二化螟热休克蛋白70基因的克隆及热胁迫下的表达分析

热休克蛋白70是已知热休克蛋白家族中最重要的一种, 它在细胞内的大量表达可以明显改善细胞的生存能力, 提高对环境胁迫的耐受性。为探讨热胁迫对二化螟Chilo suppressalis幼虫热休克蛋白70表达的影响, 采用RT-PCR及RACE技术从二化螟血淋巴细胞中克隆了热休克蛋白70基因全长cDNA序列。该基因全长2 102 bp, 开放阅读框 (open reading frame, ORF)为1 959 bp, 编码652个氨基酸; 5′非编码区（untranslated region, UTR)为81 bp, 3′UTR为62 bp。从该基因推导的氨基酸序列与其他昆虫的同源序列比较有很高的相似性（73%~97%)。实时定量PCR显示二化螟HSP70基因能被热胁迫诱导表达, 幼虫血淋巴细胞的HSP70基因在36℃时表达量最高。流式细胞术研究发现HSP70在蛋白质水平上的表达变化与在mRNA水平上高度一致, 说明二化螟HSP70基因在转录及翻译水平上受到热应激的调节。     

异色瓢虫热休克蛋白70基因的克隆与分析

本研究通过RT-PCR和RACE方法,首次克隆了异色瓢虫Harmonia axyridis(Pallas)热休克蛋白70(HSP70)cDNA全序列(GenBank登录号:EF668009).获得的cDNA全长2200 bp,其中开放阅读框1956 bp,编码一个651氨基酸的蛋白,计算分子量为70 kDa,等电点(PI)为5.32.同源序列比对结果表明,异色瓢虫HSP70与其它真核生物的HSP/HSC70有着较高的序列同源性(85%～93%).其中与赤拟谷盗Tribolium castaneum热休克蛋白70(HSP70)同源性最高,达到93%.与其它真核生物HSP70一样,该序列包含真核生物HSP70高度保守的全部三个家族标签以及ATP/GTP结合位点、一个Bipartive nuclear localization signal和细胞核定位信号.     

草鱼AMBP基因cDNA的克隆与序列分析

α1-微球蛋白和Bikunin是由同一基因翻译表达出的两种功能不相关联的血浆蛋白。本文通过快速扩增cDNA末端的方法,首次从草鱼肝脏组织克隆了α1-微球蛋白和Bikunin前体蛋白(α1-microglobulin/Bulinin precursor, AMBP）基因全长cDNA。其cDNA全长1230bp,包含5′非翻译区23 bp,3′非翻译区160 bp和开放读码框1047 bp。开放读码框编码348个氨基酸,包含182个氨基酸的α1-微球蛋白和145个氨基酸的Bikunin。草鱼AMBP与其他物种的氨基酸序列分析结果表明,它们具有较高的同源性(44.7%－84.4%),其中草鱼与斑马鱼同源性最高(84.4%)。结果表明AMBP序列结构和α1-微球蛋白与Bikunin共翻译表达特点在动物机体中具有着重要的生理意义。     

西伯利亚蓼rd22基因的克隆与序列分析

以3% NaHCO₃溶液胁迫处理48 h的西伯利亚蓼为试材,利用RACE技术,从其茎部组织克隆了脱水应答蛋白RD22的全长cDNA序列。测序后的结果分析表明,该cDNA序列全长为1 302 bp,5′非翻译区为59 bp,3′非翻译区为25 bp,开放读码框为1 218 bp,编码405个氨基酸。在氨基酸序列的C端含有一个比较保守的BURP结构域,N端含有5个重复序列THV-VGKGGV-V。信号肽检测证明该蛋白为分泌性蛋白,前21个氨基酸区域为信号肽结构。其推演的氨基酸序列与葡萄的同源性最高,达到60%。该基因已在GenBank上注册,基因序列登录号为DQ836050。     

