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 of Buffalo BMP1 Gene and its Expression Pattern Investigation in Different Tissues

Buffalo BMP1 gene was cloned in the present study, the BMP1 sequence was systemically analysed by bioinformatics techniques, and the expression level of BMP1 gene in different tissues were also assayed with Real-time fluorescence quantitative PCR (QRT-PCR).The results showed that with RT-PCR a 3 195 bp buffalo BMP1 gene was cloned and sequenced, including the whole ORF of 2 967 bp (coding 988 amino acid).The sequence multialigned results showed that buffalo BMP1 gene shared 99%, 96%, 96%, 96% and 95% of similar amino acid sequence with Bos taurus, Sus scrofa, Equus, Homa sapiens and Mus musclus, respectively.It was predicted that buffalo BMP1 protein contained a signal peptide domain, a preregion, a metalloproteinases domain, five complement-Uegf-BMP-1 domain (CUB domain) and two epitheloid growth factor-like domain (EGF-like domain).In addition, we also analyzed the expression level in different tissues through QRT-PCR, the results showed that BMP1 gene mRNA existed in all nine tissues with the most abundant expression in heart, followed by testis, ovary, genital ridge, while with lower amount of bone and other tissues, the minimal expression in liver was observed.     

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。     

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.     

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。     

水牛LPIN1基因克隆及序列分析

试验旨在克隆获得水牛脂素1（LPIN1）基因，并对其进行生物信息学分析，为揭示该基因在水牛脂肪沉积、生殖发育和泌乳调控中的作用奠定基础。本研究以水牛卵巢组织cDNA为模板，PCR扩增获得了LPIN1基因CDS区全长后测序，并结合生物信息学分析方法预测及分析蛋白质理化性质、二级结构及三级结构等。结果表明，水牛LPIN1基因编码区长2 793 bp，编码930个氨基酸。MegAlign软件分析显示，水牛LPIN1基因核苷酸序列与水牛（预测）、牦牛、黄牛、山羊、藏羚羊、绵羊、猪、骆驼、人和小鼠LPIN1基因的同源性分别为99.6%、97.9%、97.7%、97.5%、97.4%、97.1%、89.9%、89.8%、86.2%和83.5%；水牛lipin1蛋白氨基酸序列与黄牛、牦牛、山羊、藏羚羊、骆驼、猪及人的同源性分别为99%、99%、99%、99%、94%、94%及90%。应用Mega 5.0软件构建系统进化树发现，水牛与黄牛的亲缘关系最近，其次为绵羊和山羊，LPIN1基因在不同物种及进化的过程中具有高度保守性。对lipin1蛋白分析发现，其二级结构由α-螺旋、β-折叠、T-转角和无规则卷曲组成；蛋白呈弱酸性，无信号肽，亚细胞主要定位于细胞核中，存在Lipin_N、LNS2和AF1Q等结构域，其中Lipin_N、LNS2为保守结构域。     

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.     

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PNPLA8基因编码区克隆、表达分析及催乳素对该基因的调节作用

为了探究磷脂酶patatin样域包含蛋白8（patatin-like phospholipase domain containing 8,PNPLA8）在水牛乳腺脂质代谢中的作用,试验根据GenBank中公布的奶牛PNPLA8基因序列（登录号：XM_005205444.4）设计引物,应用PCR扩增并克隆水牛PNPLA8基因编码区（CDS）,应用生物信息学软件分析序列及蛋白质结构;抽提水牛不同组织及不同泌乳期乳腺组织RNA,利用实时荧光定量PCR检测PNPLA8基因在不同组织间和不同泌乳期的表达;利用不同浓度的催乳素处理水牛乳腺上皮细胞,通过定量检测催乳素对PNPLA8基因表达的影响。结果显示,水牛PNPLA8基因CDS长2 355 bp,编码784个氨基酸,与牦牛、山羊等PNPLA8基因具有较高的同源性;PNPLA8基因在所检测的水牛11个组织中有不同水平的表达,在肺脏和乳腺中表达量相对较高,在脂肪和肌肉组织中表达量较低;在整个泌乳期内PNPLA8基因的表达呈现"低-高-低"趋势;催乳素处理水牛乳腺上皮细胞结果显示,随着催乳素浓度升高,PNPLA8基因表达量逐渐下降。本研究成功克隆了水牛PNPLA8基因,并发现PNPLA8基因是参与乳腺泌乳的一个功能基因,为进一步研究PNPLA8基因在水牛乳腺中的功能奠定基础。     

Cloning,Expression Analysis and Regulation of PNPLA8 Gene by Prolactin in Buffalo

In order to investigate the role of patatin-like phospholipase domain-containing 8(PNPLA8) in lipid metabolism of mammary gland in buffalo,the coding region (CDS) was amplified and cloned by PCR based on the sequence of Bos taurus PNPLA8 gene in GenBank (accession No.:XM_005205444.4) were analyzed using bioinformatics software.Total RNA was extracted from different tissues of buffalo and mammary glands,which were harvested from different lactating buffaloes.The expression of PNPLA8 gene mRNA in different tissues and different lactation period was detected by Real-time quantitative PCR.For buffalo mammary epithelial cell treatment,different concentrations of prolactin were used and the effect of prolactin on the expression of PNPLA8 gene was detected by Real-time quantitative PCR.The results showed that the length of the PNPLA8 gene CDS was 2 355 bp,encoding 784 amino acids.The sequence showed high homology with Bos mutes and Capra hircus.PNPLA8 gene was expressed at different levels in 11 tissues examined,with a relatively high level in the lung and mammary tissues while the low level in the fat and muscle tissues.The expression abundance of the PNPLA8 gene was variable during lactation and showed a trend of "low-high-low".Prolactin treatment showed that the expression of PNPLA8 gene decreased with the increase of prolactin concentration.In this study,PNPLA8 gene of buffalo was successfully cloned,and the expression of PNPLA8 gene in different tissues and the lactation period was analyzed.Herein,the effect of prolactin on the expression of PNPLA8 gene was studied that laid a foundation for further research on PNPLA8 gene of mammary gland in buffalo.     

