黄瓜 CsCCD7 基因的克隆及表达研究

使用RACE技术克隆黄瓜(Cucumis sativus L.)腋芽生长抑制基因并进行生物信息学和半定量RT-PCR分析。结果表明：从黄瓜腋芽中成功克隆了拟南芥(Arabidopsis thaliana)AtCCD7/MAX3同源基因,命名为CsCCD7(GenBank登录号：HQ005419);CsCCD7基因序列含有1665 bp的开放阅读框(ORF),编码554个氨基酸;编码的蛋白质命名为CsCCD7,隶属于CCD蛋白家族成员,蛋白质的二级结构和三级结构预测显示其富含β折叠和β转角以及无规卷曲,是不稳定蛋白。CsCCD7在根中的表达量最高,在多分枝、矮化黄瓜D0462中的表达量最低,这说明CsCCD7蛋白可能参与调控植物分枝信号的转导及分枝相关基因的表达调控。     

毛竹PsbS基因的克隆及其原核表达特征研究

采用RT-PCR技术从毛竹(Phyllostachys edulis)叶片中克隆到1个PsbS基因,命名为PePsbS (GenBank No. FJ600727),其编码区为810 bp,编码269个氨基酸。序列分析表明,PePsbS编码的蛋白与其它单子叶植物的PsbS蛋白有很高的相似性。蛋白结构分析表明,PePsbS基因编码蛋白包含导肽部分和成熟蛋白,其中成熟蛋白包含4个跨膜结构域。将PePsbS基因编码成熟蛋白的序列构建到原核表达载体pET23a中,并转入大肠杆菌,用IPTG进行诱导表达。结果表明, 41℃下诱导4 h的表达效果最好,目的蛋白含量约占总蛋白的21.5%,分子量约为22.0 kD。这说明温度和诱导时间明显影响PePsbS基因的表达。     

麻疯树鲨烯合酶基因SQS的克隆及生物信息学分析

以麻疯树(Jatropha curcas L.)总RNA为模板,根据已报道的鲨烯合酶基因序列设计简并引物,用RACE方法克隆得到麻疯树鲨烯合酶基因全长cDNA,命名为JcSQS。JcSQS全长1609 bp,包含1个1242 bp的开放阅读框,预测麻疯树鲨烯合酶基因编码的蛋白含有413个氨基酸。JcSQS具有鲨烯合酶类的保守结构域,JcSQS 蛋白与蓖麻、柿、木榄等植物中SQS基因编码的氨基酸序列具有高度同源性。这为研究麻疯树萜烯类物质的生物合成和调控机制奠定了基础。     

当归延伸因子AsEF 1β基因的克隆及胁迫应答分析

延伸因子1β(EF 1β)是蛋白质生物合成过程中肽链延长必需的调节因子之一。该研究采用同源克隆和RACE扩增技术克隆当归EF 1β基因序列,分析该基因序列特征、蛋白结构特点及UV B辐射胁迫下的组织响应表达,以揭示当归栽培生境变迁过程中对UV B胁迫适应的分子机制。结果显示：(1)成功克隆获得当归EF 1β基因全长序列(950 bp),编码225个氨基酸,命名为AsEF 1β(GenBank登录号：MG736314);AsEF 1β蛋白的分子量为24.5 kD,理论等电点为4.48,属亲水性氨基酸,在其C末端具有一个EF 1B超蛋白家族的典型结构域和保守区,鸟嘌呤核苷酸交换结构域;其氨基酸序列与同为伞形科的胡萝卜氨基酸序列相似性最高,达93%。(2)qRT PCR分析结果显示,AsEF 1β基因在当归根部的表达量显著高于茎和叶(P<0.05);UV B辐射胁迫下,茎及叶中的表达量均上调,分别是自然光照处理的2.43和3.76倍。研究表明,AsEF 1β基因可能参与当归对UV B辐射胁迫的适应过程,为深入研究其在药用植物生长发育、逆境抗性形成及药效物质的生物合成代谢过程的生态调控奠定了基础。     

