CAT1基因在尼罗罗非鱼肌肉和肝的节律性表达研究

为了探究碱性氨基酸转运载体(CAT1)基因在尼罗罗非鱼(Oreochromis niloticus)饥饿0、7 d肌肉和肝的昼夜表达规律,实验分别在尼罗罗非鱼饥饿0,7 d 06:00、09:00、12:00、15:00、18:00、21:00和24:00、第2天03:00、06:00,分别对应区时(Zone time, ZT) ZT0、ZT3、ZT6、ZT9、ZT12、ZT15、ZT18、ZT21、ZT24采样,采样在一昼夜内完成。每组随机取3尾鱼进行解剖,取其肌肉和肝样品分析。结果表明:CAT1基因在正常投喂的尼罗罗非鱼肌肉表达具有显著性时间差异(p0.05),呈现昼低夜高的节律性振荡(p0.3),达到峰值的时间为ZT 0.72。饥饿7 d后,虽然在各时间点上CAT1基因表达有显著性差异(p0.05),但无昼夜节律性(p0.3);CAT1基因表达为在光周期先升后降,在暗周期先降后升,峰值相位在光照阶段,为ZT 7.44。在尼罗罗非鱼肝中,CAT1基因在正常投喂时表达具有显著性时间差异(p.05),呈现节律性振荡(p0.3),CAT1基因表达为在光周期先降后升,在暗周期则先升后降,峰值相位在黑暗阶段,达到峰值的时间为ZT 16.56。饥饿7 d后,在各时间点上CAT1基因表达无显著性时间差异(p0.05),且表达无昼夜节律性(p0.3);CAT1基因表达为在光周期先降后升,在暗周期则先升后降,峰值相位在黑暗阶段,为ZT 21.60。以上结果说明CAT1基因在尼罗罗非鱼肌肉和肝表达的昼夜节律不同。     

尼罗罗非鱼TCP-1-beta和TCP-1-eta的分子特征及其低温诱导表达

为了研究尼罗罗非鱼耐寒性状的分子基础并为耐寒品种选育提供参考,研究从尼罗罗非鱼中克隆了HSP60家族TCP-1-beta和TCP-1-eta基因并对其在低温诱导下的表达特征进行了分析。尼罗罗非鱼TCP-1-beta cDNA长度为1755 bp,包括1605 bp的完整开放阅读框,编码534个氨基酸;尼罗罗非鱼TCP-1-eta cDNA长度1651 bp,包括1638 bp的完整开放阅读框,编码545个氨基酸。与其他物种同源基因的蛋白序列比对结果显示,TCP-1-beta和TCP-1-eta蛋白在物种间同源性很高,且都具有保守的ATP结合结构域等,预示其在物种间功能的保守性。实时荧光定量PCR结果表明:TCP-1-beta和TCP-1-eta在各组织中呈遍在表达,但在肌肉中表达量最高;诱导温度从22℃降至12℃,不同低温诱导48h后TCP-1-beta和TCP-1-eta均呈上调表达,在18℃时表达开始上调,随着低温胁迫程度加强,表达上调幅度增大,至12℃时表达量达到最高,TCP-1-beta和TCP-1-eta上调幅度分别达到常温的12.2倍和10.7倍。这些结果预示在尼罗罗非鱼中,TCP-1-beta和TCP-1-eta是潜在的耐寒相关基因。     

LHRH-A对尼罗罗非鱼生长及生长轴相关基因表达的影响

促性腺激素释放激素(GnRH)的主要作用是刺激脑垂体促性激素(GtH)的释放, 亦可促进鱼类生长激素(GH)的释放。促黄体素释放激素类似物(LHRH-A)是哺乳类GnRH的类似物, 为了分析LHRH-A对尼罗罗非鱼生长调节的作用, 设计了长期和短期2个实验, 采用腹腔注射(剂量0.1 μg/g体重)方法, 分析LHRH-A对尼罗罗非鱼绝对生长率、特定生长率、肝体系数和肥满度的影响, 并应用荧光实时定量PCR方法检测在注射LHRH-A后不同时段(6h、12h、24h、2周)尼罗罗非鱼垂体GH、肝脏GHR和肝脏IGF-I基因的表达变化。结果表明, LHRH-A组尼罗罗非鱼的绝对生长率、特定生长率、肝体系数、肥满度均显著高于对照组(P<0.05); 注射LHRH-A后12h、24h垂体GH mRNA表达水平均显著升高(P<0.05), 2周后恢复到对照组水平; 注射LHRH-A后24h和2周肝脏GHR mRNA表达水平显著上升(P<0.05); 注射LHRH-A后6h肝脏IGF-I mRNA表达水平显著升高(P<0.05), 12h、24h和2周恢复到对照组水平。以上结果提示, LHRH-A可显著上调尼罗罗非鱼生长轴相关基因的表达, 从而促进鱼类的生长。     

