MC1R基因的表达模式,并通过Western blotting方法检测沼泽水牛和白沼泽水牛MC1R基因的蛋白表达差异。结果表明,应用PCR方法成功克隆了水牛MC1R基因,其编码区全长954 bp,共编码317个氨基酸。测序分析后发现沼泽水牛、白沼泽水牛、摩拉水牛和黄牛MC1R基因的核苷酸序列和氨基酸序列相似性很高。沼泽水牛与白沼泽水牛在476、618、881、930和931 bp位点上分别发生T→C、G→C、G→A、G→A和A→G突变,导致了沼泽水牛和白沼泽水牛第159位氨基酸由丝氨酸变成苯丙氨酸,第310位氨基酸由谷氨酸变成丙氨酸,第294位氨基酸由天冬氨酸变成丙氨酸,发生了非同义突变。QRT-PCR结果发现,MC1R基因在摩拉水牛、沼泽水牛和黄牛皮肤组织中的相对表达量均显著高于白沼泽水牛(P<0.05);Western blotting分析结果显示,沼泽水牛皮肤组织中MC1R蛋白的表达量高于白沼泽水牛。综上所述,白沼泽水牛MC1R基因的编码区发生氨基酸位点突变,且相对表达量和蛋白表达量均低于沼泽水牛,推测此为白沼泽水牛体内合成的黑色素缺失而导致毛色白化的主因。     

Cloning,Analysis and Construction of Eukaryotic Expression Vector of Buffalo MBD3 Gene

In this study,through cloning buffalo MBD3 gene and analyzing the biological information of MBD3 gene sequence,and constructing the expression vector of buffalo MBD3,to provide a basis for the function research of buffalo MBD3 gene on embryo development and iPCSs.The total RNA was extracted from buffalo fresh ovary,and MBD3 gene was amplified and sequenced,the sequence was systemically analysed with bioinformatics techniques.And the MBD3 CDS was cloned into the pEGFP-C1 vector.Then the recombinant plasmid pEGFP-C1-MBD3 was transferred into the HEK293T cells and buffalo fetal fibroblasts (BFF),the expression was analyzed by RT-PCR,Western blotting and fluorescence microscope.The results showed that 898 bp of MBD3 gene fragment including whole 774 bp CDS was cloned and sequenced,and encoded 257 amino acids.The multiple sequence alignment and analysis of phylogeny tree showed that MBD3 gene was highly conserved in the process of evolution.Especially the MBD domain,the MBD domain of buffalo MBD3 gene shared 100% of similar nucleotide sequence with Bos taurus,and shared 97% of similar nucleotide sequence with Homo sapiens,Sus scrofa and Pan troglodytes.The recombinant plasmids pEGFP-C1-MBD3 were transferred into HEK293T cells and BFF,fluorescence observation,RT-PCR and Western blotting were used to analyze the expression of buffalo MBD3.The results suggested that the expression vector of buffalo MBD3 gene was successfully constructed.The study laid the foundation for the function research of MBD3 on embryo development and inducing of iPCSs.     

水牛MBD3基因克隆分析及其真核表达载体的构建

本研究旨在克隆水牛MBD3基因,进行生物信息学分析,并构建MBD3基因的真核表达载体,为研究MBD3基因在水牛胚胎发育及诱导多能干细胞（iPSCs）中的作用奠定基础。试验从卵巢组织中提取总RNA,反转录得到cDNA,并以此为模板,应用RT-PCR克隆得到MBD3基因,测序并应用相关的生物学软件进行分析;将MBD3基因连接至真核表达载体pEGFP-C1,再将携带目的基因的重组质粒转染HEK293T细胞和水牛胎儿成纤维细胞（BFF）,利用RT-PCR及Western blotting方法分析目的基因的表达。结果表明,克隆获得了898 bp的水牛MBD3基因序列,其中编码区全长774 bp,编码257个氨基酸。通过对MBD3基因核苷酸序列的多重比对及进化树分析,MBD3基因在进化中高度保守,特别是MBD结构域,水牛与牛的同源性为100%,与人、猪、猩猩的同源性均为97%。将水牛MBD3基因真核表达载体转染HEK293T细胞和BFF,通过荧光观察、RT-PCR及Western blotting方法鉴定表明,成功构建了水牛MBD3基因的真核表达载体。本研究克隆得到了水牛的MBD3基因,并成功构建了MBD3基因的真核表达载体,为进一步研究MBD3基因在水牛胚胎发育及iPSCs诱导上的作用奠定了基础。     

