The responsive expression of heat shock protein 22 gene in zhikong scallop Chlamys farreri against a bacterial challenge

HSP22 is a member of a small HSP subfamily contributing to the growth, transformation and apoptosis of the cell as well as acting as a molecular chaperone. In the present study, CfHSP22 cDNA was cloned from Chlamys farreri by the rapid amplification of cDNA ends technique. The full-length cDNA of CfHSP22 was of 1279 bp, consisting of a 5'-terminal untranslated region (5'UTR) of 122 bp, a 3'UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly( A) tail, and an open reading frame of 576 bp encoding a polypeptide with a molecular mass of 22.21 kDa and a predicted isoelectric point of 9.69. There was an alpha-crystallin domain, a hallmark of the sHSP subfamily, in the C-terminus, and the deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. CfHSP22 was constitutively expressed in the haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in the hepatopancreas was higher than that in the other tissues. CfHSP22 transcription was up-regulated and reached a maximal level at 12 h after the bacterial challenge, and then declined progressively to the original level at 48 h. These results suggested that CfHSP22 perhaps play a critical role in response to the bacterial challenge in haemocytes of scallop C. farreri.

Cloning, characterization, and expression analysis of extracellular copper/zinc superoxide dismutase gene from bay scallop Argopecten irradians

Extracellular superoxide dismutase (ECSOD) is a major extracellular antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned a novel ECSOD from the bay scallop Argopecten irradians (AiECSOD) by 3' and 5' RACE. The full-length cDNA of AiECSOD was 893 bp with a 657 bp open reading frame encoding 218 amino acids. The deduced amino acid sequence contained a putative signal peptide of 20 amino acids, and sequence comparison showed that AiECSOD had low degree of homology to ECSODs of other organisms. The genomic length of the AiECSOD gene was about 5276 bp containing five exons and six introns. The promoter region contained many putative transcription factor binding sites such as c-Myb, Oct-1, Sp1, Kruppel-like, c-ETS, NF kappa B, GATA-1, AP-1, and Ubx binding sites. Furthermore, tissue-specific expressions of AiECSOD and temporal expressions of AiECSOD in haemocytes of bay scallops challenged with bacteria Vibrio anguillarum were quantified using qRT-PCR. High levels of expression were detected in haemocytes, but not in gonad and mantle. The expression of AiECSOD reached the highest level at 12 h post-injection with V. anguillarum and then returned to normal between 24 h and 48 h post-injection. These results indicated that AiECSOD was an inducible protein and that it may play an important role in the immune responses against V anguillarum. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians: Its gene structure, mRNA expression and recombinant protein

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Two unique and highly compartmentalized bay scallop Argopecten irradians superoxide dismutases: MnSOD and ecCuZnSOD, have been molecularly characterized in our previous study. To complete characterize the SOD family in A. irradians, a novel intracellular copper/zinc SOD from the A. irradians (Ai-icCuZnSOD) was obtained and characterized. The full-length cDNA of Ai-icCuZnSOD was 1047 bp with a 459 bp open reading frame encoding 152 amino acids. The genomic length of the Ai-icCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region containing many putative transcription factor binding sites were analyzed. Furthermore, quantitative reverse transcriptase real-time PCR (qRT-PCR) analysis indicated that the highest expression of the Ai-icCuZnSOD was detected in gill and the expression profiles in hemocytes of bay scallops challenged with bacteria Vibrio anguillarum and lipopolysaccharide (LPS) were different. The result presented an increased expression after injection with LPS whereas no significant changes were observed after V. anguillarum injection. A fusion protein containing Ai-icCuZnSOD was produced in vitro. The rAi-icCuZnSOD is a stable enzyme, retaining more than 80% of its activity between 10 and 60 degrees C and keeping above 88% of its activity at pH values between 5.8 and 9. Ai-icCuZnSOD is more stable under alkaline than acidic conditions. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

The manganese superoxide dismutase gene in bay scallop Argopecten irradians: Cloning, 3D modelling and mRNA expression