B型烟粉虱和温室白粉虱热激蛋白90基因（hsp90）的全长cDNA克隆与系统发育分析

B型烟粉虱Bemisia tabaci (Gennadius) biotype B和温室白粉虱Trialeurodes vaporariorum均为全球普遍发生的重要害虫。本研究以其他昆虫热激蛋白90基因（hsp90）保守区域设计兼并引物扩增两种粉虱hsp90中间片段, 然后利用RACE技术获得全长cDNA。温室白粉虱hsp90全长cDNA的开放性阅读框2 166 bp, 编码722个氨基酸; 烟粉虱hsp90全长cDNA的开放性阅读框2 160 bp, 编码720个氨基酸。两种粉虱HSP90的完整氨基酸序列相似性高达92.94％, 并均具有定义HSP90家族签名序列的5个氨基酸保守区域和末尾基序“MEEVD”。通过real-time PCR技术, 探测到两个基因在mRNA水平上皆能高温诱导表达。采用昆虫纲所有完整HSP90氨基酸序列进行Kimura双参数遗传距离分析并构建NJ进化树, 结果显示hsp90在昆虫纲低级阶元水平和高级阶元水平系统进化上能得到一个较理想结果。本研究结果为B型烟粉虱和温室白粉虱抗逆适应性研究提供基础, 并进一步验证保守的功能基因hsp90可以作为研究生物系统发育的手段之一     

大菱鲆组成型热休克蛋白70的全长cDNA克隆及表达分析

根据感染哈维氏弧菌(Vibrio harveyi)的大菱鲆(Scophthalmus maximus)的差减cDNA文库中hsp70EST序列设计引物,用RACE方法首次克隆到长2188bp的大菱鲆HSC70(Heat-shock cognate protein 70)全长cDNA序列,包括1956bp的开放阅读框及5′和3′非翻译区。在预测的651个氨基酸序列中发现了Dnak特征性基序,胞质HSP70特征基序以及四肽简并重复序列。与其他真核生物HSP70家族成员进行同源性比较,发现大菱鲆HSC70与牙鲆(Paralichthys olivaceus)HSC71、虹鳟(Oncorhynchus mykiss)HSC71、人(Homo sapiens)HSC70、家鼠(Mus musculus)HSC70、烟草天蛾(Manduca sexta)HSC70的氨基酸相似性分别是97%,95%,94%,93%,86%,表现出较高的保守性。表达分析显示,hsc70mRNA在大菱鲆正常肝脏、鳃、肠、脾脏、头肾、肾等组织中以不同的水平存在,呈组成型表达;被哈维氏弧菌感染后,大菱鲆肝脏和脾脏组织hsc70mRNA表达水平分别在24h和12h出现上调(2.5-fold和1.6-fold);注射生理盐水组与未注射组之间差异不显著。       

柽柳金属硫蛋白基因的克隆及序列分析

用木麻黄(Casuarina glauca)的金属硫蛋白基因(metallothionein 1)氨基酸序列对柽柳ESTs序列本地数据库进行tBlastn检索,获得了柽柳金属硫蛋白基因全长cDNA序列,去除polyA后该基因全长366 bp,其中5′非翻译区97 bp,3′非翻译区59 bp,开放读码框(ORF)长210 bp,编码70 个氨基酸组成的多肽,蛋白分子量为6.793 kD,理论等电点为4.99,含10个Cys,集中分布在肽链的N端和C端。BlastP同源性分析表明该基因与花生同源性最高,与小豆同源性最低。该基因的EST序列在GenBank登录(登录号：CV792539)。     

大口黑鲈脂代谢相关基因NPY、UCP2、LPL、HL克隆与分子进化分析

采用RT-PCR和RACE方法克隆了大口黑鲈NPY基因cDNA全序列及UCP2、LPL、HL基因cDNA核心片段。序列分析结果表明,大口黑鲈NPY基因cDNA全序列长664 bp,其中5′端非翻译区(5′-UTR)长53 bp,3′端非翻译区(3′-UTR)长311 bp,开放阅读框(ORF)长300 bp,编码99个氨基酸,即前体NPY。大口黑鲈前体NPY包括三个部分,28个氨基酸组成的信号肽、36氨基酸组成的成熟NPY以及32个氨基酸组成的由Gly-Lys-Arg指示的NPY C端肽（CPON）。大口黑鲈UCP2、LPL、HL基因cDNA核心片段长度分别为737 bp、509 bp和666 bp,各自编码245个氨基酸、169个氨基酸和222个氨基酸。将4个基因的氨基酸序列分别与其他物种的氨基酸序列进行同源性比较,并通过MEGA3构建系统树,对这4个脂代谢相关基因的分子进化特征进行了探讨。     

