阿坝藏猪蒲江种群SLA-DRB基因片段SNP检测及其与生长发育性状关联分析

猪Sus scrofa domesticus的白细胞抗原复合体(SLA)基因的单核苷酸多态性(SNP)与猪生长发育等性状有关。为了开发藏猪品系选育的遗传标记,本文测定了阿坝藏猪蒲江种群89头个体的SLA-DRB基因部分序列,共鉴定出14个SNP位点。根据位点间的连锁强度分为3个组合(组合1:g. 11470、g. 11945、g. 11997、g. 11387、g. 11369与g. 11829;组合2:g. 11532与g. 11838;组合3:g. 11911与g. 11708)和4个独立多态位点(g. 11468、g. 11440、g. 11478和g. 11944)。将基因型与体质量、体长等14项生长发育性状进行关联性分析,结果表明,组合3的基因型与体高、体长、胸围等11项体尺指标间的相关性均显著(FDR 0. 05),基因型为TT/CG的个体较小,表明SLA-DRB基因可用于藏猪小型化选育的遗传标记。管围和12项管围率(除体质量以外)与SLA-DRB基因多态性的相关性最高,受组合1、组合2和位点g. 11468、g. 11478基因型的极显著影响(FDR 0. 001),其中,基因型为组合1=GGGAAC/GGGAAC、组合2=GT/GT、位点g. 11468=C/C和g. 11478=C/C的个体拥有更大的管围和管围率。管骨骨髓是SLA基因发挥免疫功能的主要场所之一。本研究为基于管围的猪种筛选提供了理论依据和可用的SNP位点,也为藏猪抗病遗传筛选提供了基础数据和参考资料。     

应用寡核苷酸芯片并行检测CYP1A1和 GSTM1基因多态性

利用寡核苷酸芯片检测方法分析CYP1A1单核苷酸多态性 (SNP)和GSTM1缺失与否 ,实验结果证明了寡核苷酸芯片技术可并行、准确、高效地检测基因的单核苷酸多态性和其他类型的基因多态型 ,可为疾病遗传易感性及单体型的研究提供强有力的研究工具。采用该寡核苷酸芯片 ,检测了 84份正常人的血液DNA样本 ,其中GSTM1基因缺失率达到 4 7 6 % ,接近报道数值。统计分析发现 ,CYP1A1m1 m2的 3种基因型组合TT AG、TT GG和TC GG的发生频率都为 0 ,而根据实验得到的m1和m2各自基因型数据计算 ,它们的发生频率应是11 4 %、2 6 %和 3 1% ,所以推测在所检测的样本中没有T(m1位点 )和G(m2位点 )的连锁组合 ,即m1和m2位点的组合只有 3种单体型 :T A、C A和C G ,其发生频率分别是 6 9 6 %、7 7%和 2 2 6 %。     

应用寡核苷酸芯片并行检测CYP1A和GSTM1基因多态性

利用寡核苷酸芯片检测方法分析CYP1A单核苷酸多态性(SNP)和GSTM1缺失与否,实验结果证明了寡核苷酸芯片技术可并行、准确、高效地检测基因的单核苷酸多态性和其他类型的基因多态型,可为疾病遗传易感性及单体型的研究提供强有力的研究工具。采用该寡核苷酸芯片,检测了84份正常人的血液DNA样本,其中GSTMl基因缺失率达到47．6％,接近报道数值。统计分析发现,CYP1A m1-m2的3种基因型组合TT-AG、TT-GG和TC-GG的发生频率都为0,而根据实验得到的m1和m2各自基因型数据计算,它们的发生频率应是11．4％、2．6％和3．1％,所以推测在所检测的样本中没有T(m1位点)和G(m2位点)的连锁组合,即m1和m2位点的组合只有3种单体型：T-A、C-A和C-G,其发生频率分别是69．6％、7．7％和22．6％。     