奥利亚罗非鱼髓样分化因子(MyD88)的克隆及其在组织中的表达

髓样分化因子（myeloid differentiation factor 88,MyD88）是TLR（toll-like receptor）信号通路的关键接头蛋白,在先天性免疫中具有重要作用。通过RACE-RCR技术克隆了奥利亚罗非鱼（Oreochromis aureus）MyD88基因cDNA全长序列（GenBank登录号：JN032017）。序列分析表明,奥利亚罗非鱼MyD88 基因全长为1 611 bp,其中包括155 bp的5’非编码区,589 bp的3’非编码区和867 bp的编码区,编码288个氨基酸残基。MyD88蛋白N端具有死亡结构域,C端具有TIR结构域。同源性分析表明,奥利亚罗非鱼MyD88氨基酸序列与鳜鱼（Siniperca chuats）相似性最高,为85.8%,与其他鱼类相似性为70%~82%,与哺乳动物相似性为63%~66%;系统进化树分析表明,奥利亚罗非鱼MyD88与同属鲈形目的鳜鱼、大黄鱼（Larimichthys crocea）聚在一起。采用实时定量PCR方法检测MyD88在奥利亚罗非鱼各组织中的表达情况。结果显示,MyD88在所有被测组织中都有表达,其中表达量最高的是卵巢,其次在小肠、脾、肝、肾、鳃和血液中有较高的表达量,肌肉、精巢组织中表达量最低。本研究可为进一步探讨MyD88在奥利亚罗非鱼TLR信号通路中的作用奠定一定的基础。     

黄瓜叶绿素降解关键酶基因CsPAO的克隆与分析

该研究以黄瓜“津春2号”cDNA为模板,采用RT PCR方法克隆得到黄瓜叶绿素降解关键酶(pheophorbide a oxygenase,PAO)基因(CsPAO),对其进行亚细胞定位观察,并采用实时荧光定量PCR技术和生物信息学技术,分析了CsPAO基因的表达模式及其编码蛋白的特性。结果表明：(1)CsPAO编码545个氨基酸,理论等电点为6.09,蛋白相对分子质量为61.02 kD。蛋白预测发现,黄瓜CsPAO属于不稳定蛋白,具有2个蛋白结合位点,且存在跨膜现象。(2)荧光定量PCR结果表明,CsPAO基因表达响应水杨酸(SA)、茉莉酸 (JA)和赤霉素(GA₃)的调控,在高温(42 ℃)和低温(4 ℃)处理下CsPAO基因的表达量显著上升并达到最高,但黑暗处理对CsPAO基因表达没有影响;在黄瓜不同组织中花的表达显著高于根、茎、叶、萼、须、果。(3)亚细胞定位结果表明,CsPAO蛋白定位于叶绿体内。(4)系统进化树分析显示,黄瓜CsPAO与葫芦科植物苦瓜、西葫芦、南瓜、笋瓜等亲缘关系较近。本研究结果为进一步揭示黄瓜叶绿素降解的分子机制奠定了基础。     

龋齿致病菌变形链球菌蛋白 Smu.260 的 制备、结晶及初级晶体学分析

变形链球菌 (Streptococcus mutans) 是最主要的龋齿致病菌,其基因 Smu.260 编码一个约 23 ku (200 个氨基酸 ) 的蛋白质. Smu.260的 DNA 片段被克隆到表达载体 pET28a 后在大肠杆菌 BL21(DE3) 菌株中表达得到很好的产量. 产物 Smu.260 蛋白通过 Ni²⁺亲和柱和分子筛两步法纯化,并发现纯化后的蛋白以两种形式存在,二聚体 (约46 ku) 和四聚体,前者呈亮黄色,后者无色. 采用悬滴气象扩散法得到了二聚体形式的晶体. 晶体的 X 射线衍射分辨率达到 2.3埃,晶体属正交空间群 P2₁2₁2₁,晶格参数为a=89.88埃, b=90.91埃, c=105.17埃. 晶胞不对称单元内估计含有一个二聚体,溶剂含量为 53％ .     

巨桉EgrGBF1基因的克隆和表达模式分析

G-box结合蛋白(GBF)是一类能够识别并结合G-box的转录因子,广泛参与植物基因响应外界刺激的表达调控。通过巨桉(Eucalyptus grandis)初生生长到次生生长的转录组测序筛选出差异表达基因EgrGBF1,为探讨其在桉树生长发育中的功能,从巨桉中克隆了该基因,并进行了结构和进化分析。结果表明,EgrGBF1编码区长度为984 bp,编码327个氨基酸, 存在2个转录本,分别命名为EgrGBF1α和EgrGBF1β。实时荧光定量PCR结果表明,EgrGBF1α和EgrGBF1β在不同组织中,不同激素、胁迫处理下的表达模式不同,EgrGBF1α主要在茎尖表达,沿节间向下表达量逐渐降低,而EgrGBF1β在韧皮部高表达,在节间的表达量无显著差异。在水杨酸和缺硼处理下,EgrGBF1α和EgrGBF1β的表达趋势相反。EgrGBF1α在缺磷处理168 h的表达量最高,而EgrGBF1β在处理6 h的表达量最高。因此,EgrGBF1在桉树生长发育以及响应胁迫中发挥着重要作用,且转录本EgrGBF1α和EgrGBF1β可能具有不同的功能。     