飞蝗热休克蛋白70cDNA片段的克隆和序列分析

采用R-PCR方法对海南、河北和辽宁3个飞蝗(Locusta migratoria L．)种群的热休克蛋白70(HSP70)基因cDNA片段进行克隆。在事先优化的条件下,通过简并性上游引物和下游引物扩增出了河北种群飞蝗HSP70基因的604bp cDNA片段(GenBank登录号为AY299637),推导的氨基酸序列包含201个氨基酸残基。分析表明,由飞蝗该片段推导的氨基酸序列与其他昆虫的同源性较高;3个种群该片段的核苷酸序列相似性更高达98．75％。由此推测,飞蝗种群间抗寒性的差异可能不是HSP70的序列变异引起的,而与HSP70的诱导表达有关。     

Cu2+胁迫后青海湖裸鲤DNAJC2及其相关基因的表达分析

为进一步明确青海湖裸鲤(Gymnocypris przewalskii)应对胁迫反应的分子机制,并挖掘有重要功能的基因,本研究克隆了青海湖裸鲤中一个编码J结构域的基因DNAJC2,并分析了其相关基因对Cu²⁺胁迫的应答反应。研究发现,青海湖裸鲤DNAJC2基因的开放阅读框为1 866 bp,共编码621个氨基酸,5′-UTR非翻译区为108 bp, 3′-UTR非翻译区为130 bp。DNAJC2在青海湖裸鲤肝脏、脑和鳃组织中高表达。与空白对照组相比,随着Cu²⁺浓度和胁迫时间的增加,在鳃和肾脏组织中,Grp78、ID1和DNAJC2基因以表达上调的方式参与应激反应,而Hspa14、Hyou1、RARα和XPC基因以表达下调或者上调后又恢复至正常水平的方式参与应激反应;在肝脏中,Hspa14和ID1基因以表达上调的方式参与应激反应,而Grp78、DNAJC2、CDK8和XPC基因以表达上调或下调后又下调或上调的方式参与应激反应,Hyou1和RARα基因在低浓度下以表达上调的方式参与应激反应,但在高浓度下以表达下调后又上调的方式参与应激反应。以...     

稀有鮈鲫HSP70基因启动子的克隆及功能分析

热休克蛋白70(HSP70)作为一种分子伴侣,在环境毒理学中受到广泛研究。前期研究表明稀有鮈鲫HSP70基因(GrHSP70)表达量与五氯酚(pentachlorophenol, PCP)处理的浓度和时间在肝脏中呈现剂量/时间-依赖效应。为探究启动子在热休克蛋白70表达调控中的作用,根据已知的GrHSP70 cDNA序列,采用染色体步移技术克隆了GrHSP70的5'侧翼区的核苷酸序列。生物信息学分析从预测的转录起始位点(C)起的5'侧翼区域共1 487bp,潜在的转录因子结合位点包括雌激素响应元件(ERE)、Sp1结合位点(Sp1)、糖皮质激素响应元件(GRE)、TATA结合蛋白(TBP)、CCAAT/增强子蛋白结合位点(C/EBP)、Oct-1结合位点(Oct-1)、GATA转录因子结合位点(GATA-1)等。实验构建了含有启动子缺失片段的萤火虫萤光素酶(firefly luciferase)和海肾萤光素酶(renilla luciferase)报告基因表达载体,瞬时转染HeLa细胞后,利用双荧光活性检测确定获得的GrHSP70启动子具有启动活性,其核心启动位点位于转录起始点上游-1 487~-1 093bp。同时,用不同浓度PCP暴露成功转染了重组质粒(pGL-HSP70 promoter-Luc+)的HeLa细胞,培养24h后检测双荧光活性,与对照相比,随PCP浓度的增加,荧光活性均显著增加。说明在稀有鮈鲫肝脏中PCP会通过激活GrHSP70启动子来诱导GrHSP70表达,但PCP在稀有鮈鲫体内通过何种机制来调节HSP70的合成,仍然需要进一步研究。     

Influence of co-cations on biosorption of lead and zinc – a comparative evaluation in binary and multimetal systems

