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MYF5基因克隆分析及真核表达载体构建

本研究旨在对广西沼泽型水牛MYF5基因进行克隆和分析,并构建其真核表达载体。应用RT-PCR方法从水牛肌肉组织中扩增MYF5基因,测序鉴定后对其核苷酸序列和蛋白质序列进行生物信息学分析,同时构建该基因真核表达载体,并在细胞水平验证所构建载体的正确性。结果表明:水牛MYF5基因编码区序列长度为768 bp,编码255个氨基酸,其核苷酸序列与牛、山羊、猪、人、猩猩和狼相应序列的相似性分别为99%、98%、94%、91%、90%和89%;水牛MYF5基因在不同物种及进化过程中具有高度保守性;MYF5蛋白为膜外蛋白,具有MYOD家族标志性MYOD结构域;构建水牛MYF5基因真核表达载体pMXs-MYF5,转染HEK293T、水牛颗粒细胞后产生绿色荧光信号,表明细胞表达MYF5基因。     

水牛KDM4D基因克隆分析及其真核表达载体的构建

旨在克隆水牛组蛋白去甲基化酶KDM4D (lysine K-specific demethylase 4D)基因,对其进行生物信息学分析,并构建KDM4D基因的真核表达载体,为研究KDM4D基因在水牛体细胞核移植(somatic cell nuclear transfer,SCNT)胚胎发育中的作用奠定基础。试验从水牛睾丸组织中提取总RNA,反转录cDNA后, RT-PCR技术克隆KDM4D基因片段,并运用生物信息学软件对序列进行分析;随后将KDM4D基因片段连接至真核表达载体pEGFP-C1,再把重组质粒转染进HEK293T细胞。结果表明,克隆所得的水牛KDM4D基因片段编码区全长1 155 bp,共编码384个氨基酸。水牛KDM4D基因与黄牛、山羊、绵羊、犬、猫、猪、人、小鼠、大鼠的同源性分别为98.4%,96.6%,96.3%,84.9%,84.1%,81.4%,80.4%,77.4%,77.3%。多重比较和进化树分析结果显示,KDM4D基因在不同物种及生物进化中具有较高的保守性。蛋白结构域分析显示,克隆所得的KDM4D蛋白结构特殊,只具有1个JmjN (Jumonji N)和1个JmjC (jumonji C)结构域,与KDM4家族其他成员相比缺少PHD (plant homeodomain)和Tud (tudor)功能域。KDM4D蛋白质三级结构预测结果显示,水牛、黄牛和人3种物种间具有较高的相似性。转染结果显示,构建的重组质粒可以在HEK293T细胞中表达。本试验成功克隆水牛KDM4D基因,对其进行生物信息学分析,构建了真核表达载体pEGFP-C1-KDM4D,并在HEK293T细胞中成功表达。     

猪JHDM2A基因的克隆及表达分析

试验旨在克隆猪JHDM2A基因,并研究其在猪卵巢组织中的表达情况。首先克隆猪JHDM2A基因,并构建pLVX-IRES-ZsGreen1-JHDM2A真核表达载体,同时对JHDM2A基因在猪卵泡发育过程中的表达情况进行分析。结果显示,克隆得到的猪JHDM2A基因编码区长度为3 945 bp,编码1 315个氨基酸。通过多重氨基酸序列比对发现,猪JHDM2A基因与黄牛、水牛、绵羊和人相应氨基酸序列的同源性分别为93.5%、94.7%、94.7%和93.8%。蛋白质分子系统进化树分析结果表明,JHDM2A基因在物种进化过程中高度保守。通过脂质体转染法将构建的pLVX-IRES-ZsGreen1-JHDM2A真核表达载体导入HEK293T细胞,可观察到清晰的绿色荧光蛋白表达。免疫组化结果显示,JHDM2A蛋白在不同发育阶段猪卵泡中均有表达。本试验通过克隆获得猪JHDM2A基因序列,JHDM2A蛋白在猪卵巢中高度表达,表明其功能可能与猪卵泡发育密切相关。     

水牛HSD17B1基因克隆分析及其真核表达载体构建

为了阐明水牛17β-羟类固醇脱氢酶1 (17 beta-hydroxysteroid dehydrogenase 1,HSD17B1)基因对水牛繁殖性能的影响,本试验采用了3'-RACE克隆获得HSD17B1基因,并对其核苷酸序列和蛋白质序列进行了生物信息学分析,通过构建其真核表达载体并转染293T细胞验证所构建载体的准确性。结果表明,水牛HSD17B1基因编码区长954 bp,3'-UTR区长58 bp,编码317个氨基酸。BLAST分析显示水牛HSD17B1核苷酸序列与牛、绵羊、猪、马、犬、非洲象和人的相似性分别为100%、100%、92%、94%、87%、87%和87%,系统进化树分析结果表明,HSD17B1基因在不同物种及进化的过程中具有高度保守性。蛋白质分析结果表明HSD17B1蛋白呈弱酸性,无信号肽,亚定位于细胞质,存在type1_17beta-HSD-like_SDR_c、PRK05993、LPOR和FabG等结构域。试验成功构建了水牛HSD17B1基因真核表达载体pEGFPN1-HSD17B1,转染293T后,产生较强的绿色荧光信号,表明能够形成HSD17B1-EGFP融合蛋白。水牛HSD17B1基因的克隆及其真核表达载体的成功构建,为今后阐明HSD17B1基因在水牛卵泡及胚胎发生过程中的作用及分子机制奠定了理论基础。     