萨罗罗非鱼与尼罗罗非鱼正反杂交子代间的分子遗传学差异

用耐盐能力强的萨罗罗非鱼和生长性能优的尼罗罗非鱼进行人工正反杂交,对两者正反交F1代微卫星分子遗传学差异进行了研究,结果如下:(1)两亲本及其正反杂交子代间都有特异的微卫星分子标记.(2)群体遗传多样性指标大都是尼萨罗非鱼>尼罗罗非鱼>萨尼罗非鱼>萨罗罗非鱼.(3)群体内平均遗传相似指数为:萨罗罗非鱼>萨尼罗非鱼>尼罗罗非鱼>尼萨罗非鱼.(4)萨罗罗非鱼对杂交子代的影响要远大于尼罗罗非鱼;正交子代尼萨岁非鱼可能在生产上更有利用价值和具有更高的选育潜力.     

“灭非灵”对外来鱼类尼罗罗非鱼的毒杀效果

尼罗罗非鱼(Oreochromis niloticus)是全球性的外来入侵鱼类之一,已经入侵到全世界多个自然水域,严重威胁着土著鱼类的生存和水生态系统的结构与功能。为了有效控制自然水域中尼罗罗非鱼的数量,本实验室筛选出一种对尼罗罗非鱼具有特异性杀灭作用的有机磷类药物"灭非灵",用于野外逃逸尼罗罗非鱼的应急防治。本文通过急性毒性试验方式,验证"灭非灵"对尼罗罗非鱼和多种常见鱼类的毒杀效果,并测定了"灭非灵"对尼罗罗非鱼幼鱼(体长4.0±0.23 cm,体重2.5±0.44 g)的24、48、72和96 h半致死浓度和其在环境中的安全浓度。结果表明:"灭非灵"对尼罗罗非鱼的毒杀效果非常显著(P0.01),暴露在0.10、0.17和0.20 mg·L-1药液中的尼罗罗非鱼在96 h内全部死亡,而草鱼,鲮,鲫,斑马鱼、唐鱼和泥鳅均能正常生长;"灭非灵"对尼罗罗非鱼幼鱼的24、48、72和96 h半致死浓度分别为0.167、0.077、0.057和0.043 mg·L-1,安全浓度为0.0043 mg·L-1。结果证明,"灭非灵"对尼罗罗非鱼的应急防治有积极的效果,可应用于小水域内入侵尼罗罗非鱼的特异性灭除。     

用RAPD技术对罗非鱼遗传变异的研究及应用

应用随机扩增多态性DNA（RAPD）技术检测了一个奥利亚罗非鱼（au）和湘湖（nx)、美国（nm)、沙市（np）三个尼罗罗非鱼养殖群体（Table1)。在20外引物（Table2）中筛选到12个引物,它们的扩增物显示了罗非鱼和尼罗罗非鱼二者在群体内或群体间存在遗传差异。其中（Fig.1),OPZ06、OPZ16、OPZ12和OPZ19四个引物分别有一个扩增片段具有种的特异性。它们的大小分别是900、1500、1700和730bp。可以作为鉴别罗非鱼和尼罗罗非鱼二者的分子遗传标记,湘湖（nx）、美国（nm）和沙市（np）三个尼罗罗非鱼群体都保留了较高水平的遗传变异。而奥利亚罗非鱼（au）的群体内遗传变异最小。奥利亚罗非鱼（au）与湘湖（nx）、美国（nm)、沙市（np）三个尼罗罗非鱼群体之间的遗传距离分别是0．285、0．262和0．344（Table3）,说明奥利亚罗非鱼（au）和沙市尼罗罗非鱼（np）杂交将可能产生较强的杂种优势。     