不同月龄BALB/c小鼠感染甲型H1N1型流感病毒的炎性调控差异研究

为对比不同月龄BALB/c小鼠感染甲型H1N1型流感病毒后的淋巴细胞相对含量和炎症因子调控差异,提供流感类病毒性疾病防控的基础研究证据,本研究选用2月、8月龄雌性BALB/c小鼠,分别感染1×105TCID50/0.1mL的甲型H1N1型流感病毒PR8毒株,每天记录体重、肛温变化,于感染第7d处死解剖取材,计算脏器指数,qPCR技术检测肺组织流感病毒M基因拷贝数,流式细胞术检测全血CD3+、CD4+、CD8+淋巴细胞含量及肺组织中IL-6、IFN-γ、MCP-1等炎症因子的分泌水平。结果显示：(1)感染相同剂量病毒7d后,2月龄小鼠有1例样本死亡,生存质量下降较大,8月龄小鼠全部存活,体重较第0d相比仅下降12.35%,感染后2月龄组小鼠胸腺指数明显下降（P<0.01）,8月龄组变化不明显。(2)2月龄组染毒小鼠肺组织M基因拷贝数显著低于8月龄染毒组（P<0.05）。(3)染毒后,2月龄组小鼠体内IFN-γ、IFN-β、IL-6等促炎性细胞因子含量增长显著（P<0.05）,IL-6、MCP-1、IL-23含量显著高于8月龄组（P<0.05）;2月龄染毒组小鼠杀伤...     

不同海拔猪种EPO基因核苷酸多态性研究

青藏高原低氧胁迫响应下,藏猪在长期进化过程中形成了独特的高原低氧适应性。本研究对藏猪(合作猪,甘孜藏猪,迪庆猪,西藏藏猪)、八眉猪、烟台黑猪及长白猪、杜洛克猪、大白猪促红细胞生成素(erythropoietin,EPO)基因全序进行了测定分析。所得数据表明:9个猪群体的37个核苷酸序列共得到14个单倍型,25个变异位点包括8个转换,3个颠换和一个连续14 bp的缺失,其中有义突变3个;引入猪种仅分布在两个单倍型H1和H2中,我国地方猪种则分布在13个单倍型中;分析发现藏猪"TTGGGAAGTCTCAT"的一个序列片段缺失,这一特征在低海拔群体中不存在。EPO基因编码的194个氨基酸中发现3个突变位点,变异后的单倍型及氨基酸序列在藏猪中占优势。因此,在适应高寒低氧环境的生理学和分子学机制方面,藏猪EPO基因在长期进化过程中可能起着关键性遗传作用。     

雌性藏猪不同生长发育阶段肌肉生长和脂肪沉积相关基因的表达模式

本研究利用包含140个与猪肌肉生长和脂肪沉积密切相关基因的Oligo功能分类基因芯片检测了藏猪在2、4、6和8月龄间背最长肌中这些基因的表达量变化,并在2月龄时与脂肪型的太湖猪和瘦肉型的长白猪进行比较.ANOVA分析结果表明:2-8月龄间藏猪分别有10和 7个基因的表达差异达极显著(P<0.01)和显著水平(P<0.05);2月龄时藏猪体重极显著低于长白猪(P<0.01)和显著低于太湖猪(P<0.05),而藏猪肌纤维面积却为最大,但品种间差异未达显著水平(P>0.05);2月龄时3个品种间分别有15和13个基因的表达差异达极显著 (P<0.01)和显著水平(P<0.05).STEM聚类分析结果表明:直线下降和上升是藏猪在2 -8月龄间最具代表性的基因表达模式(P<0.01).另外,5个差异表达基因的荧光定量RT -PC R验证结果与基因芯片结果的Person相关系数平均高达0.856±0.109.提示:藏猪在2-8月龄间骨骼肌生长发育强度较肌内脂肪合成沉积占优势,2月龄时藏猪脂肪酸合成相关基因的表达水平较其他两品种低,而脂肪酸β氧化和肌纤维生长相关基因的表达水平较高,与其在高原独特的自然生态环境和全放牧散养的饲喂方式下长期形成的品种特性相符 [动物学报 54(3):442-452,2008].     