The influence of co-cations (cadmium, copper, cobalt and nickel) on lead and zinc biosorption by Streptoverticillium cinnamoneum and Penicillium chrysogenum in binary and multimetal systems was evaluated. The metal sorption capacity of S. cinnamoneum was higher than P. chrysogenum for all the metals tested. Both the biomasses exhibited preferential uptake of lead in a multimetal situation. Even though mutual inhibition was seen for all binary systems containing zinc, systems containing lead exhibited unequal inhibition. The extent of metal sorption was dependent on metal chemistry, affinity for binding sites and the type of metal binding. In multimetal systems, S. cinnamoneum and P. chrysogenum exhibited preferential sorption orders: Pb²⁺ > Zn²⁺=Cu²⁺ > Cd²⁺ > Ni²⁺ > Co²⁺ and Pb²⁺ > Cu²⁺ > Zn²⁺ > Cd²⁺ > Ni²⁺ > Co²⁺. The order of metal biosorption in a multimetal system could be predicted well on the basis of Langmuir parameters evaluated in binary metal systems.     

来源于嗜碱芽孢杆菌N16-5甘露聚糖利用基因簇的乙酰酯酶AesA的克隆及性质分析*

天然来源的多糖底物上常存在乙酰基取代,特异性的乙酰酯酶能够切割这些底物上的乙酰基,从而有利于聚糖底物的进一步降解.对Bacillus sp. N16-5甘露聚糖利用基因簇上编码的乙酰酯酶AesA进行了基因克隆和异源表达,并对其酶学性质进行了研究.aesA基因长957bp,编码318个氨基酸,属于碳水化合物酯酶第7家族.AesA对4-甲基伞形酮乙酸酯(4-methylumbelliferyl-acetate)表现出较好的催化活性,金属离子Fe³⁺,Fe²⁺,Mn²⁺及Cu²⁺对AesA活性均有不同程度的促进作用.AesA与甘露聚糖酶ManA对乙酰化的甘露聚糖底物具有显著的协同作用.此项研究有助于理解嗜碱芽孢杆菌Bacillus sp.N16-5对甘露聚糖的水解机制,并且在甘露聚糖降解中具有潜在的应用前景.     

Enzymatic properties of a novel highly active and chelator resistant protease from a Pseudomonas aeruginosa PD100

Pseudomonas aeruginosa PD100 capable of producing an extracellular protease was isolated from the soil collected from local area (garbage site) from Shivage market in Pune, India. The purified protease showed a single band on native and SDS-PAGE with a molecular weight of 36 kDa on SDS-PAGE. The optimum pH value and temperature range were found to be 8 and 55–60 °C, respectively. The enzyme exhibited broad range of substrate specificity with higher activity for collagen. The enzyme was inhibited with low concentration of Ag²⁺, Ni²⁺, and Cu²⁺. β-Mercaptoethanol was able to inactivate the enzyme at 2.5 mM, suggesting that disulfide bond(s) play a critical role in the enzyme activity. Studies with inhibitors showed that different classes of protease inhibitors, known to inhibit specific proteases, could not inhibit the activity of this protease. Amino acid modification studies data and pK_a values showed that Cys, His and Trp were involved in the protease activity. P. aeruginosa PD100 produces one form of protease with some different properties as compared to other reported proteases from P. aeruginosa strains. With respect to properties of the purified protease such as pH optimum, temperature stability with capability to degrade different proteins, high stability in the presences of detergents and chemicals, and metal ions independency, suggesting that it has great potential for different applications.     

Purification and characterization of UDPG:ginsenoside Rd glucosyltransferase from suspended cells of Panax notoginseng

Heterogeneity of ginsenosides is an interesting and important issue because those structure-similar secondary metabolites have different or even totally opposite pharmacological activities. In this work, a new enzyme UDP-glucose:ginsenoside Rd glucosyltransferase (UGRdGT), which catalyzes the formation of ginsenoside Rb₁ from ginsenoside Rd [Biotechnol. Bioeng. 89: 444–52, 2005], was purified approximately 145-fold from suspended cells of Panax notoginseng with an overall yield of 0.2%. Purification to apparent homogeneity, as judged by SDS-PAGE, was successfully achieved by using sequential ammonium sulphate precipitation, anion-exchange chromatography and native PAGE. The enzyme had a molecular mass of 36 kDa, and its activity was optimal at pH 8.5 and 35 °C. The enzyme activity was enhanced by Mn²⁺, Ca²⁺ and Mg²⁺, but strongly inhibited by Zn²⁺, Hg²⁺, Co²⁺, Fe²⁺ and Cu²⁺. The apparent K_m value for UDP-glucose and ginsenoside Rd was 0.32 and 0.14 mM, respectively. The biotransformation yield from ginsenoside Rd to Rb₁ by UGRdGT in 50 mM Tris–HCl buffer at pH 8.5 and 35 °C was over 80%. This work provides a basis for further molecular study on the ginsenoside Rb₁ biosynthesis by P. notoginseng cells and it is also useful for potential application to in vitro biotransformation from ginsenoside Rd to Rb₁.     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