A novel manganese superoxide dismutase (MnSOD) was cloned from bay scallop Argopecten irradians by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of MnSOD was of 1207 bp with a 678 bp open reading frame encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the MnSOD of A. irradians shared high identity with MnSOD in invertebrates and vertebrates, such as MnSOD from abalone Haliotis discus discus (ABG88843) and frog Xenopus laevis (AAQ63483). Furthermore, the 3D structure of bay scallop MnSOD was predicted by SWISS-MODEL Protein Modelling Server and compared with those of other MnSODs. The overall structure of bay scallop MnSOD was similar to those of zebrafish Danio rerio, fruit fly Drosophila melanogaster, Chinese shrimp Fenneropenaeus chinensis, human Homo sapiens, and had the highest similarity to scallop Mizuhopecten yessoensis and abalone H. discus discus. A quantitative real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of MnSOD in different tissues and the temporal expression in haemocytes following challenge with the bacterium Vibrio anguillarum. A higher-level of mRNA expression of MnSOD was detected in gill and mantle. The expression of MnSOD reached the highest level at 3 h post-injection with V. anguillarum and then slightly recovered from 6 to 48 h. The results indicated that bay scallop MnSOD was a constitutive and inducible protein and thus could play an important role in the immune responses against V anguillarum infection. (c) 2008 Elsevier Ltd. All rights reserved.

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631 bp, consisting of a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 573 by with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968 bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96 h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12 h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response. (c) 2006 Elsevier Ltd. All rights reserved.

青海家牦牛HIF-1α基因组织特异性表达

[目的]探究青海家牦牛HIF-1α基因组织的特异性表达。[方法]应用半定量反转录PCR和实时定量反转录PCR（SYBRGreen）技术对青海家牦牛HIF-1α基因的组织特异性表达进行检测。通过提取不同组织总RNA，经DNase I消化后，用随机引物进行反转录合成cDNA，采用特异性引物分别对HIF-1α和β-actin基因进行RT-PCR和Real Time RT-PCR扩增。[结果]结果表明，HIF-1α基因在心、肝、脾、肺、肾、脑、肌肉、睾丸组织中均有表达，其中以睾丸和脾中HIF-1α基因表达量最高，肌肉的表达量最低。[结论]该研究为进一步揭示HIF-1α在高原土著动物低氧适应过程中的分子机制有着重要的意义。

高原鼠兔ob基因的组织表达特征

ob基因编码的leptin蛋白在调节生物体能量平衡中起到重要作用。本研究应用Taqman探针real time PCR技术对高原鼠兔ob基因的组织分布进行检测。通过提取不同组织总RNA，经DNase I消化后，用随机引物进行反转录合成cDNA，采用特异性Taqman探针和引物分别对ob基因及β-actin基因进行实时定量PCR扩增，对不同组织中ob基因和β-actin基因的初始拷贝数之比进行比较。结果表明ob基因在脑、心脏、肺、肝脏、脾脏、肾脏、骨骼肌、脂肪组织中均有表达，其中以白色脂肪组织中ob基因表达量最高，其次为心脏和肺，表达量最低的是肝脏和肾脏。

马铃薯Alfin1-like锌指蛋白基因克隆与序列分析

利用紫花苜蓿盐胁迫相关锌指结构蛋白Alfin1基因cDNA序列,通过电子克隆在GenBank中对马铃薯同源EST序列进行查询比较和拼接,获得了1个含有完整编码区的cDNA序列,并通过RT-PCR成功获得了该序列.获得的全长cDNA序列中包含1个747 bp的最大读码框,编码248个氨基酸,将其命名为Stfin1.氨基酸序列分析表明存在典型的Cys4-His-Cys3锌指结构,与Alfin1一致性达93.09%.从结构分析结果推测Stfin1与Alfin1在功能上具有一定的相关性.

常氧下高原鼠兔HIF-1α mRNA的表达

低氧诱导因子-1(HIF-1)是一种由低氧诱导因子-1α(HIF-1α)和芳香烃核受体转录因子(ARNT)组成的二聚体转录因子，在生物体氧平衡调节中起关键作用。高原鼠兔(Ochotona curzoniae)是生活在青藏高原海拔3000 m以上区域，有着极强的低氧、低温耐受能力的特有自然种群。它通过高的基础代谢率和高的氧利用率来适应高原低温、缺氧环境。将高原鼠兔带到北京，常氧室温条件下适应7 d。 RT-PCR扩增高原鼠兔HIF-1αcDNA片断，~（32）p标记作为特异性探针，Northern杂交检测了常氧条件下高原鼠兔HIF-1α的组织特异性表达。结果显示，HIF-1α mRNA在高原鼠兔多种组织中均有表达，并且表现出明显的组织差异性。其中脑的表达量最大，肾其次，心、脾和肝的表达相对较少。