高能量膳食对中老年食蟹猴糖尿病相关基因表达的影响

本研究选择空腹血糖值(FPG)在正常范围内(3.20 mmol/L≤FPG<5.50 mmol/L)的中老年食蟹猴(Macaca fascicularis)60只,高能量膳食诱导12个月后,将其分为正常血糖组和诱高血糖组(FPG≥5.50 mmol/L)。采用荧光定量PCR技术对2组中36个糖尿病相关基因在诱导前后外周血白细胞中的mRNA表达量进行分析。结果表明,高能量膳食诱导后,诱高血糖组FPG和甘油三酯(TG)显著高于正常血糖组(P<0.05),而胆固醇(TCHO)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)与分组无显著相关性(P>0.05)。基因表达水平上,诱高血糖组和正常血糖组均有血管紧张素转换酶基因(ACE)、肝糖原磷酸化酶基因(PYGL)、水通道蛋白基因(AQP2)等19个基因的mRNA表达量在高能量膳食诱导前后存在显著差异(P<0.05),且基因的表达模式变化一致,但诱高血糖组的mRNA表达量变化大,且三磷酸腺苷柠檬酸裂解酶(ACLY)、选择素L(SELL)、突触相关蛋白23(SNAP23)、突触融合蛋白(STX4)这4个基因的mRNA表达水平仅在诱高血糖组高能量膳食诱导前后呈差异表达(P<0.05)。     

甘薯IbGL3的克隆和表达分析

紫色甘薯富含花青素,具有较高的食用和药用价值。花青素的生物合成受到结构基因和调节基因的控制。bHLH(basic helix-loop-helix protein)转录因子能够调节多个花青素结构基因的表达,在花青素生物合成途径中具有重要的调控作用,但目前在甘薯中还没有关于bHLH调控花青素生物合成的相关报道。为进一步了解IbGL3基因在甘薯花青素生物合成的功能和作用机理,该研究根据甘薯转录组数据,利用RT-PCR技术在甘薯中克隆了一个2 120 bp的bHLH基因IbGL3,该基因包含一个1 878 bp的开放阅读框,编码625个氨基酸,蛋白质分子量69.08 kD,理论等电点(pI)5.20。IbGL3蛋白和其他植物中类黄酮合成相关的bHLH蛋白具有较高的同源性,都包含保守的MIR区、bHLH结构域和ACT类似结构域。系统发育进化树分析结果显示,IbGL3与其他植物类黄酮相关bHLH蛋白聚为一类,属于Ⅲf 亚类成员。表达结果显示,IbGL3基因在紫色甘薯中的表达量最高,在浅紫色甘薯中的表达量次之,在白色甘薯中表达量最低, 与花青素积累正相关,因此推测其在甘薯花青素生物合成途径中具有重要的调控作用。     

巨桉扩展蛋白EgrEXPA8和EgrEXPA10基因的克隆和表达特性分析

为探讨扩展蛋白在桉树生长发育中的作用,以在桉树初生生长到次生生长转换转录组测序中筛选出的差异表达基因EgrEXPA8和EgrEXPA10为基础,从巨桉(Eucalyptusgrandis)中克隆了2个扩展蛋白基因EgrEXPA8和EgrEXPA10,分别编码249和244个氨基酸,属于亲水蛋白,但Egr EXPA8稳定性高于Egr EXPA10。q RT-PCR分析表明,Egr EXPA8和Egr EXPA10基因均在幼叶和茎尖组织中表达量较高,在木质部和韧皮部表达量较低;且在茎顶端初生生长阶段表达量较高,而在下部次生生长节间表达量较低,可能其主要参与巨桉的初生生长或者负调控次生生长;另外在盐胁迫、茉莉酸甲酯处理下其均被抑制表达;而在水杨酸、缺硼、缺磷处理下均上调表达。这说明EgrEXPA8和EgrEXPA10在巨桉响应逆境胁迫时起到重要作用,且呈现出相似的调控方式。     

Gene structure and chromosomal localization of mouse cyclin G2 (Ccng2)

Cyclins are essential activators of cyclin-dependent kinases (Cdk) which, in turn, play pivotal roles in controlling transition through cell-cycle checkpoints. Cyclin G2 is a recently discovered second member of the G-type cyclins. The two members of the G-type cyclins, cyclin G1 and cyclin G2, share high structural similarity but their function remains to be defined. Here we characterize the structure of the mouse cyclin G2 gene by first cloning and sequencing the full-length mouse cyclin G2 cDNA. The cyclin G2 cDNA was used to isolate the cyclin G2 gene from a BAC library and to establish that the gene was transcribed from eight exons spanning a total of 8604 bp. The cyclin G2 gene was mapped by fluorescence in situ hybridization (FISH) to mouse chromosome 5E3.3.–F1.3. This region is syntenic to a region on human chromosome 4. The expression of cyclins G1 and G2 was examined in various tissues, but no correlation between expression patterns of the two genes was observed. However, during hepatic ontogenesis the cyclin G2 expression level decreased with age, whereas cyclin G1 expression increased. Transient expression of cyclin G2-green fluorescent protein (GFP) fusion protein in NIH3T3 cells showed that cyclin G2 is essentially a cytoplasmic protein, in contrast to the largely nuclear localization of cyclin G1. Our data suggest that, despite the close structural similarity between mouse cyclins G1 and G2, these proteins most likely perform distinct functions.     