Thermal stress and neural function: adaptive mechanisms in insect model systems

Neural circuit function is vulnerable to hyperthermic failure but can be protected by stress pretreatments, such as exposure to a brief, sub-lethal high temperature (heat shock, HS), by increasing the time to failure and decreasing the time to recover.

Insects provide excellent model systems to investigate potential mechanisms underlying thermotolerant operation.

Induced thermotolerance is mediated by increased expression of heat shock proteins, HSPs, notably HSP70. Enhanced expression of HSP70 by increasing the gene dosage does not improve HS-induced thermotolerance of larval locomotion or locomotor central pattern generation in Drosophila.

Prior stress down-regulates neuronal K⁺ currents and this is associated with adaptive increases in the duration of action potentials.

Hyperthermic failure and recovery of the ventilatory central pattern generator in locusts is tightly correlated with a catastrophic increase in extracellular K⁺ concentration and its subsequent restoration.

These, and other data, suggest that neural circuit function can be protected by a stress-induced upregulation of HSPs that stabilize the cytoskeleton and preserve the operation of important membrane proteins such as ion channels, receptors and the Na⁺/K⁺-ATPase.     

Cloning and expression of HSP70 gene of sipuncula Phascolosoma esculenta

In this study the gene encoding HSP70 was isolated from Phascoloma esculenta by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length of cDNA (2520 bp) consists of a 5′-terminal untranslated region (UTR) (125 bp), a 3′-terminal UTR (421 bp) with a canonical polyadenylation signal sequence (AATAAA), a poly (A) tail, and an open reading frame (ORF) (1974 bp). The predicted molecular mass and isoelectric point for HSP70 is 71.6 kDa and 5.15, respectively. BLAST analysis showed that P. esculenta HSP70 gene shared high similarity. Classical HSP signature motifs, ATP/GTP-Binding Site Motif A, Bipartite Nuclear Targeting Sequence, the cytosolic HSP70 could be expressed in Escherichia coli BL21. After purification, the recombinant pET-HSP70 protein was used to produce the polyclonal antibody in mice and the specificity of the antibody was confirmed by Western blot analysis. Fluorescent real-time quantitative PCR analysis showed that expression of Hsp70 in sipuncula was increased significantly after exposure to 10 mM Zn for12 h, Cd for 24 h, Cu for 48 h, and was exposure to 37 °C for 24 h sea water.     

花群珊瑚对两种微藻的摄食

以花群珊瑚为材料,采用碳清除率测定、PCR分子营养标记和组织学观察的方法,研究花群珊瑚是否摄食亚心形扁藻和球等鞭金藻。结果表明: 花群珊瑚对球等鞭金藻的碳清除率显著高于亚心形扁藻,分别为0.44和0.11 pg·mL^-1·polyp^-1·h^-1。球等鞭金藻烯酰基载体蛋白还原酶基因片段作为分子标记在投喂组珊瑚组织中可扩增出目的片段。亚心形扁藻18S rRNA基因的引物在相应投喂组珊瑚组织中也可扩增出目的片段。组织学观察发现: 投喂组珊瑚水螅体内部隔膜较宽,隔膜与体壁的胃层都存在大量食物泡。亚心形扁藻投喂组在体壁胃层的食物泡中和口道沟处均发现未消化完全的亚心形扁藻。球等鞭金藻投喂组则在隔膜胃层和体壁胃层内的食物泡中发现所摄食金藻。因此,碳清除率、分子营养标记以及组织学观察均表明花群珊瑚对亚心形扁藻与球等鞭金藻存在摄食现象。