水牛Smad4基因的克隆及其真核表达载体的构建

为了阐明Smad4基因在水牛卵巢颗粒细胞增殖及胚胎发育中的分子机制,试验采用RT-PCR扩增并克隆水牛Smad4基因,对其核苷酸序列和蛋白质序列进行生物信息学分析,构建Smad4基因的真核表达载体,通过脂质体转染法转染体外培养的牛卵巢颗粒细胞。结果表明,试验克隆得到水牛Smad4基因编码序列,编码区全长为1 662 bp,编码553个氨基酸。通过BLAST对水牛Smad4基因的核苷酸序列进行同源性比对,结果显示,水牛Smad4基因与牛的同源性为99%,与绵羊、猪、马、人的同源性分别为98%、96%、96%和95%。系统进化树分析表明,Smad4基因在不同物种的进化过程中具有高度的保守性。试验成功构建了水牛Smad4基因的真核表达载体pEGFP-N1-Smad4,并在水牛卵巢颗粒细胞中表达出较强的绿色荧光蛋白Smad4-EGFP融合蛋白。本研究成功克隆了水牛Smad4基因,并成功构建了Smad4基因的真核表达载体,为进一步研究Smad4基因在水牛胚胎发育过程中的分子机制奠定基础。     

Cloning and Construction of Eukaryotic Expression Vector of Buffalo Smad4 Gene

This study was aimed to elucidate the molecular mecbanisms of Smad4 gene on granulose cells proliferation and embryonic development in buffalo. The Smad4 gene was amplified by RT-PCR, analyzed by bioinformatics, studied with eukaryotic vector construction, and used liposome transfection skill to transfect into the buffalo granulose cells. The results showed that Smad4 gene was cloned, the coding region was 1 662 bp, and encoded 553 amino acids. BLAST analysis showed that the buffalo Smad4 gene shared 99% of similar nucleotide sequence with Bos taurus, and shared 98%, 96%, 96% and 95% of similar nucleotide sequence with Ovis aries, Sus scrofa, Equues caballus and Homo sapiens, respectively. Phylogenetic tree analysis showed that Smad4 gene was highly conserved in different species. The buffalo pEGFP-N1-Smad4 eukaryotic expression vector was successfully constructed, and transferred into the buffalo granulose cells, and the Smad4-EGFP fusion protein was detected in the cells. The results suggested that the success cloning and construction vector of buffalo Smad4 gene laid the foundation for the research of Smad4 gene on embryo development.     

水牛水通道蛋白9基因克隆及其表达分析

本研究旨在对水牛水通道蛋白9 (aquaporins 9,AQP9) 基因进行克隆,并对其在水牛不同组织中的表达规律及其在水牛卵巢和睾丸组织中的表达差异进行探索。根据GenBank上黄牛AQP9基因序列(登录号:NM_001205833.1)设计特异性引物,以水牛睾丸组织cDNA为模板,应用RT-PCR方法扩增AQP9基因编码区片段;运用生物信息学方法分析其核苷酸序列的保守性和氨基酸的理化性质;应用实时荧光定量PCR技术分析AQP9基因在水牛组织中的表达情况;免疫组织化学方法分析AQP9蛋白在不同发育阶段水牛卵泡及睾丸组织中的表达差异。结果表明,克隆获得了888 bp的水牛AQP9基因编码区序列,其编码295个氨基酸。多重序列比较显示,水牛AQP9核苷酸序列与牛、猪、绵羊和人相应序列相似性分别为99%、90%、97%、88%;氨基酸序列的同源性分别为99%、86%、97%、83%,系统进化树分析结果推测,AQP9基因在物种进化过程中具有高度保守性。实时荧光定量PCR结果显示,AQP9基因在水牛肝脏、肺脏、大脑、皮肤、睾丸和卵巢组织中有不同程度的表达,在肝脏组织中表达最高,皮肤和睾丸次之,肺脏和卵巢表达较低。免疫组化结果显示,在卵巢组织中,AQP9蛋白表达随卵泡发育时期的不同而变化,并随着卵泡发育其表达逐渐增强;在睾丸组织中,AQP9蛋白在各级精母细胞和间质细胞中均有表达。结果提示,成功克隆得到水牛AQP9基因序列;AQP9在水牛卵巢和睾丸中的表达及其功能可能与水牛卵泡发育和精子发生有重要的关联。     