梅花鹿Smad2与Smad4基因的克隆及其在鹿茸顶端组织的表达

为了从梅花鹿鹿茸顶端组织中克隆Smad2与Smad4基因的编码区(CDS)序列,分析其分子特性及在鹿茸顶端不同组织中的表达情况,试验采用TRIzol法提取鹿茸顶端组织总RNA,以PCR方法获得Smad2与Smad4基因,并利用生物信息学软件对其进行生物信息学分析,免疫组化法检测Smad2与Smad4基因在鹿茸顶端不同组织中的表达水平。结果表明:Smad2基因完整的CDS序列长度为1 404 bp,编码467个氨基酸,与牛、人、马和猪的Smad2核苷酸序列同源性分别为98.29%、94.52%、95.30%和95.51%;Smad4基因完整的CDS序列长度为1 662 bp,编码553个氨基酸,与牛、人、马和猪的Smad4核苷酸序列同源性分别为98.26%、94.89%、95.85%和95.97%;Smad2与Smad4蛋白的分子质量分别为52.24 ku与60.50 ku,理论等电点分别为6.13与6.50,均具有较强的亲水性;梅花鹿Smad2与Smad4基因在茸皮层、间充质层和软骨层中均有表达,在软骨层中表达水平较高。说明梅花鹿Smad2与Smad4基因在鹿茸软骨层中表达水平较高,对鹿茸再生发育具有一定的调节作用。     

水牛p21基因在卵母细胞体外成熟过程中的表达及其真核表达载体构建

旨在检测p21基因在水牛卵母细胞体外成熟过程中的表达变化,并克隆摩拉水牛p21基因CDs序列,构建真核表达载体。收集体外成熟培养不同时期(0、6、12、24h)和不同形态[有第1极体(first polar body,PB1)PB1和无PB1]的水牛卵母细胞,通过RT-qPCR检测其p21mRNA相对表达量。同时利用RTPCR技术从摩拉水牛卵巢颗粒细胞中扩增出p21基因编码序列,插入pEGFP-N1真核表达载体中,构建出重组质粒。将重组质粒pEGFP-N1-p21转染水牛颗粒细胞,并于培养24h和48h后观察转染细胞的荧光表达情况。收集转染48h后的颗粒细胞,通过RT-qPCR检测p21mRNA相对表达量。结果显示,成熟培养6h的卵母细胞p21mRNA表达量显著高于成熟培养0、12、24h的表达量(P0.05),有PB1的卵母细胞中p21mRNA的表达量显著高于无PB1的卵母细胞(P0.05);重组质粒pEGFP-N1-p21转染颗粒细胞后,有绿色荧光蛋白表达,且与转染pEGFP-N1和空白组相比,pEGFP-N1-p21转染组的p21mRNA表达量显著升高。结果表明,在体外成熟培养过程中均能检测到水牛卵母细胞p21mRNA表达量,成功构建了摩拉水牛p21基因真核表达载体,为下一步研究p21基因在水牛卵母细胞成熟和胚胎发育过程中的功能和调控奠定了基础。     

水牛PTHLH基因克隆分析及其真核表达载体构建

为揭示甲状旁腺激素样激素(parathyroid hormone-like hormone,PTHLH)基因对水牛繁殖性能的影响,本研究对水牛PTHLH基因进行克隆,并对其核苷酸和氨基酸序列进行生物信息学分析。以牛PTHLH基因为种子序列(GenBank登录号:NM_001290949),应用CE Design软件设计引物序列,运用PCR扩增和测序技术获得水牛完整编码区序列,使用DNAMAN、ProtParam、SOPMA、PSORTⅡPrediction等在线软件分析PTHLH蛋白的一级结构、二级结构、三级结构与理化性质,并进行同源性比对分析及系统进化树构建。结果显示,试验克隆了水牛PTHLH基因完整编码区序列,该序列长为534bp,可编码177个氨基酸。水牛PTHLH基因编码区核苷酸序列与黄牛、猪、马、山羊、绵羊和骆驼的同源性分别为98.3%、90.4%、90.1%、98.1%、97.5%和89.2%,物种之间同源性较高,系统进化树分析结果与其亲缘关系远近一致,表明水牛PTHLH基因编码区在进化过程中比较保守。蛋白理化性质分析显示,水牛PTHLH蛋白分子式为C895H1451N271O266S2,分子质量为2 885u,半衰期为30h,理论等电点(pI)为10.00,水溶液在280nm处的消光系数为23 950,肽链N端为蛋氨酸(Met),不稳定系数为60.04,属于碱性不稳定蛋白;脂肪系数为72.15,总平均亲水性为-0.928,该蛋白属于不可溶性蛋白,亚细胞定位于细胞核、细胞质和线粒体。结构域预测结果显示,水牛PTHLH蛋白包含有1个PTH区域,同时还包含有1个低复杂度区域。二级结构分析显示,水牛PTHLH蛋白包含83个α-螺旋(46.89%)、17个延伸链(9.60%)、10个β-转角(5.66%)和67个无规则卷曲(37.85%),与三级结构预测结果相一致。试验构建了PTHLH基因真核表达载体pcDNA3.1-PTHLH,并通过电泳和测序验证了载体的准确性。PTHLH基因的成功克隆及其真核表达载体的成功构建为今后研究水牛PTHLH基因的功能和遗传特性提供了材料。     