The F1 genes of the F1F0 ATP synthase from the acidophilic bacterium Thiobacillus ferrooxidans complement Escherichia coli F1unc mutants

Abstract Because of the allelic variations within the M protein gene ( emm gene) of group A streptococci, reliable typing of this important human pathogen can be accomplished by the use of emm gene-specific oligonucleotide probes. Two technical modifications (a reverse dot blot and a reverse line blot hybridization assay) of a novel approach for the type-specific identification of emm genes have been developed. Both procedures involved amplification of an emm gene by polymerase chain reaction. The non-radioactively labeled amplicon was subsequently hybridized to a membrane carrying an array of immobilized emm gene-specific oligonucleotide probes, thus allowing the simultaneous analysis of the gene polymorphism in a single hybridization reaction. The feasibility of these rapid and easy to perform methods was shown for the unequivocal identification of reference strains and clinical isolates belonging to 16 different M serotypes.     

nif Gene expression in a Nif+, Fix−Bradyrhizobium japanicum variant

Abstract Bradyhizobium japonicum USDA 110 has been shown to contain several genetically similar, naturally occurring colony morphology variants. One of these variants, L2-110, although devoid of symbiotic nitrogen fixation, retains significant levels of explanta nitrogen fixation ability relative to other symbiotically competent USDA 110 variants (MN-110 and I-110). Interestingly, Northern blot analyses revealed that L2-110 nodules, despite their lack of symbiotic nitrogen fixation, contained 65% the level of mRNA for dinitrogenase ( nif DK) and 64% the level of dinitrogenase reductase ( nif H) mRNA relative to MN-110 nodules. Western blot analyses of tissue from the same nodules detected 32% the level of dinitrogenase and 31% the level of dinitrogenase reductase in L2-110 relative to MN-110. L2-110 appears to be a new class of mutant based on the complete absence of symbiotic nitrogen fixation (Fix⁻ and the presence of significant exanta nitrogen fixation (Nif⁺).     

The chorion genes of the medfly. II. DNA sequence evolution of the autosomal chorion genes s18, s15, s19 and s16 in Diptera

We present a total of approximately 15 kb of DNA sequences, encompassing four chorion genes Ccs18, Ccs15, Ccs19, Cc16 and their flanking DNA in the medfly C. capitata. Comparison of coding regions, introns and intergenic sequences in five Dipteran species, D. melanogaster, D. subobscura, D. virilis, D. grimshawi and C. capitata documented an extensive divergence in introns and coding regions, but few well conserved elements in the proximal 5′ flanking regions in all species. These elements are related to conserved regulatory features of three of the genes, including tissue- and temporal regulation. In the fourth, gene s15, significant alterations in the 5′ flanking region may be responsible for its changed temporal regulation in C. capitata. One long intergenic sequence, located in the distal 5′ flanking region of gene s18, is homologous to ACE3, a major amplification control element and contains an 80-bp A/T-rich sequence, known to stimulate strong binding of the origin recognition complex (ORC) in D. melanogaster. Analysis of the nucleotide composition of all chorion genes in C. capitata and D. melanogaster showed that C. capitata exhibit less biased representation of synonymous codons than does D. melanogaster.     

Interorganellar gene transfer in bryophytes: the functional nad7 gene is nuclear encoded in Marchantia polymorpha

The nad7 gene, encoding subunit 7 of NADH dehydrogenase, is mitochondrially encoded in seed plants. In the liverwort, Marchantia polymorpha, only a pseudogene is located in the mitochondrial genome. We have now identified the functional nad7 gene copy in the nuclear genome of Marchantia, coding for a polypeptide of 468 amino acids. The nuclear-encoded nad7 has lost the two group II introns present in the mitochondrial pseudogene copy. Instead, a typical nuclear intron is found to split an exon encoding the presumptive mitochondrial targeting signal peptide and the mature subunit 7 of NADH dehydrogenase. These results suggest that RNA-mediated gene transfer from the mitochondrial into the nuclear genome occurs not only in seed plants but also in bryophytes. Received: 11 March 1997 / Accepted: 20 August 1997