棉铃虫单粒包埋型核型多角体病毒极早期基因(ie-1)分析(英文)

克隆了棉铃虫Helicoverpaarmigera单粒包埋型核型多角体病毒 (HaSNPV)C1株基因组DNA ,并通过随机测序的方法测定了经XbaI酶切后的H片段的核苷酸全序列。序列比较和分析发现该片段中ORF1 3与苜蓿丫纹夜蛾Autographacalifornica多粒包埋型核型多角体病毒 (AcMNPV)基因组ORF1 47(ie 1 )同源。ie 1基因编码区全长 1 986bp ,根据推测的氨基酸序列 ,可编码 6 6 1个氨基酸残基组成的多肽 ,预计分子量为 76 .5kD。将所推导的HaSNPVIE 1氨基酸序列与其它已知的杆状病毒IE 1氨基酸序列进行比较 ,结果表明 ,HaSNPV和谷实夜蛾H .zea单粒包埋型核型多角体病毒IE 1氨基酸序列最为相似 ,同源性高达 98%。与AcMNPV、家蚕Bombyxmori核型多角体病毒 (BmNPV)、云杉卷叶蛾Choristoneurafu miferana多粒包埋型核型多角体病毒 (CfMNPV)、舞毒蛾Lymantriadispar多粒包埋型核型多角体病毒(LdMNPV)、黄杉毒蛾Orgyiapseudotsugata多粒包埋型核型多角体病毒 (OpMNPV)、甜菜夜蛾Spodopteraex igua多粒包埋型核型多角体病毒 (SeMNPV)、小菜蛾Plutellaxylostella颗粒体病毒 (PxGV)和Xestiac ni grum颗粒体病毒 (XcGV)的IE 1氨基酸序列同源性较低 ,分别为 2 3 %、2 3 %、2 3 %、2 5 %、2 3 %、1 4%、2 7%和 7%。根据氨基酸序列由GENETYX     

中国福建汉族14个YSNPs位点的研究

本研究对中国福建汉族的 80份DNA样品中的 14个YSNPs位点及YAP位点进行了基因分型。结果发现 ,14个YSNPs位点中除M7、M4 5、M10 3位点未发现变异外 ,其余M9、M5 0、M110、M15、M134、M12 2、M95、M89、M119、M111、M88等 11个YSNPs位点和YAP位点均有不同程度的变异。单体型分析结果显示 :在福建省汉族中共发现H1、H3、H4、H5、H6、H8、H9七种单体型 ,其频率分别为 2 2 .5 %、1. 3%、1. 3%、1 .3%、30. 0 %、2 8. 8%、15 .0 %.。     

PLCE1基因及rs2274223和rs3765524单体型的克隆与表达

目的：克隆人PLCE1基因,构建PLCE1 rs2274223和rs3765524 GT（PLCE1 Minor）和AC（PLCE1 Major）单体型真核表达重组载体,研究PLCE1基因多态性及单体型与基因表达的相关性。方法：利用重叠延伸PCR、In-Fusion技术构建PLCE1真核表达载体;基于同源重组的双点突变技术改造目的基因;利用基因转染、实时定量PCR和蛋白印迹等技术实现PLCE1表达载体在真核细胞中的过表达及表达水平的鉴定。结果：成功扩增并获得了全长6 927bp的人PLCE1 cDNA并经过DNA测序证实,与NCBI数据库PLCE1参考序列（NM_016341）比较,发现编码区3 554~3 572bp处存在18bp连续碱基插入,其余序列基本匹配。成功构建了PLCE1 Minor和PLCE1 Major两种单体型重组真核表达载体,转染细胞揭示PLCE1 Minor型的mRNA转录和蛋白质表达水平均高于Major型。结论：发现了一种新的PLCE1 mRNA转录本,成功构建了2种单体型真核表达载体;发现PLCE1 rs2274223G-rs3765524T单体型能够促进自身mRNA和蛋白质表达,为进一步揭示PLCE1 SNPs与癌症易感性的关系奠定了基础。     