湖羊Sp1基因CDS区克隆及其对颗粒细胞增殖和凋亡的影响

旨在探讨湖羊Sp1基因序列特征及其对颗粒细胞增殖及凋亡的影响。采用克隆、测序获得湖羊Sp1基因CDS区并用生物信息学方法分析其序列特征;NJ方法构建基于Sp1氨基酸序列的系统进化树;RT-PCR方法检测Sp1基因湖羊组织表达谱;克隆方法构建湖羊Sp1过表达载体;试剂盒检测Caspase-3活性和CCK-8值;利用流式细胞术检测人卵巢颗粒细胞(KGN)凋亡率。结果表明,湖羊Sp1CDS区长2 340bp,编码779个氨基酸残基,与人的核苷酸序列和氨基酸序列的同源性分别为94.28%和95.75%;湖羊与牛的亲缘关系最近;Sp1基因在湖羊卵巢、卵泡等组织中广泛表达。本研究成功构建了湖羊Sp1过表达载体并转染人卵巢颗粒细胞(KGN);试验组Caspase-3活性显著高于对照组(P0.05),CCK-8值检测结果相反。表明过表达Sp1基因抑制KGN-细胞增殖(P0.05)、促进KGN-细胞凋亡(P0.05)。Sp1基因在哺乳动物中高度保守,湖羊Sp1基因可抑制人卵巢颗粒细胞(KGN)增殖、诱导其凋亡。     

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.     

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 Construction of Eukaryotic Expression Vector of Buffalo HSD17B1 Gene

In order to clarify the effect of 17 beta-hydroxysteroid dehydrogenase 1 (HSD17B1) gene on the reproductive performance of buffalo,in this study,buffalo HSD17B1 gene was cloned by 3'-RACE,analyzed by bioinformatics,and studied with eukaryotic vector construction and cell transfection technology.The results showed that the coding region of buffalo HSD17B1 was 954 bp,3'-UTR was 58 bp,encoded 317 amino acids.The buffalo HSD17B1 gene shared 100%,100%,92%,94%,87%,87% and 87% of similar nucleotide sequence with that of Bos taurus,Ovis aries,Sus scrofa,Equusca ballus,Canis lupus,Loxodonta africana and Homo sapiens,respectively.Phylogenetic tree analysis showed that HSD17B1 gene was highly conserved in different species and evolution.HSD17B1 protein was weakly acidic,without signal peptide,located inthe cytoplasmic,and with the presence of type1_17beta-HSD-like_SDR_c,PRK05993,LPOR and FabG domains.The buffalo HSD17B1 eukaryotic expression vector was successfully constructed,after transfected into 293T cell lines,HSD17B1-EGFP fusion protein was detectable.In a word,the success cloning and construction of eukaryotic expression vector of buffalo HSD17B1 gene,provided an important reference for surveying the regulation mechanism of HSD17B1 gene during buffalo folliculogenesis and embryogenesis.     

水牛水通道蛋白8基因克隆及表达分析

In this study, buffalo AQP8 gene was cloned and its eukaryotic expression vector was constructed, the expression pattern of AQP8 gene in buffalo ovary tissue was also assayed. The results showed that the CDS length of cloned buffalo AQP8 gene was 732 bp, and it shared 100% homology of amino acid sequence with cattle and mouse. AQP8 protein was detected in different developmental stages of buffalo follicles, it had significantly higher expression in the secondary follicles than that of in the primordial and the primary follicles (P＜0.05), and it mainly expressed in the granulosa cells of the secondary follicles. Clear EGFP green fluorescent was observed in transfected cell groups with transfection of the pEGFP-N1-AQP8 eukaryotic expression plasmid into HEK293T cells by Lipofectamine^R LTX and PLUS^TM reagent. The above results lay foundation to further investigate the function of AQP8 gene in the buffalo follicle development and granulosa cell apopotosis.     

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。