Sequence of GDF 8 (Myostatin) Gene in Bubalus Bubalis

The sequence of myostatin gene (growth differentiation factor 8 [GDF 8]) in Indian riverine buffalo (Bubalus bubalis) is reported. The genomic DNA as well as mRNA were sequenced. The sequence is conserved across all the livestock species. Five nonsynonymous changes as compared to cattle were found in this study and were also confirmed by mRNA sequence. Two intronic single nucleotide polymorphisms (SNPs) were identified in buffalo.     

Isolation and Sequence Characterization of Mammary Derived Growth Inhibitor Gene of Riverine Buffalo (Bubalus Bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5′ UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3′ UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI//fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

BRCA-1 Gene Expression and Comparative Proteomic Profile of Primordial Follicles from Young and Adult Buffalo (Bubalus bubalis) Ovaries

In our previous study, we demonstrated that the repair efficiency of DNA double-strand breaks declines with increasing age in rat primordial follicles. In the present study, we extended our studies to buffalo (Bubalus bubalis) wherein we studied the expression of BRCA-1 related DNA repair genes in primordial follicles of young (12 months-22 months) and adult (72–96 months) buffaloes. The relative expression of selected genes, as determined by RT-PCR, revealed a significant (p?相似文献   

Molecular Cloning and Characterization of the Myostatin Gene in Croceine Croaker, Pseudosciaena crocea

Myostatin (MSTN) is a negative regulator of skeletal muscle mass and has a potential application in aquaculture. We reported the characterization of the myostatin gene and its expression in the croceine croaker, Pseudosciaena crocea. The myostatin gene had three exons encoding 376 amino acids. The cDNA was 1,906 bp long with a 5′-UTR and 3′-UTR of 108 bp and 667 bp, respectively. A microsatellite sequence, CA₃₀ and CA₂₆ separated by TA, existed in the 3′-UTR. Intron I and II were 343 bp and 758 bp in length, respectively. The deduced amino acid sequence was highly conserved, and had more than 90% identical to shi drum, gilthead seabream, striped sea-bass, white perch, and white bass proteins. The myostatin of croceine croaker had a putative amino terminal signal sequence (residues 1–22), a transforming growth factor-beta (TGF-β) propeptide domain (residues 41–256), a RXXR proteolytic processing site (RARR, residues 264–267, matching the RXXR consensus site), and a TGF-β domain (residues 282–376). There were 13 conserved cysteine residues in croceine croaker myostatin, nine of which are common to all TGF-β superfamily members. The most conserved region of vertebrate myostatins is the TGF-β domain, which was the mature bioactive domain of the myostatin protein. The myostatin gene was expressed not only in the skeletal muscle, but also in the other tissues.     

Molecular Characterization and Comparison of Phospholipase C zeta (PLCZ1) Gene Between Swamp (Bubalus carabanensis) and Riverine (Bubalus bubalis) Buffaloes: Its Implications and Future Perspectives

Phospholipase C zeta, a novel sperm-specific protein which is widely known to induce oocyte activation following fertilization, had already been characterized in various mammalian species, but not in water buffaloes thus far. The present study was conducted to initially characterize and compare the sequences of PLCZ1 gene of swamp and riverine buffaloes. Semen samples were collected; total RNA was extracted and reverse-transcribed. PLCZ1 cDNA was then amplified, and submitted for sequencing. Buffalo PLCZ1 gene yielded a sequence of 1905 base pair nucleotides translated into 634?bp amino acids. In general, the buffalo PLCZ1 gene was found to have high sequence identity with cattle and other domestic species. Similarly, significant residues and motifs in PLCZ1 gene sequence are found conserved in water buffaloes. However, there are variations in sequences identified between types of water buffaloes that may play a role in species-specific differences in terms of gene and protein expression, physiological mechanisms, and biological functions. The molecular information on buffalo PLCZ1 gene is highly valuable in subsequent works such as correlation studies on the identified gene variations with semen quality and fertility, and the development of biomarkers for bull fertility.     