INCENP基因功能性单倍型可调控启动子活性并影响公牛精液品质

为研究内着丝粒蛋白(Inner centromere protein, INCENP)基因启动子区单核苷酸多态性(SNPs)与精液品质的相关性,本文利用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测了250头中国荷斯坦公牛INCENP基因的基因型。在INCENP基因启动子区鉴定出两个SNPs (g.-556 G>T,rs 136823901和g.-692 C>T,rs 211010999),发现了3种单倍型(CG、TT、TG)。分析两个SNP位点的基因型频率和等位基因频率,各SNP及单倍型组合与中国荷斯坦公牛精液品质的相关性,结果表明SNP位点g.-556 G>T GT基因型个体的鲜精活力显著高于GG基因型个体(P<0.05),单倍型组合H1H1(CCGG)、H1H3(CTGT)、H2H3(TTGT)和H3H3(TTTT)个体的鲜精活力和冻精解冻后活力均显著高于H1H2个体(P<0.05)。为进一步研究g.-556 G>T和g.-692 C>T影响精液品质的可能机理,本文将3种单倍型质粒分别转染小鼠睾丸间质细胞(MLTC-1),结果显示含TG单倍型的载体荧光素酶活性最高。由此推测,g.-556 G>T和g.-692 C>T为启动子区功能性突变位点,可通过调节启动子活性来调控INCENP基因表达,进而影响精液品质。     

LINE-1编码蛋白L1-ORF1的原核表达纯化和多克隆抗体制备

目的: 制备具有肿瘤组织特异性表达的L1-ORF1蛋白多克隆抗体并进行初步应用研究。方法:采取基因工程表达方法制备L1-ORF1蛋白,免疫家兔制备多克隆抗体,间接ELISA检测抗体效价,Western blot和细胞免疫荧光方法检测抗体特异性,免疫检测验证其识别肿瘤细胞内L1-ORF1蛋白的特异性。结果:制备的抗L1-ORF1蛋白多克隆抗体具有很高的敏感性与特异性,免疫学检测表明该抗体不仅能检测出正常细胞中瞬时表达的L1-ORF1蛋白,而且可检测出肿瘤细胞中天然表达的L1-ORF1蛋白。结论:制备的多克隆抗体具有较高的敏感性与特异性,为以后该抗体的进一步应用奠定了基础。     

Role of the ASK1-SEK1-JNK1-HIPK1 signal in Daxx trafficking and ASK1 oligomerization

Overexpression of JNK binding domain inhibited glucose deprivation-induced JNK1 activation, relocalization of Daxx from the nucleus to the cytoplasm, and apoptosis signal-regulating kinase 1 (ASK1) oligomerization in human prostate adenocarcinoma DU-145 cells. However, SB203580, a p38 inhibitor, did not prevent relocalization of Daxx and oligomerization of ASK1 during glucose deprivation. Studies from in vivo labeling and immune complex kinase assay demonstrated that phosphorylation of Daxx occurred during glucose deprivation, and its phosphorylation was mediated through the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. Data from immunofluorescence staining and protein interaction assay suggest that phosphorylated Daxx may be translocated to the cytoplasm, bind to ASK1, and subsequently lead to ASK1 oligomerization. Mutation of Daxx Ser667 to Ala results in suppression of Daxx relocalization during glucose deprivation, suggesting that Ser667 residue plays an important role in the relocalization of Daxx. Unlike wild-type Daxx, a Daxx deletion mutant (amino acids 501-625) mainly localized to the cytoplasm, where it associated with ASK1, activated JNK1, and induced ASK1 oligomerization without glucose deprivation. Taken together, these results show that glucose deprivation activates the ASK1-SEK1-JNK1-HIPK1 pathway, and the activated HIPK1 is probably involved in the relocalization of Daxx from the nucleus to the cytoplasm. The relocalized Daxx may play an important role in glucose deprivation-induced ASK1 oligomerization.     

Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Regulation of PCTAIRE1 protein stability by AKT1, LKB1 and BRCA1

PCTAIRE1, also known as CDK16, is a cyclin-dependent kinase that is regulated by cyclin Y. It is a member of the serine-threonine family of kinases and its functions have primarily been implicated in cellular processes like vesicular transport, neuronal growth and development, myogenesis, spermatogenesis and cell proliferation. However, as extensive studies on PCTAIRE1 have not yet been conducted, the signaling pathways for this kinase involved in governing many cellular processes are yet to be elucidated in detail. Here, we report the association of PCTAIRE1 with important cellular proteins involved in major cell signaling pathways, especially cell proliferation. In particular, here we show that PCTAIRE1 interacts with AKT1, a key player of the PI3K signaling pathway that is responsible for promoting cell survival and proliferation. Our studies show that PCTAIRE1 is a substrate of AKT1 that gets stabilized by it. Further, we show that PCTAIRE1 also interacts with and is degraded by LKB1, a kinase that is known to suppress cellular proliferation and also regulate cellular energy metabolism. Moreover, our results show that PCTAIRE1 is also degraded by BRCA1, a well-known tumor suppressor. Together, our studies highlight the regulation of PCTAIRE1 by key players of the major cell signaling pathways involved in regulating cell proliferation, and therefore, provide crucial links that could be explored further to elucidate the mechanistic role of PCTAIRE1 in cell proliferation and tumorigenesis.     

Antagonism between Cry1Ac1 and Cyt1A1 Toxins of Bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC₅₀s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC₅₀s were obtained. All of these LC₅₀s were significantly higher than the expected LC₅₀s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC₅₀ of Cry1Ac1 was 2.46 ng/cm² of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC₅₀s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm² of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm² of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Isolation and Characterization of Ubiquitin-activating Enzyme E1-domain Containing 1, UBE1DC1

Ubiquitin and other ubiquitin-like proteins play important roles in post-translational modification. They are phylogenetically well-conserved in eukaryotes. Activated by other proteins, ubiquitin and ubiquitin-like proteins can covalently modify target proteins. The enzymes responsible for the activation of this modification have been known to include UBA1, SAE2, UBA3, SAE1 and ULA1. Here we report a new ubiquitin activating enzyme like cDNA, named ubiquitin activating enzyme E1-domain containing 1 (UBE1DC1), whose cDNA is 2654 base pairs in length and contains an open reading frame encoding 404 amino acids. The UBE1DC1 gene consists of 12 exons and is located at human chromosome 3q22. The result of RT-PCR showed that UBE1DC1 is expressed in most of human tissues. These two authors contributed equally to this paper. The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY253672.     

Characterization of Chrysanthemum ClSOC1-1 and ClSOC1-2, homologous genes of SOC1

The MADS-box gene SOC1/TM3 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/ Tomato MADS-box gene 3) is a main integrator in the Arabidopsis flowering pathway; its structure and function are highly conserved in many plant species. SOC1-like genes have been isolated in chrysanthemum, one of the most well-known ornamental plants, but it has not been well characterized thus far. We isolated and characterized ClSOC1-1 and ClSOC1-2, two putative orthologs of Arabidopsis SOC1, from the wild diploid chrysanthemum, Chrysanthemum lavandulifolium, to investigate the regulatory mechanisms of flowering time control in chrysanthemum. Expression analysis indicated that ClSOC1-1 and ClSOC1-2 were expressed in all examined organs/tissues (leaves, shoot apices, petioles, stems and roots) with different expression levels, and with high expression in the shoot apices and leaves during the early stage of floral transition. The expression levels of ClSOC1-1 and ClSOC1-2 in the shoot apices increased at different developmental stages with the highest expression levels after 7 days of short-day treatment. Overexpression of ClSOC1-1 and ClSOC1-2 in wild-type Arabidopsis resulted in early flowering, which was coupled with the upregulation of one of the flowering promoter genes LEAFY. Our results suggested that the ClSOC1-1 and ClSOC1-2 genes play an evolutionarily conserved role in promoting flowering in Chrysanthemum lavandulifolium and could serve as a vital target for the genetic manipulation of flowering time in the chrysanthemum.