Cloning and Tissue Expression Characterization of the Chicken APOB Gene

Apolipoprotein B (APOB) serves an essential role in the assembly and secretion of triglyceride-rich lipoproteins and lipids transport. This study was designed to clone the full-length cDNA of the chicken APOB gene, to characterize the expression profile, and investigate the differential expression between layer and broiler of the chicken APOB gene. The full-length cDNA sequence (14,150-bp) that contained a 13,896-bp ORF encoding 4,631 amino acids was obtained by RT-PCR, RACE, and bioinformatics analysis. qReal-Time PCR analysis showed that the chicken APOB gene was highly expressed in kidney, liver, and intestine. The results of differential expression showed that the APOB gene was more highly expressed in intestine and kidney in Bai'er layer than in broiler, but there was no significant difference in liver between the two breeds. The results of this study provided basic molecular information for studying the role of APOB in the energy transportation in avian species.     

军曹鱼催乳素受体PRLR1基因的克隆及其在不同盐度条件下mRNA表达差异

催乳素受体通过结合催乳素,能调节鱼体渗透压。为研究催乳素受体1(PRLR1)在高盐水体和低盐水体中对军曹鱼(Rachycentron canadum)的渗透调节作用,利用cDNA末端快速扩增(RACE-PCR)技术,获得了军曹鱼PRLR1全长cDNA序列。该基因全长为2629 bp,包含1953 bp的开放阅读框ORF,可编码650个氨基酸。氨基酸序列包含了2个纤维连接蛋白3型结构域(FN3)、保守的WS区和box1。采用qRT-PCR技术,检测不同盐度(10‰、30‰和35‰)条件下鳃、肠、体肾中PRLR1基因mRNA表达情况。结果显示,PRLR1基因在军曹鱼的各个组织中均有表达,其中鳃表达量最高,其次是肌肉、体肾和肠,而在胃、脾、脑和心脏中则微量表达。低盐组、正常组和高盐组中,PRLR1基因的表达量均为鳃最高;肠次之;体肾最低。随着盐度提高,PRLR1基因的鳃、肠和体肾组织表达量变化规律均呈逐步下降趋势。以上结果反映了军曹鱼PRLR1在渗透压器官中的功能差异性,说明PRLR1在军曹鱼渗透压调节上具有重要作用。     

鸡PPARγ基因的表达特性及其对脂肪细胞增殖分化的影响

为分析鸡PPARγ基因的组织表达特性及其在脂肪细胞增殖和分化过程中的功能,文章以东北农业大学高、低腹脂双向选择品系肉鸡为实验材料,利用Western blotting方法,检测PPARγ基因的组织表达特性及其在高、低脂系肉鸡腹部脂肪组织间的表达差异;采用RNAi技术,在鸡原代脂肪细胞中抑制PPARγ基因的表达后,通过MTT和油红O提取比色的方法,研究鸡PPARγ基因对脂肪细胞增殖和分化的调控作用;利用Real-timePCR和Western blotting技术,分析PPARγ基因表达下调后,其他脂肪细胞分化转录因子以及与脂肪细胞分化相关的重要基因的表达变化情况。结果表明,PPARγ基因在7周龄高脂系肉鸡腹部脂肪组织、肌胃、脾脏、肾脏组织中表达量较高,在心脏中表达量较低,在肝脏、胸肌、腿肌、十二指肠中未检测到表达信号;与高脂系相比,PPARγ基因在5和7周龄低脂系肉鸡腹部脂肪组织中的表达量较低(P<0.05);PPARγ基因的表达量下降后,鸡脂肪细胞的增殖能力增强,分化能力减弱;同时,C/EBPα、SREBP1、A-FABP、Perilipin1、LPL、IGFBP-2基因的表达量均下降(P<0.05)。由此可见,PPARγ基因的表达可能与肉鸡腹部脂肪的沉积有一定的关系,该基因可能是调控鸡脂肪细胞增殖与分化的关键因子。     