大黄鱼MSTN成熟肽区基因的原核表达

根据本实验室克隆的大黄鱼肌肉生长抑制素(MSTN)基因编码序列设计引物,通过RT-PCR法扩增大黄鱼MSTN成熟肽区域的基因,将目的片段插入pMD18-T克隆质粒,并测序验证。验证后用BamHⅠ和HindⅢ限制性内切酶酶切含目的片段的pMD18-T克隆质粒和pET-28 a表达质粒,构建MSTN-pET-28 a重组表达质粒。将重组表达质粒转化至BL21菌中,经IPTG诱导后表达蛋白的大小约17 kD。     

Ontogenic Development of Corticotrophs in Fetal Buffalo (Bubalus Bubalis) Pituitary Gland

To evaluate the subpopulation of corticotrophs in developing buffalo (Bubalus bubalis) fetus, pituitary glands were recovered (n=6 per group) from late first, second and third gestational female buffalo dams. The corticotrophs were identified by using specific antibodies against proopiomelanocortin (POMC) and adrenocorticotrophic hormone (ACTH) through immunohistochemistry. There was a significant (P≤0.05) increase of immunoreactive (ir) ir-ACTH cells during late 2^nd trimester while, ir-POMC cells were more (P≤0.05) at late 3^rd trimester of gestation as compared to other age groups. The quantity of co-localized cells for POMC and ACTH was significantly (P≤0.05) greater at the end of 1^st gestation rather than 2^nd and 3^rd gestational fetal adenohypophyseal cells. This study is the first to demonstrate co-localization of POMC+ACTH and the affect of gestational age on the expression of these cells in buffalo fetus adenohypophysis.Key words: buffalo, corticotrophs, fetus, pituitary gland, immunohistochemistry     

Water Buffalo (Bubalus bubalis) as a spontaneous animal model of Vitiligo

Vitiligo is a multifactorial acquired depigmenting disorder. Recent insights into the molecular mechanisms driving the gradual destruction of melanocytes in vitiligo will likely lead to the discovery of novel therapies, which need to be evaluated in animal models that closely recapitulate the pathogenesis of human vitiligo. In humans, vitiligo is characterized by a spontaneous loss of functional melanocytes from the epidermis, but most animal models of vitiligo are either inducible or genetically programmed. Here, we report that acquired depigmentation in water buffalo recapitulates molecular, histological, immunohistochemical, and ultrastructural changes observed in human vitiligo and hence could be used as a model to study vitiligo pathogenesis and facilitate the discovery and evaluation of therapeutic interventions for vitiligo.     

绵羊肌肉生长抑制素MSTN原核表达及其纳米抗体文库的构建与鉴定

克隆绵羊肌肉生长抑制素（myostatin,MSTN）基因并在大肠杆菌中诱导表达,纯化重组蛋白免疫健康的双峰驼（Bactrian camel）,分离其外周血淋巴细胞提取总RNA,利用RT-PCR扩增骆驼重链抗体IgG2、IgG3的可变区（VHH）基因片段,将VHH片段与pCANTAB5E连接后电转入大肠杆菌TG1构建纳米抗体文库。结果显示,纳米抗体文库容量为9.5×10⁵,挑取部分克隆进行测序分析,所获得的纳米抗体文库具有良好的多态性,为进一步筛选绵羊MSTN的高特异性纳米抗体片段奠定了基础。     

军曹鱼肌生成抑制素基因的克隆与序列分析

肌生成抑制素(Myostation,MSTN)是一种骨骼肌生长的负调控因子,其生物功能主要是抑制骨骼肌的生长。肌生成抑制素的活性降低或丧失,可使肌肉与其他组织的比例大大提高,因此在动物育种和医疗上有很大的潜在应用价值。目前包括鱼类在内的20多种脊椎动物的MSTN cDNA已经得到克隆和测序。本实验依据已知的鱼MSTN cDNA的保守区域设计一对特异引物,利用PCR技术分别从军曹鱼基因组中扩增出一个约1000bp的特异片段和300bp片段,所得目的片段回收纯化,将其酶切产物连接到pMDl8-T克隆载体上,转化入JM109感受态细胞中,挑取阳性克隆进行转化子鉴定,其质粒测序结果与文献报道的一致,证明成功地克隆了军曹鱼肌生成抑制素基因。     

Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis)

In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β(1) domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.     