“灭非灵”对尼罗罗非鱼鳃、肝和肾组织结构的影响

通过"灭非灵"对尼罗罗非鱼(Oreochromis niloticus)(体重为34.65±5.69 g)的急性毒性试验及对鳃、肝、肾的组织学研究,从组织学角度探讨了"灭非灵"对尼罗罗非鱼的致死机理。结果表明:"灭非灵"对尼罗罗非鱼的24、48、72和96 h-LC50分别为0.148、0.103、0.048和0.032 mg·L-1;其组织病理学损伤表现为鳃小片萎缩、卷曲、坏死、脱落和融合,鳃间隙分泌大量的粘液细胞;肝细胞肿大,空泡化,细胞界限模糊,细胞核固缩;肾细胞肿大,充血;"灭非灵"对3种组织的损伤程度为鳃肝脏肾脏,3种组织的损伤很有可能是造成尼罗罗非鱼死亡的主要原因。     

大绒鼠及高山姬鼠体温调节和蒸发失水的日节律

为比较横断山区同域分布物种大绒鼠(Eothenomys miletus)和高山姬鼠(Apodemus chevrieri)的日节律特征,对两种鼠在24 h中4个时间段(04:00～06:00时、10:00～12:00时、16:00～18:00时和22:00～24:00时)的体温和蒸发失水进行了测定.结果显示,大绒鼠、高...     

吉丽罗非鱼(尼罗罗非鱼♀×萨罗罗非鱼♂)及其两亲本遗传变异的微卫星分析

吉丽罗非鱼是由耐盐性较强的萨罗罗非鱼做父本与生长速度较快的尼罗罗非鱼做母本进行杂交、杂交后代自交产生,2009年全国水产原良种审定委员会审定为养殖新品种。为了分析吉丽罗非鱼及其两亲本遗传特性,选择有代表性的6对微卫星引物,对这3种罗非鱼遗传变异进行研究分析。研究结果表明:(1)6对微卫星引物扩增产物片段大小为180～350bp,共发现21个等位基因,鱼类群体间、微卫星座位间及等位基因间都存在极显著差异。(2)有效等位基因数(Ne)、Nei's基因多样指数(H)和多态信息含量(PIC)值等群体遗传多样性指标都是吉丽罗非鱼>尼罗罗非鱼>萨罗罗非鱼,吉丽罗非鱼PIC值达到了0.657,属于高度多态性。(3)吉丽罗非鱼与萨罗罗非鱼的遗传距离要比与尼罗罗非鱼的近,萨罗罗非鱼对吉丽罗非鱼的遗传影响要大于尼罗罗非鱼。     

四个罗非鱼选育品种抗链球菌病能力差异研究

为筛选出抗病力优良的罗非鱼品种, 以奥利亚罗非鱼“夏奥1号”、尼罗罗非鱼“99”埃及品系、吉富罗非鱼“中威1号”和奥尼罗非鱼为研究对象, 33℃水温暂养7d后分别进行无乳链球菌人工感染实验, 连续7d统计累计死亡率, 并于人工感染后0、24h、48h和72h采集血液和组织样本, 研究这4个罗非鱼选育品种抗链球菌病能力的差异。结果显示: 感染7d后奥尼罗非鱼的累计死亡率最低; 奥尼罗非鱼的谷草转氨酶(AST)感染前后始终都低于其余3个品种罗非鱼(P<0.05); 埃及尼罗和奥尼在感染72h后球蛋白(GLO)分别显著升高1.13倍和1.41倍; 奥尼罗非鱼白蛋白/球蛋白(A/G)在感染前后没有显著性变化(P>0.05), 而其余3个品种罗非鱼A/G比值在感染后都显著性降低(P<0.05); 埃及尼罗的碱性磷酸酶(AKP)在感染72h后显著降低(P<0.05), 奥利亚和吉富的AKP表现为先上升后下降, 奥尼的AKP感染前后没有显著性变化(P>0.05); 各品种罗非鱼血清中的乳酸脱氢酶(LDH)感染后都显著升高(P<0.05); 奥利亚、吉富和奥尼罗非鱼的超氧化歧化酶(SOD)感染48h时都显著升高(P<0.05); 奥尼罗非鱼在感染前后溶菌酶(LZM)活性都显著高于其余3个品种罗非鱼(P<0.05)。组织病理学结果显示:吉富和奥尼肝细胞水肿变性, 而奥利亚和埃及尼罗出现大面积肝细胞脂肪变性; 每个品种罗非鱼均呈现严重的脾炎, 奥利亚、埃及尼罗和吉富的脾脏中大量铁血黄素沉积; 每种罗非鱼呈现不同程度的肾小球萎缩, 肾小管上皮细胞变性、坏死。研究表明奥尼罗非鱼抗链球菌病能力最强, 感染后血清中AST水平与肝受损程度呈一定的正相关, LZM水平和罗非鱼抗链球菌病能力呈一定的正相关。     

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.