高原鼠兔低氧诱导因子-1α的初步研究

高寒和缺氧是高原地区的两个重要的限制生态因子，高原土著动物在长期的适应进化过程中必将形成自己独特的低温、低氧适应策略。高原鼠兔（Ochotona curzoniae）是生活在青藏高原海拔以上地区的特有物种，具有极强的低温、低氧耐受能力。它主要通过高的基础代谢率和增加非颤抖性产热以及高的氧利用率来适应高寒、缺氧环境[1-4] 。低氧诱导因子-l（Hypoxia-Induced FacHIF一l）是 1992年Semanza等在低氧的肝癌细胞株Hep3B中发现的一种特异性结合于红细胞生成素（EPO）增强子寡核昔酸序列的转录因子[5]，它HIF-la和HIF-lβ/ARNT两个亚单位组成［6〕。HIF-l。受低氧诱导，属功能性亚基，HIF-1侧ARNT在细胞中构建型表达，不受低氧诱导[7]。为进一步揭示原鼠兔低氧适应的分子机理，我们正在克隆高原鼠兔HIF-1。基因的CDNA全长，并试图对其在低氧适应中的作用进行研究，本文报道了高原鼠兔低氧诱导因子-la编码区部分片段的克隆。

Molecular cloning, expression and biochemical property analysis of AtKP1, a kinesin gene from Arabidopsis thaliana

Kinesins are common in a variety of eukaryotic cells with diverse functions. A cDNA encoding a member of the Kinesin-14B subfamily is obtained using X-RACE technology and named AtKP1 (for Arabidopsis kinesin protein 1). This cDNA has a maximum open reading frame of 3.3 kb encoding a polypeptide of 1087 aa. Protein domain analysis shows that AtKP1 contains the motor domain and the calponin homology domain in the central and amino-terminal regions, respectively. The carboxyl-terminal region with 202 aa residues is diverse from other known kinesins. Northern blot analysis shows that AtKP1 is widely expressed at a higher level in seedlings than in mature plants. 2808 bp of the AtKP1 promoter region is cloned and fused to GUS. GUS expression driven by the AtKP1 promoter region shows that AtKP1 is mainly expressed in vasculature of young organs and young leaf trichomes, indicating that AtKP1 may participate in the differentiation or development of Arabidopsis thaliana vascular bundles and trichomes. A truncated AtKP1 protein containing the putative motor domain is expressed in E. coli and affinity-purified. In vitro characterizations indicate that the polypeptide has nucleotide-dependent microtubule-binding ability and microtubule-stimulated ATPase activity.

Resumos...

Análise proteômica de raízes de feijão (Phaseolus vulgaris) inoculadas com Rhizobium tropici. Teste para comprovar a eficiência do método de extração e determinação de ácido fítico em sementes de soja utilizado na Embrapa Soja. Auditoria de comunicação: avaliando os veículos de comunicação interna da Embrapa Soja. Utilização da técnica de espectrofotometria do infravermelho próximo (NIR) para análise discriminante dos ácidos graxos oléico e linoléico de genótipos de girassol. Análise da disponibilidade hídrica para a cultura da soja nas safras 2004/05 e 2009/10 em Londrina, PR . Calibração de psicrômetros para avaliações de potencial hídrico foliar. Avaliação de ácidos graxos da soja: grão inteiro, casca, cotilédones e hipocótilo. Variabilidade temporal da produtividade da soja após conversão do preparo convencional para o sistema plantio direto. COMUT na biblioteca da Embrapa Soja. EM DIA: Boletim interno compartilhando informações. Sistema Web para estimativas de perdas por seca na cultura da soja. Portais Web desenvolvidos no laboratório de Bioinformática da Embrapa Soja. Validação de um método para detecção e quantificação de soja cultivance® tolerante a herbicidas imidazolinonas por PCR . Índice de vegetação por diferença normalizada (NDVI) de cultivares de soja sob três níveis de disponibilidade hídrica no solo. Avaliação do fluxo de seiva em cultivares de soja em três níveis de disponibilidade hídrica no solo. A importância do controle de qualidade dos inoculantes. Comportamento exploratório de ninfas recém eclodidas de Edessa meditabunda (F.) (Heteroptera: Pentatomidae) sobre a superfície dos córions. Produção de brotos de soja da cultivar BRS 216. Teste de aceitabilidade de brotos de soja da cultivar BRS 216. Análise de lignina com diferentes massas de tegumento de soja utilizando método gravimétrico. Desenvolvimento e caracterização físico-química de biscoitos com farinha de soja orgânica de cultivares especiais para a alimentação humana. Análise sensorial de biscoitos com farinha de soja orgânica de cultivares especiais para a alimentação humana. Avaliação de genótipos de soja de diferentes grupos de maturação e resistência aos percevejos. Métodos químicos para extração de boro no solo. Abordagem computacional para a identificação de elementos cis-regulatórios no genoma da soja. Quebra de dormência em sementes de girassol silvestre utilizando ácido giberélico. Teor relativo de água em cultivares de soja sob três níveis de disponibilidade hídrica no solo. Soybean gene express: plataforma para análise de expressão diferencial em bibliotecas subtrativas de CDNA. Biologia e exigências térmicas do ácaro vermelho Tetranychus gigas. Características biológicas de Telenomus remus em diferentes hospedeiros após serem criados em de ovos de Anticarsia gemmatalis e Spodoptera frugiperda por uma geração. Enfoque sobre o plano de saúde dos empregados da Embrapa no contexto do pacote de benefícios sociais oferecidos pela empresa. Dinâmica populacional do ácaro verde Mononychellus planki em cultivares de soja. Envelhecimento, trabalho e tempo livre: elaborando projetos de vida. Teor de isoflavonas em vinte cultivares de soja semeadas em Londrina e Ponta Grossa. Utilização de Pseudomonas fluorescens no controle biológico de Macrophomina phaseolina. Algoritmo computacional para determinar o perfil mínimo de marcadores moleculares que discriminam um conjunto de cultivares. Qualidade física do solo em um sistema de integração lavoura-pecuária com diferentes pressões de pastejo. Diversidade de estirpes do gênero Burkholderia em solos do cerrado brasileiro baseado no sequenciamento do gene ribossomal 16S. Estudo da diversidade genética de isolados de Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei.