鳜肌肉生长抑制素Myostatin cDNA克隆与组织表达分析

利用RT-PCR和cDNA末端快速扩增法(RACE)克隆了鳜(Siniperca chuatsi)肌肉生长抑制素(myostatin,MSTN)cDNA序列,并分析了该基因的结构特征和亲缘关系。鳜MSTN cDNA序列全长2627bp,包括5′端非翻译区117bp、3′端非翻译区1376bp和开放阅读框(ORF)1134bp,共编码377个氨基酸,含22个氨基酸的信号肽。鳜MSTN具有脊椎动物MSTN的共同序列特征,含有1个蛋白酶水解位点RARR和9个保守的半胱氨酸残基。脊椎动物MSTN氨基酸序列的亲缘关系分析表明,鳜与其他鱼类聚为一支。RT-PCR分析表明,鳜MSTN在成体不同组织中的表达情况不同,其中,卵巢、肾、眼、肌肉、心、脑、皮肤和胃中有表达,肝胰脏未见表达。     

Water buffalo (Bubalus bubalus Arnee) allotypes: Identification of a multiple allelic system

This paper describes two allotypes of water buffaIo controlled by two codominant allelic genes. The third allele is a null allele and behaves as a recessive one. The two detectable serum antigens are termed A1 and A2 aild the third one (as yet undetectable) A0. The A1 antigen was recovered in the third peak and A2 antigen in the first peak following gel filtration through Sephadex G-200. The A1 antigen is common to water buffalo and cattle; the frequency of the corresponding gene (A¹) was the same in both species.     

双须骨舌鱼卵黄蛋白原基因vtg的克隆、组织表达和原核表达分析

为探究双须骨舌鱼(Osteoglossum bicirrhosum)卵黄蛋白原vtg基因的结构和表达特征,本研究运用RACE技术克隆双须骨舌鱼vtg全长c DNA序列。序列分析发现,双须骨舌鱼vtg的c DNA全长为5 325 bp,开放阅读框(ORF)为5 121 bp,其5′和3′非编码区(UTR)分别为46 bp和159 bp,共编码1 706个氨基酸,预测蛋白质分子量约为186.79 ku。同源性分析发现,双须骨舌鱼与同科的美丽仆骨舌鱼(Scleropages formosus)相似度达到84.78%,与鲤形目、鲈形目、鲱形目、鲽形目鱼类的相似度均高于50%。系统进化树分析结果显示,本文获得的双须骨舌鱼vtg基因与骨舌鱼目聚为一类,与鳗鲡目硬骨鱼类亲缘关系较近,与双须骨舌鱼进化地位相符。荧光定量PCR检测结果表明,在雌雄鱼性腺和肝中vtg均有表达,且在雌鱼肝中的相对表达量显著高雄鱼(P0.05),卵巢和精巢表达量无显著差异(P0.05);在雌雄鱼鳃、脾、肾、肌肉、心、头肾、脑等7个组织中均极微量表达,且无显著差异(P0.05)。在Ⅱ、Ⅲ、Ⅳ期卵巢中vtg相对表达量依次增加,Ⅳ期显著高于Ⅱ和Ⅲ期(P0.05),也显著高于精巢Ⅳ期(P0.05);在雌鱼肝组织中呈先增后减趋势,且Ⅱ、Ⅲ、Ⅳ期之间无显著差异(P0.05),但每个时期都显著高于雄性(P0.05)。成功构建了原核表达载体p EASY-Blunt E2-vtg,并转入Transetta(DE3)表达感受态细胞中成功诱导出融合蛋白。Western-blotting检测显示,融合蛋白可被抗His标签特异性识别。综上表明,vtg表达水平高低与性别相关,并与性腺发育程度有关。此外,本研究结果也为后续深入研究卵黄蛋白原的生理功能打下基础。     