东北七鳃鳗TRAF6基因克隆、表达分析及亚细胞定位研究

为研究TRAF6在TLR信号介导的先天免疫中的调控作用, 研究通过克隆技术获得了东北七鳃鳗(Lethenteron morii)TRAF6基因的cDNA全长, 命名为LmTRAF6。利用实时荧光定量方法(qPCR)分析了LmTRAF6在幼鱼和成鱼中各组织的表达情况以及在铜绿假单胞菌(Pseudomonas aeruginosa)感染后脂肪体、鳃、肠和肾组织在不同时间点的表达量变化。利用双酶切技术构建pEGFP-TRAF6重组质粒并转染HEK293T细胞, 48h后进行荧光观察并拍照。结果表明, LmTRAF6的cDNA全长为2751 bp, 开放阅读框(ORF)为1785 bp, 编码594个氨基酸。其蛋白结构域高度保守, 具有RING结构、两个锌指结构、环-环(Coiled-coil)α螺旋结构域和MATH结构域。通过系统进化树分析, 发现LmTRAF6与哺乳类以及鱼类TRAF6的亲缘关系较近, 与果蝇、中国对虾TRAF6的亲缘关系较远。qPCR结果显示, LmTRAF6在各组织中均有表达, 在幼鱼的心脏、皮肤、鳃、肝中的表达量相对较高, 而在肠中表达量相对较低。在成鱼的肾、鳃、肌肉中的表达量相对较高, 而在心脏中表达量相对较低。成鱼LmTRAF6在铜绿假单胞菌感染后, 鳃、肠和肾的表达量在24h达到峰值。细胞定位显示, LmTRAF6在HEK293T的细胞质和细胞核中均有表达。以上结果为进一步探究七鳃鳗中TRAF6在TLR信号通路中的作用奠定了理论基础。     

Cloning, identification and accurate normalization expression analysis of PPARα gene by GeNorm in Megalobrama amblycephala

Megalobrama amblycephala suffers from serious liver diseases recently and PPARα gene has been reported to play an important role in the immune system of animal liver. On the basis of these facts, we have cloned and identified full-length cDNA of PPARα and examined its expression patterns at different embryo developmental stages and in different tissues of adult and young fish in order to improve liver disease immunity of M. amblycephala. We also accurately normalized seven reference genes by GeNorm and calculated their gene expression normalization factors. The total length of PPARα cDNA was 2021 bp, comprising of 214-bp 5'-untranslated region; 1404-bp open reading frame (encoding 467-amino acids); and 403-bp 3'-untranslated region. PPARα peptide was predicted to consist of 4 domains, i.e. A/B, C, D, and E/F. PPARα mRNAs were detected in different tissues of adult and young fish including adipose tissue, gill, heart, liver, spleen, kidney, white muscle, intestine, brain and gonad. In adult fish, the expression of PPARα in white muscles was highest followed by liver and it was lowest in gonads. Its expression in male gonads was significantly higher than female gonads. In young fish, the expression of PPARα was highest in brain, followed by intestines and it was lowest in spleen. At different embryo developmental stages, the expression of PPARα was highest at 2 cells stage and it was lowest at gastrula stage, but it increased on first day after hatching. In unfertilized spermatozoa, the expression of PPARα was higher than unfertilized ovum.     

利用电子差异展示方法克隆人类睾丸高表达新基因SPATA11

利用NCBI中的电子差异展示(digital differential display,DDD)软件,比较来自睾丸(包括睾丸癌)与来自其它组织的EST文库,从筛查人类睾丸中高表达而在其他组织中不表达或低表达的差异ESTs入手,成功克隆了一个在人类睾丸中高表达的新基因SPATA11．RT-PCR实验证实其在成人睾丸高表达．序列分析表明该基因含4个外显子,基因组跨越2．6kb,定位于19pl3．3．cDNA编码一个含221个氨基酸,相对分子质量为24．5kD的新蛋白．Northern杂交结果显示：该基因含有1．1kb大小的唯一转录本,主要在睾丸中强表达．肝脏、肺、卵巢和肾脏中有微弱表达．而其他组织中该基因无表达．     

Molecular cloning of a human MafF homologue, which specifically binds to the oxytocin receptor gene in term myometrium.

