Molecular cloning,sequence characterization,polymorphism and association analysis of porcine ROPN1 gene

The full-length cDNA sequence of one porcine gene, ROPN1, was isolated using the rapid amplification of cDNA ends (RACE) method based on one pig EST sequence which was highly homologous to the coding sequence of human ROPN1 gene. The porcine ROPN1 gene encodes a protein of 212 amino acids which shares high homology with the rhophilin associated protein 1 (ROPN1) of eight species: gray short-tailed opossum (96%), horse (95%), cattle (94%), mouse (93%), rat (92%), chimpanzee (85%), human (85%) and rhesus monkey (85%). Phylogenetic analysis revealed that the porcine ROPN1 gene has a closer genetic relationship with the ROPN1 gene of gray short-tailed opossum. Polymorphism analysis showed that there was a T/C mutation at the position of 536 bp of mRNA and this leaded to the amino acid alteration from the Arg residue to the Cys residue. PCR-Hae III-RFLP was established to detect this T/C mutation and eight pig breeds display obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and results demonstrated that this polymorphic locus was significantly associated with the litter size of first parity (P < 0.01) and all parities (P < 0.05) in Large White sows, and also significantly associated with the litter size of all parities in Landrace sows (P < 0.01). Therefore, ROPN1 gene could be a useful candidate gene in selection for increasing litter size in pigs. These data serve as a foundation for further insight into this novel porcine gene.     

Molecular cloning and characterization of canine metalloproteinase-9 gene promoter

This paper describes the cloning and characterization of the canine matrix metalloproteinase-9 (MMP-9) gene promoter. The 5' untranslated region was obtained by genome walking upstream of the canine MMP-9 translation start site using canine genomic DNA as template. A DNA fragment of 1894 bp was isolated and on analysis demonstrated regions of sequence homology with the MMP-9 promoter sequences already determined for other species. In general, conserved regions correlated with DNA binding motifs such as a TATA-like box, AP-1 sites, GC boxes and a nuclear factor-kappaB binding domain. The DNA promoter fragment was sufficient to drive basal expression of a luciferase reporter gene in Madin Darby canine kidney (MDCK) cells and to a lesser extent in feline embryonic fibroblast (FEA) cells. Activity of the promoter was enhanced by the treatment of transfected MDCK cells with phorbol 12-myristate 13-acetate but no effect was observed in the FEA cells. Promoter deletion studies revealed that regions of promoter were necessary for induction of reporter gene expression.     

Molecular characterization, expression pattern, polymorphism and association analysis of bovine ADAMTSL3 gene

A disintegrin-like and metalloprotease domain with thrombospondin type I motifs-like 3 (ADAMTSL3) is an important candidate gene for body measurement traits through marker-assisted selection (MAS). The objective of this study was to analyze SNP of bovine ADAMTSL3 gene and its specific expression in tissues to explore its possible correlation with body measurement traits in Bos taurus. Our genomic structural analysis showed that bovine ADAMTSL3 shares much similarity with human ADAMTSL3. Gene expression analysis indicated that the order of specific tissue expression patterns of bovine ADAMTSL3 was the testis, heart, fat, stomach, small intestine, liver, lung, trachea, kidney, spleen, large intestine and muscle. Allele frequencies demonstrated significant variance in different local cattle breeds. Also, the T1532C SNP in exon 13 and C1899T SNP in exon 15 were significantly correlated with Body Length (BL), Chest Depth (CD), Heart Girth (HG) and Pin Bone Width (PBW). Furthermore, C1899T SNP marker had significant correlation with the PBW (P = 0. 0235) based on 867 individual analysis. In the total population, combination of T1532C and C1899T SNPs significantly affected on the BL (P = 0.0028), CD (P < 0.0001), HG (P = 0.0002) and PBW (P = 0.0049). These results suggest that the ADAMTSL3 gene, as one of target genes for body measurement traits in cattle breeds, could be used as a genetic marker to select excellent body type of cattle population in the animal breeding program.     

LRP16基因启动子克隆及特征分析

克隆LRP16基因启动子分子,并对启动子特征进行分析,预测启动子区调控元件,为深入研究LRP16基因的表达调控机制奠定基础。在NCBI的人类基因组数据库中截取并下载LRP16基因转录起始位点5′侧翼区2.7kb的基因组序列,设计PCR引物,从健康外周血单个核细胞中扩增,利用Genomatix程序对5′侧翼区近1000bp进行启动子特征分析,获得了与GenBank序列一致,长度为2.7kb的LRP16基因启动子DNA序列,该序列具有典型的真核生物RNA聚合酶Ⅱ启动子特征及多个核受体结合位点,如α视黄酸受体及RAR相关孤生受体。     

Molecular gene cloning, expression, and characterization of bovine brain glutamate dehydrogenase

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The Km and Vmax values for NAD+ were 0.1 mM and 1.08 micromol/min/mg, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - 100 microM, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.     

Molecular cloning and characterization of the promoter region of the inducible nitric oxide synthase gene of the rat

To investigate the mechanism by which inducible nitric oxide synthase (iNOS) within vascular smooth muscle cells (VSMC) is expressed following exposure to proinflammatory cytokines, we cloned the promoter region of the rat iNOS gene by a single specific primer PCR cloning strategy. The 5;-flanking region of the rat iNOS gene contains interferon-gamma (IFN-gamma)- and tumor necrosis factor-alpha (TNF-alpha)-responsive elements and an NF-kappaB-binding consensus sequences. The position and alignment of these consensus sequences are distinct from those in the mouse iNOS and human iNOS genes. It was shown that some nuclear factor(s) which bound specifically to the fragment of the rat iNOS gene containing several consensus sequences were produced after VSMC were treated with interleukin 1 (IL-1) and IFN-gamma.     