斑鳜myostatin基因及其启动子的克隆与序列分析

采用Tail-PCR和常规PCR技术首次克隆出斑鳜myostatin基因及其启动子序列。经生物信息学分析发现,斑鳜myostatin基因由3个外显子和2个内含子组成,编码区1131bp,共编码376个氨基酸。斑鳜myostatin启动子区域大小为840bp,存在1个TATAA-box、4个E-box和1个CAAT-box作用元件。利用软件CLUSTALW和MEGA3.1构建14种硬骨鱼的myostatin启动子的系统进化树结果表明:斑鳜同大口黑鲈亲缘关系最近,与鲤鱼和缨野鲮亲缘关系较远,其结果与传统形态学分类中的亲缘关系一致。斑鳜myostatin基因及其启动子克隆与特征分析,将为进一步研究鱼类myostatin基因的表达调控及其功能分析提供参考。     

水牛皮肤成纤维细胞的分离与体外培养

探讨水牛成纤维细胞的分离与传代培养方法。组织块培养法培养的成纤维细胞原代生长较慢,需12天左右方可汇合形成单层,而酶消化法培养的成纤维细胞原代生长相对生长快,仅需8天便可汇合形成单层。两种方法传代细胞的生长速度相似,仅需4-5天就可汇合形成单层。通过体细胞的核型分析发现,成纤维细胞在传代培养过程中的核型变化不大,66．67％～81．67％的细胞具有正常的二倍体核型,各代之间无显著差异。结果表明,水牛成纤维细胞均能稳定地进行传代培养。     

查尔酮合酶基因的克隆、全序列分析及在大肠杆菌中的高效表达

类黄酮是植物中的一种重要的次级代谢产物,它与植物的花色形成有关。查尔酮合酶是类黄酮合成途径中的一个关键酶,在植物体内,ＣＨＳ表达量的增加或减少都可能改变花的。从矮牵牛花瓣的ｃＤＮＡ中克隆到了ＣＨＳ－Ａ基因,进行了全序列分析,并与国外已报道的ＣＨＳ－Ａ－序列进行了同源性比较。     

Polymorphism of Mitochondrial DNAs of Yunnan Domestic Water Buffaloes, Bubalus bubalis, in China, Based on Restriction Endonuclease Cleavage Patterns

Restriction endonuclease cleavage patterns of mitochondrial DNA(mtDNA) of three local types of Yunnan native water buffalo were analyzed using 18 enzymes which recognize six nucleotides. Among the 12 animals analyzed, 3 of 18 enzymes, BamHI, EcoRI, and ScaI, revealed polymorphisms. Three mtDNA types were identified. The results indicate that a relatively low level of mtDNA variation exists in Yunnan domestic water buffaloes. The origin of Chinese buffalo derived from Yunnan province of China is discussed.     

尾悬吊大鼠比目鱼肌‘肌肉生长抑制素’基因的表达水平

目的确定尾悬吊大鼠比目鱼肌组织中‘肌肉生长抑制素’(myostatin,MSTN)基因的表达水平。方法在测定尾悬吊14、30 d大鼠后肢比目鱼肌相对湿重的基础上,采用real-time RT-PCR方法,在基因水平对尾悬吊14、30 d大鼠比目鱼肌中MSTN mRNA水平进行检测。结果尾悬吊14、30 d后比目鱼肌的相对湿重分别下降了11%和19%。real-time RT-PCR结果表明,大鼠尾悬吊14、30 d后,比目鱼肌中MSTN mRNA表达水平均有所提高,分别是对照大鼠的2.2倍和3.5倍。结论尾悬吊大鼠后肢去负荷后可诱导抗重力肌-比目鱼肌组织中MSTN mRNA表达水平的上调。     

Water buffalo (Bubalus bubalus Arnee) allotypes: Identification of two specificities controlled by independent genes

Two water buffalo allotypes (Bl and CI) are described, which are located on distinct low molecular weight molecules. Bl is common to water buffalo and cattle. These two markers are inherited in a simple Mendelian manner and controlled by two independent genes.