The US-2 DNA-binding element (ggaatgattactcagctaga) in the promoter of the human oxytocin receptor (OTR) gene has been shown to bind specifically nuclear proteins from human myometrium at parturition. To elucidate the molecular mechanisms involved in OTR gene upregulation at term, the US-2 element was used in a yeast one-hybrid system to screen a cDNA library derived from term human myometrium. Positive clones were further screened by electrophoretic mobility shift assay for their ability to bind the human OTR gene promoter, containing the US-2 motif. A 2.3-kb full-length cDNA encoding a human homologue of chicken MafF (hMafF) was isolated. hMafF represents an 18-kDa protein and contains an extended leucine zipper structure, but lacks a transactivation domain. Furthermore, Northern hybridization showed strong hMafF mRNA expression in the kidney and in term myometrium only, but not in nonpregnant myometrium. The hMafF protein is also preferentially expressed in term myometrium, as shown by specific binding to the OTR promoter. The highly specific binding of hMafF to the US-2 motif in the human OTR gene, together with its pattern of expression, supports a role for hMafF in OTR gene upregulation at term.     

牛肝辅酶Ⅱ依赖性视黄醇脱氢酶cDNA的克隆及组织表达

迄今为止的研究证明 ,维生素A亦称视黄醇(retinol)的生理功能是通过其两步氧化代谢产物视黄醛与视黄酸 (亦称维甲酸 )来完成的 .视黄醛通过其光学异构体 1 1 顺式视黄醛与视觉细胞内的视蛋白 (opsin)结合组成视色素 .感光时 ,1 1 顺式视黄醛转变成全反式视黄醛从视蛋白脱落 ,这一过程同时传导到大脑产生视觉[1 ] .全反式维甲酸 (all transretinoicacid)则通过与其在核内受体 (RARα ,β ,γ)结合调节基因的转录来发挥其许多重要的生理功能 ,包括正常胚胎的发育 ,形态、神经系统的形成 ,成体动物的生长、发育、繁殖等 ,并通过调解组织及…     

A novel splice variant of human gene NPL, mainly expressed in human liver, kidney and peripheral blood leukocyte.

From the human fetal brain cDNA library constructed by our lab, a novel variant cDNA of a human gene was successfully cloned and identified. Because the gene has been named N-acetylneuraminate pyruvate lyase (NPL), accordingly we term our splice variant NPL_v2. The cDNA of NPL_v2 has a full-length open reading frame (ORF) from the nucleotide position 320 to 1225 that encodes a protein comprising 301 amino acids. SMART analysis showed that our hypothetical protein has one dihydrodipicolinate synthase (DHDPS) domain. Phosphorylation analysis of the deduced protein show that there are five phosphorylation sites including three "serine" and two "threonine" at the region that are not found in other splice variant. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human placenta, liver, kidney, pancreas, spleen, thymus, ovary, small intestine and peripheral blood leukocyte.     

北京油鸡α1-AGP基因结构与表达特征研究

提取北京油鸡心、肝、脾、肺、肾、脑、腿肌与胸肌等不同组织的总RNA, 利用RT-PCR方法检测a1-酸性糖蛋白基因(a1-AGP)mRNA的差异表达。将a1-AGP基因完整开放阅读框定向插入pEGFP-C1, 构建带有GFP报告基因的重组表达载体pEGFP-α1-AGP, 将其导入北京油鸡体外培养细胞中, 并进行G418药物筛选和克隆化培养。结果表明: a1-AGP基因开放阅读框长度为612 bp, 编码203个氨基酸, 在北京油鸡肝、肺、腿肌和胸肌中有表达; 转染后24、48和72 h, pEGFP- a1-AGP转染率在31.3%~47.6%之间, 绿色荧光主要集中在细胞核中, 随着表达量的增加, 绿色荧光在细胞核中聚集成团块状或颗粒状, 经药物筛选和克隆化培养, 获得表达pEGFP-a1-AGP融合蛋白的阳性克隆细胞株, 利用RT-PCR与Western blotting检测确认pEGFP-α1-AGP已整合到北京油鸡成纤维细胞的基因组中, 在优化的条件下, 阳性细胞凋亡率、形态、生长与增殖状况与对照组比较差异不显著(P >0.05)。     