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。     

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.     

Cloning and Bioinformatics Analysis of FADS2 Gene in Buffalo

This study was aimed to clone the buffalo Δ6-fatty acid desaturases (FADS2) gene using in-Silico cloning and analyze its genetic struction with bioinformatics, which provide a foundation for investigating the milk performance in buffaloes. Primers were designed according the sequence of FADS2 gene in dairy cow (GenBank accession No.:NM_001083444.1). The FADS2 gene was amplified by RT-PCR, and its sequence was analyzed by bioinformatics. Sequence analysis revealed that the buffalo FADS2 gene had 36 600 bp in length and consisted of 12 exons and 11 introns, containing an open reading frame (ORF) of 1 335 bp which encoding 444 amino acids. Sequence homology analysis indicated that the buffalo FADS2 protein gene showed 98.88%, 98.88%, 89.66%, 90.79%, 90.85%, 92.35% and 87.11% identity with that of Bos mutus, Bos taurus, Homo sapiens, Sus scrofa, Oryctolagus cuniculus, Orcinus orca and Rattus norvegicus, respectively. Protein prediction analysis showed that the molecular weight and isoelectric point (pI) of buffalo FADS2 were 52.51 ku and 8.75, respectively, and the FADS2 protein was weak alkali and the hydrophobicity protein without signal peptide. Phylogenetic tree analysis showed that FADS2 gene was highly conserved in different species and evolutionary processes,buffalo was close to Bos mutus and Bos taurus, and was far from Rattus norvegicus. This study suggested that FADS2 gene was successfully cloned in buffalo, which laid a foundation for clarifying the mechanism of milk performance in buffalo.     

水牛FADS2基因的电子克隆及序列分析

试验旨在利用电子克隆法对水牛Δ6脂肪酸脱氢酶（Δ6-fatty acid desaturases,FADS2）基因进行克隆和生物信息学分析,为探究FADS2基因对水牛泌乳性能的作用机制奠定基础。以奶牛FADS2基因序列（GenBank登录号：NM_001083444.1）为探针设计引物,利用电子克隆法克隆水牛FADS2基因,并通过RT-PCR验证,对FADS2基因的序列特征进行生物信息学分析。测序结果表明,水牛FADS2基因序列全长为36 600 bp,由12个外显子和11个内含子组成,包含一个长1 335 bp的开放阅读框,可编码444个氨基酸。序列同源性分析显示,水牛FADS2基因编码序列与牦牛、黄牛、人、猪、家兔、虎鲸和褐家鼠序列的同源性分别为98.88%、98.88%、89.66%、90.79%、90.85%、92.35%和87.11%。蛋白质预测分析表明,水牛FADS2蛋白分子质量为52.51 ku,理论等电点（pI）为8.75,呈弱碱性,属于亲水性蛋白,无信号肽。系统进化树分析结果表明,FADS2基因在不同物种及进化的过程中具有高度保守性,其中水牛与牦牛、黄牛亲缘关系较近,与褐家鼠亲缘关系较远。水牛FADS2基因的成功克隆为今后阐明水牛泌乳性能的作用机制奠定了基础。     

三穗麻鸭Cofilin2基因序列分析及其组织表达研究

为探究三穗麻鸭丝切蛋白2（Cofilin2）基因特征及其在鸭组织中的表达规律,试验根据GenBank中鸡Cofilin2基因序列设计特异性引物,利用RT-PCR方法扩增获得目的基因,利用生物信息学软件分析三穗麻鸭Cofilin2基因特征,实时荧光定量PCR方法检测Cofilin2基因在三穗麻鸭不同组织中的表达情况。结果显示,三穗麻鸭Cofilin2基因编码区大小为498 bp,可编码166个氨基酸;三穗麻鸭Cofilin2基因序列与鸡、猕猴、牛、小鼠、安大略鲑、人和猪相应序列的同源性分别为94.4%、91.6%、91.6%、87.6%、77.7%、70.5%和69.5%;系统进化树显示,Cofilin2基因在不同物种进化过程中高度保守;Cofilin2基因编码蛋白的二级结构由α-螺旋、延伸链、β-转角和无规则卷曲组成,三级结构呈弯曲螺旋结构;实时荧光定量PCR检测显示,三穗麻鸭Cofilin2基因在心脏、肝脏、脾脏、肺脏、肾脏、脑、胸腺、十二指肠、腿肌和法氏囊等组织器官中均有不同程度表达,其中心脏中表达量最高,法氏囊中表达量最低。本试验结果为三穗麻鸭抗病分子机理研究奠定了基础。