Cloning of Buffalo AQP9 Gene and Analysing its Expression in the Buffalo Tissues

Cloning buffalo AQP9 gene and analyzing its expression in buffalo tissues.A pair of primers was designed according to the released bovine AQP9 sequences in GenBank,which was used to clone buffalo AQP9 gene.The AQP9 gene was amplified by RT-PCR,whose nucleotide sequence and protein structure were analyzed by bioinformatics methods.The expression of AQP9 in buffalo tissues was assayed by Real-time quantitative PCR.The expression of AQP9 gene in buffalo ovary and testis tissue was detected by immunohistochemical staining method.The results showed that the cloned ORF length of buffalo AQP9 gene was 888 bp,which coded 295 amino acids.The results of multiple sequence comparison showed that the nucleotide sequence of buffalo AQP9 shared 99%,90%,97% and 88% homologeous compared with that of Bos taurus,Sus scrofa,Ovis ariessis and Homo sapiens,respectively,while shared 99%,86%,97%,83% homologeous for amino acids,respectively.Phylogenetic tree analysis indicated that AQP9 gene was highly conservative in the evolutionary process.Real-time quantitative PCR results showed that AQP9 gene expressed in buffalo liver,lung,brain,skin,testis and ovary tissues with different levels,had the most abundant expression in liver,followed by in skin and testis,less observed in lung and ovary.The results of immunohistochemical staining showed that the expression of AQP9 protein varied with the development of buffalo ovarian tissue,and gradually enhanced with follicle development.In testicular tissue,AQP9 protein expressed in spermatocyte and leydig cells of developmental stage testis.These results indicated that we had successfully cloned buffalo AQP9 gene sequences.The expression and its function of AQP9 in buffalo ovaries and testes might play an important role in follicle development and spermatogenesis.     

奶水牛固醇携带蛋白2基因克隆、生物信息学分析及其组织表达检测

本研究旨在对水牛固醇携带蛋白2（sterol carrier protein 2,SCP2）基因进行克隆及生物信息学分析,并检测其在水牛不同组织中的表达。以黄牛SCP2基因（登录号：NM_001033990.3）为种子序列成功克隆了水牛SCP2基因完整CDS区,该序列长1 632 bp,可编码543个氨基酸;其与黄牛、绵羊、山羊、白鲸、人、家犬和家猫的同源性分别为95.9%、93.4%、92.4%、89.4%、88.3%、86.3%和86.9%,说明SCP2基因CDS区在不同物种间具有较高的保守性。聚类分析则表明水牛与黄牛的分子进化关系最近;氨基酸序列分析表明,SCP2蛋白的分子式为C₂₆₀₂H₄₁₃₁N₇₀₉O₇₇₄S₂₉₈,分子质量为58.66 ku,理论等电点（pI）为8.59,不稳定系数为27.94,平均亲水性为-0.215,属于碱性、稳定、亲水蛋白质;二级结构分析表明水牛SCP2蛋白由α-螺旋、无规则卷曲和延伸链构成,其中α-螺旋占35.54%,无规则卷曲占48.99%,延伸链占15.47%,与三级结构预测结果一致;亚细胞定位分析表明,水牛SCP2蛋白分布在细胞质（43.5%）、过氧化物酶体（21.7%）、线粒体（17.4%）、细胞核（13.0%）和细胞骨架（4.4%）;跨膜结构和信号肽预测分析表明,水牛SCP2蛋白不含跨膜结构和信号肽;磷酸化位点分析发现,水牛SCP2蛋白有13个Ser、3个Thr和2个Tyr可能成为蛋白激酶磷酸化位点;蛋白质结合位点预测结果显示,水牛SCP2蛋白含有12个蛋白质结合位点和1个多核苷酸结合位点;实时荧光定量PCR结果表明,水牛SCP2基因在肝脏中表达量最高,其他组织中表达量从高到低依次为乳腺、淋巴、肾脏、大肠、胃、肺脏、脾脏、卵巢、垂体、大脑和心脏。本试验为今后进一步探讨SCP2基因的功能奠定了基础。     

水牛TRAIL基因克隆及生物信息学分析

【目的】对水牛肿瘤坏死因子(tumor necrosis factor,TNF)相关凋亡诱导配体(TNF-related apotosis-inducing ligand,TRAIL)基因CDS序列进行克隆及序列分析,并对其编码的蛋白进行生物信息学分析,为后期TRAIL蛋白调控水牛卵巢卵泡发育、颗粒细胞增殖及凋亡的研究奠定基础。【方法】利用RT-PCR方法克隆水牛TRAIL基因CDS序列,对所获序列进行核苷酸序列、氨基酸序列相似性比对,构建系统进化树,并通过生物信息学软件分析TRAIL基因编码蛋白的结构和功能。【结果】试验成功克隆水牛TRAIL基因CDS序列,长864 bp,编码287个氨基酸;水牛TRAIL基因与牦牛、普通牛、山羊、绵羊、野猪、马、人、黑猩猩和家鼠的核苷酸序列相似性分别为99.2%、99.3%、95.9%、96.3%、84.7%、84.8%、81.3%、81.3%和70.0%。系统进化树结果表明,水牛与牦牛、普通牛的亲缘关系最近,与家鼠亲缘关系最远。氨基酸序列比对结果表明,在不同物种间,其跨膜结构域和TNF结构域序列保守性较高。TRAIL蛋白属于亲水性蛋白,存在1个跨膜结构域,140―285位氨基酸处为TNF区,具有29个磷酸化位点,无信号肽和糖基化位点,主要定位于细胞质中。TRAIL蛋白二级结构主要以无规则卷曲为主,约占51.57%,其次为延伸链(24.39%)和α-螺旋(24.04%)。TRAIL蛋白三级结构与二级结构一致,且与模型蛋白人TRAIL蛋白的相似性为75.53%。【结论】本试验克隆得到水牛TRAIL基因CDS区序列,大小为864 bp,编码287个氨基酸,水牛与牦牛、普通牛亲缘关系最近,TRAIL蛋白跨膜结构域和TNF结构域在不同物种间序列保守性较高,这可能与其功能有关。     