Cloning and analysis of cDNA clones for rat kidney alpha-spectrin

We have isolated a 3922-base pair (bp) cDNA clone for rat nonerythroid alpha-spectrin from a rat kidney lambda gt11 cDNA library. Sequence analysis revealed that this cDNA contains an open reading frame of 3090 bp encoding for the C-terminal 1030 amino acid sequence of rat kidney alpha-spectrin. The 3'-untranslated region (including a 38-bp poly(A+) tail) contains an 832-bp sequence. A single mRNA of about 8 kilobase pairs was detected in rat liver, kidney, brain, heart, intestine, lung, testis, stomach, spleen, and muscle with varying abundances, which is consistent with and further confirms the presence of spectrins in nonerythroid tissues as demonstrated previously by immunoblot analysis. Southern blot analysis suggested that there is a single gene for nonerythroid alpha-spectrin. The derived amino acid sequence contains sequence from the spectrin 106-residue internal repeat 12 to the C terminus of rat kidney alpha-spectrin. Sequence comparison with human and chicken nonerythroid alpha-spectrin showed that nonerythroid alpha-spectrin is well conserved during evolution. The rat kidney alpha-spectrin sequence, when compared to rat brain alpha-spectrin, contains an extra 76-amino-acid sequence at the C terminus. Sequence comparison of all the internal repeats available revealed that the internal repeat 3, 4, 5, 6, 7, and 8 has highest sequence similarity with internal repeat 12, 13, 14, 15, 16, and 17, respectively. Therefore, internal repeats 3-8 and 12-17 are most likely derived from an ancestral gene through gene duplication, suggesting that the spectrin gene is derived from a half-spectrin gene by gene duplication and divergence during evolution.     

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi （Brassica campestris L. ssp. chinensis Makino）

For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only fulllength cDNA from the differential fragment BcMF-A 18T 16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and largesized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed ofa 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2gene on microspore development is discussed in the present paper.     

Molecular characterization and expression profiling of BMP 3 gene in broiler and layer chicken

A study was carried out to characterize and explore the expression profile of BMP 3 gene in control broiler and control layer chicken. The total open reading frame of BMP 3 (1389 bp) was cloned and sequenced. The control broiler and control layer chicken showed variation at nucleotide and amino acid level with reference gene (Gallus gallus, NCBI Acc. No. NM_001034819). When compared to reference gene, the control broiler showed four nucleotide differences (c.192A>G, c.519C>T, 903G>A and 960C>G), while, control layer showed variation at c.33G>C, 192A>G, 858G>A, 904G>A, 960C>G and 1257C>T making six differences in total. However, between control broiler and control layer lines, nucleotide differences was observed at c.33G>C, 519T>C, 858G>A, 903A>G, 904G>A and 1257C>T. The change at amino acid level between reference and control broiler was p.D320N and with control layer chicken, it was p.D302N and p.D320N. On the other hand, a single amino acid difference (p.D302N) was observed between the control broiler and control layer chicken lines. The phylogenetic study displayed a close relationship between broiler and layer lines and reference gene and also with other avian species resulting in a cluster formation. These cluster in turn displayed a distant link with the mammalian species. The expression profile of BMP 3 gene exhibited a variation at different stages of embryonic development and also at post embryonic period among the lines with control layer showing higher expression than that of broiler chicken. The protein was also detected in bone marrow tissue of broiler and layer lines by western blotting. It is concluded that the BMP 3 gene sequence differed at nucleotide and amino acid level among the lines and the gene expressed differentially at different periods of embryonic development and also at post hatch period.     

Cloning and tissue expression of chicken heart fatty acid-binding protein and intestine fatty acid-binding protein genes

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.     

Cloning and Tissue Expression of Chicken Heart Fatty Acid-Binding Protein and Intestine Fatty Acid-Binding Protein Genes

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.