Cloning and structure analysis of hydrogenase gene from Chlamydomonas reinhardtii SE

The cDNA of Chlamydomonas reinhardtii SE encoding hydrogenase (HydA2) was obtained from the total RNA of C reinhardtii SE by RT-PCR. The DNA of hydrogenase was amplified by PCR from the genomic DNA of C reinhardtii SE. The cDNA and DNA of hydrogenase were sequenced, respectively. The structure of hydrogenase gene was analyzed by biology software. The open reading frame predicts that the hydrogenase is composed of 3584 bp encoding 505 amino acids in length with a predicted M.W. of 53.69 kDa. Ten exons (including 1518 bp) and nine introns (including 2066 bp) have been found in the hydrogenase, and there were two potential N-glycosylate sites, eight protein kinase C phosphorylation site, eight casein kinase H phosphorylation site and one sulphorylation in the sequence. The theory pI was 6.15. Total number of negatively charged residues (Asp + Glu) and positively charged residues (Arg + Lys) were 55 and 61, respectively. (c) 2005 Elsevier Ltd. All rights reserved.

Taking advantage of an old concept, "illegitimate transcription", for a proposed novel method of genetic diagnosis of McArdle disease

McArdle disease is a metabolic disorder caused by pathogenic mutations in the PYGM gene. Timely diagnosis can sometimes be difficult with direct genomic analysis, which requires additional studies of cDNA from muscle transcripts. Although the "nonsense-mediated mRNA decay" (NMD) eliminates tissue-specific aberrant transcripts, there is some residual transcription of tissue-specific genes in virtually all cells, such as peripheral blood mononuclear cells (PBMCs).We studied a subset of the main types of PYGM mutations (deletions, missense, nonsense, silent, or splicing mutations) in cDNA from easily accessible cells (PBMCs) in 12 McArdle patients.Analysis of cDNA from PBMCs allowed detection of all mutations. Importantly, the effects of mutations with unknown pathogenicity (silent and splicing mutations) were characterized in PBMCs. Because the NMD mechanism does not seem to operate in nonspecific cells, PBMCs were more suitable than muscle biopsies for detecting the pathogenicity of some PYGM mutations, notably the silent mutation c.645G>A (p.K215=), whose effect in the splicing of intron 6 was unnoticed in previous muscle transcriptomic studies.We propose considering the use of PBMCs for detecting mutations that are thought to cause McArdle disease, particularly for studying their actual pathogenicity.

Cloning and characterization of a putative fructosyltransferase and two putative invertase genes from the temperate grass Lolium temulentum L.

J. A. Gallagher, A. J. Cairns and C. J. Pollock (2004). Cloning and characterization of a putative fructosyltransferase and two putative invertase genes from the temperate grass Lolium temulentum L. Journal of Experimental Botany, 55 (397) pp.557-569 Sponsorship: BBSRC RAE2008