Molecular cloning,sequence identification,and gene expression analysis of bovine ADCY2 gene

Adenylyl cyclase 2 (ADCY2), a class B member of adenylyl cyclases, is important in accelerating phosphor-acidification as well as glycogen synthesis and breakdown. Given its distinct role in flesh tenderization after butchering, we cloned and sequenced the ADCY2 gene from Yanbian cattle and assessed its expression in bovine tissues. A 2947 bp nucleotide sequence representing the full-length cDNA of bovine ADCY2 gene was obtained by 5′ and 3′ remote analysis computations for gene expression. Analyses of the putative protein sequence showed that ADCY2 had high homology among species, except with the non-mammal Oreochromis niloticus. Gene structural domain analyses in humans and rats indicated that the ADCY2 protein had no flaw; only the transmembrane domain was reduced and the CYCc structure domain was shortened. Assessment of ADCY2 expression in bovine tissues by real-time PCR showed that the highest expression was in the testes, followed by the longissimus dorsi, tensor fasciae latae, and latissimus dorsi. These data will serve as a foundation for further insight into the cattle ADCY2 gene.     

Molecular cloning, expression and characterization of bovine UQCC and its association with body measurement traits

Ubiquinol-cytochrome c reductase complex chaperone (UQCC) involved in the development and maintenance of bone and cartilage is an important candidate gene for body measurement traits selection through marker-assisted selection (MAS). The expression of UQCC is upregulated in many human and animal models of height as well as other stature indexes. We have cloned the cDNA sequence coding UQCC gene in bovine. Genomic structural analysis indicated that bovine UQCC shares a high similarity with human UQCC. Furthermore, Real-Time PCR analysis show that the expression of bovine UQCC is remarkably different in diverse tissues, including high level expression in the spleen, heart and windpipe, and relatively low expression in other tissues. We also analyzed allele frequencies in different cattle breeds and an association study on the selected SNPs. SNP DraI A2691T in intron 1 and SNP Bsh1236I A3150G in intron 8 are significantly associated with Body Length (BL), Rump Length (RL), Chest Depth (CD) and Pin Bone Width (PBW). For the A2691T SNP marker, there are significant effects on the RL (p = 0.0001), CD (p = 0.0059) and PBW (p < 0.0001) in 679 individuals; with A3150G SNP marker, there are significant effects on the BL (p = 0.0047) and CD (p = 0.0454. Regarding association analysis of combination of the two SNPs, there are significant effects on the BL (p = 0.0215), CD (p = 0.0282) and PBW (p = 0.0329) in the total population. The results suggest that the UQCC gene is a candidate gene of body measurement traits in bovine reproduction and breeding, and provide data for establishing of an animal model using cattle to study big animal body type.     

Molecular cloning and SNP association analysis of chicken PMCH gene

The pre-melanin-concentrating hormone (PMCH) gene is an important gene functionally concerning the regulations of body fat content, feeding behavior and energy balance. In this study, the full-length cDNA of chicken PMCH gene was amplified by SMART RACE method. The single nucleotide polymorphisms (SNPs) in the PMCH gene were screened by comparative sequence analysis. The obtained non-synonymous coding SNPs (ncSNPs) were designed for genotyping firstly. Its effects on growth, carcass characteristics and meat quality traits were investigated employing the F₂ resource population of Gushi chicken crossed with Anak broiler by AluI CRS-PCR–RFLP. Our results indicated that the cDNA of chicken PMCH shared 67.25 and 66.47 % homology with that of human and bovine PMCH, respectively. The deduced amino acid sequence of chicken PMCH (163 amino acids) were 52.07 and 50.89 % identical to those of human and bovine PMCH, respectively. The PMCH protein sequence is predicted to have several functional domains, including pro-MCH, CSP, IL7, XPGI and some low complexity sequence. It has 8 phosphorylation sites and no signal peptide sequence. gga-miR-18a, gga-miR-18b, gga-miR-499 microRNA targeting site was predicted in the 3′ untranslated region of chicken PMCH mRNA. In addition, a total of seven SNPs including an ncSNP and a synonymous coding SNP, were identified in the PMCH gene. The ncSNP c.81 A > T was found to be in moderate polymorphic state (polymorphic index = 0.365), and the frequencies for genotype AA, AB and BB were 0.3648, 0.4682 and 0.1670, respectively. Significant associations between the locus and shear force of breast and leg were observed. This polymorphic site may serve as a useful target for the marker assisted selection of the growth and meat quality traits in chicken.     

大黄鱼KLF6基因分子克隆和特征分析

KLF6具有抗细胞增殖的特性,在抑制肿瘤细胞方面起着重要作用.在获得KLF6基因EST序列的基础上,克隆到了KLF6基因,全长1185 bp,其中5′端198 bp,3′端333 bp,开放阅读框654 bp,编码217个氨基酸.与KLF转录因子家族其它成员一样,大黄鱼KLF6的C-端含有3个连续的C2H2型锌指结构域.其氨基酸序列高度保守,与其它鱼类的同源性在90%以上.在检测的大黄鱼骨骼肌、肝脏、眼、脑、脾、心、鳃、肠和肾等9种组织中,KLF6都有不同程度的表达,其中肾、肝脏、鳃、脾脏和脑等组织中的表达量较高.KLF6表达范围的广泛性提示其在多种组织中参与细胞功能活动的调控.