水牛浮舰蛋白2基因克隆及序列分析

试验旨在克隆水牛浮舰蛋白2(Flotillin 2,FLOT2)基因并对其进行生物信息学分析,为揭示该基因在水牛发育繁殖中的作用奠定基础。本研究通过PCR扩增获得了FLOT2基因全长并进行克隆测序,结合生物信息学分析方法,预测及分析其蛋白质结构。结果表明,水牛FLOT2基因编码区长1 287 bp,编码428个氨基酸,3'UTR区长277 bp。多重序列比较分析显示,水牛FLOT2基因核苷酸序列与牛、山羊、绵羊、小鼠、马、猫和人相应序列的相似性分别为98%、98%、97%、90%、93%、92%和92%,系统进化树分析结果表明,FLOT2基因在不同物种以及进化的过程中具有高度保守性。对FLOT2蛋白质的分析表明,该蛋白呈弱酸性,无信号肽,细胞亚定位于细胞质中,存在SPFH_flotillin和SPFH_hflk等结构域。microRNA预测结果表明,bta-miR-2438、bta-miR-2379和bta-miR-1777a可能是其3'UTR潜在靶标。研究结果为今后阐明FLOT2基因在水牛繁殖性能中的功能,尤其是在配子及胚胎发生过程中的作用及分子机制奠定了基础。     

Cloning and Sequence Analysis of Buffalo FLOT2 Gene

In order to clarify the effect of Flotillin 2 (FLOT2) gene on the reproductive performance of buffalo, buffalo FLOT2 gene was cloned and analyzed by bioinformatics.FLOT2 was cloned by PCR, cloned and sequenced, then its protein structure was predicted and analyzed by bioinformatics tools.The results showed that the coding region of buffalo FLOT2 gene was 1 287 bp, 3'UTR was 277 bp, encoded 428 amino acids.The buffalo FLOT2 gene shared 98%, 98%, 97%, 90%, 93%, 92% and 92% of similar nucleotide sequence with that of Bos taurus, Ovis aries, Capra hircus, Mus musculus, Equus caballus, Felis catus and Homo sapiens, respectively.Phylogenetic tree analysis showed that FLOT2 gene was highly conserved in different species and evolution.FLOT2 protein was weakly acidic, without signal peptide, located in the cytoplasmic, and with the presence of SPFH_flotillin and SPFH_hflk domain.microRNA prediction showed bta-miR-2438, bta-miR-2379 and bta-miR-1777a maybe target the 3'UTR of buffalo FLOT2.In a word, this study provided an important reference for surveying the regulation mechanism of FLOT2 gene in buffalo reproductive performance, especially, during buffalo gametogenesis and embryogenesis.     

水牛TLR4 cDNA全长的克隆及其生物信息学分析

本研究利用RT-PCR方法分段克隆水牛TLR4 cDNA后拼接成全长,并克隆于pMD20-T载体中,同时运用生物信息学软件对其核苷酸序列及编码蛋白质的结构进行分析和预测。结果表明,克隆的TLR4 cDNA ORF全长2526 bp,共编码841个氨基酸,N端具有由25个氨基酸组成的信号肽,该蛋白预测的分子质量为95.98 ku,等电点为6.37;水牛TLR4与GenBank中登录的水牛TLR4(DQ857349)核苷酸序列同源性达99.01%,与黄牛、绵羊、野猪、马、人和黑猩猩的同源性超过80%,与小鼠和狗的同源性次之,分别为72.17%和61.30%,与鸡的最低,仅为53.94%;该蛋白是由胞外区(1-634位氨基酸)、跨膜区(635-657位氨基酸)和胞内区(658-841位氨基酸)3部分构成的跨膜蛋白,胞外区含有12个LRR串联重复结构域、胞内区具有TIR结构域。本试验成功克隆了水牛TLR4 cDNA全长,并获得了核苷酸及其蛋白质的生物信息学分析数据,为进一步研究其功能奠定了基础。