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Generation and analysis of drought stressed subtracted expressed sequence tags from safflower (Carthamus tinctorius L.)

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PLANT GROWTH REGULATION 2013年第1期69卷 29-41页

作者： Thippeswamy, M. Sivakumar, M. Sudhakarbabu, O. Reddy, P. Chandraobul Veeranagamallaiah, G. Pandurangaiah, M. Ramya, M. Nareshkumar, A. Kirankumar, T. Sudhakar, Chinta Sri Krishnadevaraya Univ Dept Bot Anantapur 515003 Andhra Pradesh India Indian Inst Sci Dept Biochem Bangalore 560012 Karnataka India

Drought is the most crucial environmental factor that limits productivity of many crop plants. Exploring novel genes and gene combinations is of primary importance in plant drought tolerance research. Stress tolerant genotypes/species are known to express novel stress responsive genes with unique functional significance. Hence, identification and characterization of stress responsive genes from these tolerant species might be a reliable option to engineer the drought tolerance. Safflower has been found to be a relatively drought tolerant crop and thus, it has been the choice of study to characterize the genes expressed under drought stress. In the present study, we have evaluated differential drought tolerance of two cultivars of safflower namely, A1 and Nira using selective physiological marker traits and we have identified cultivar A1 as relatively drought tolerant. To identify the drought responsive genes, we have constructed a stress subtracted cDNA library from cultivar A1 following subtractive hybridization. Analysis of similar to 1,300 cDNA clones resulted in the identification of 667 unique drought responsive ESTs. Protein homology search revealed that 521 (78 %) out of 667 ESTs showed significant similarity to known sequences in the database and majority of them previously identified as drought stress-related genes and were found to be involved in a variety of cellular functions ranging from stress perception to cellular protection. Remaining 146 (22 %) ESTs were not homologous to known sequences in the database and therefore, they were considered to be unique and novel drought responsive genes of safflower. Since safflower is a stress-adapted oil-seed crop this observation has great relevance. In addition, to validate the differential expression of the identified genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), and northern blot analysis. We showed that these clones were differentially expressed under different

关键词： Carthamus tinctorius cDNA library EST macroarray Safflower Subtractive hybridization

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Clues to apoptosis pathway involvement in hemolysis, elevated liver enzyme, and low platelet (HELLP) syndrome and intrauterine growth restriction (IUGR)

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JOURNAL OF MATERNAL-FETAL & NEONATAL MEDICINE 2013年第1期26卷 26-31页

作者： Cecati, Monia Sartini, Davide Pozzi, Valentina Giannubilo, Stefano R. Ferretti, Filippo Stortoni, Piergiorgio Saccucci, Franca Tranquilli, Andrea L. Emanuelli, Monica Univ Politecn Marche Dipartimento Sci Clin Specialist & Odontostomatol I-60131 Ancona Italy

Objective: The neurotrophin family comprises molecules involved in growth, differentiation, survival, regeneration, normal functions of the neuronal system, and in angiogenesis. We have investigated the expression pattern of neurotrophic signaling molecules in pregnancies complicated by elevated liver enzyme, and low platelet (HELLP) syndrome and intrauterine growth restriction (IUGR). Methods: Placentas from normal and pathological pregnancies were collected. macroarray analysis was performed and the data were confirmed by real-time PCR. Results: Real-time PCR analyses (pathological vs. normal pregnancies) confirmed a significant down-regulation for IL-6, STAT3 alpha, STAT3 beta, and Bcl-2. The expression of Mcl-1 isoform 1 (long) was significantly increased. Conclusions: We suggest that decreased expression of IL-6 could mean that abnormalities in the immunological system function involve inflammatory cytokines other than IL-6 in examined pathological pregnancies. The STAT3 alpha and STAT3 beta down-regulation lead to a marked reduction of cellular transcriptional activity. Decreased expression of IL-6 is associated with a down-regulation of Bcl-2 but not of Mcl-1 isoform 1, suggesting that these two antiapoptotic proteins may function independently and that Mcl-1 may have a distinct role in controlling apoptotic pathway.

关键词： Cytokine gene expression macroarray neurotrophin pregnancy diseases

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Pythium cederbergense sp nov and related taxa from Pythium clade G associated with the South African indigenous plant Aspalathus linearis (rooibos)

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MYCOLOGIA 2013年第5期105卷 1174-1189页

作者： Bahramisharif, Amirhossein Lamprecht, Sandra C. Spies, Christoffel F. J. Botha, Wilhelm J. McLeod, Adele Univ Stellenbosch Dept Plant Pathol ZA-7602 Matieland South Africa Agr Res Council Plant Protect Res Inst ZA-7599 Stellenbosch South Africa ARC PPRI ZA-0121 Pretoria South Africa

The genus Pythium consists of more than 120 species and is subdivided into 11 phylogenetic clades (A-K) based on internal transcribed spacer (ITS) region sequence data. Pythium clade G contains only seven known species, with most not being well described. Our study characterized 12 Pythium isolates from Aspalathus linearis (rooibos) that fit into clade G. Phylogenetic analyses of the ITS region and a combined phylogeny of four gene regions (ITS, beta-tubulin, COX1 and COX2 [cytochrome c oxidase subunits I, II]) identified five clade G subclades. The rooibos isolates formed two groups, Pythium Rooibos I (RB I) and II (RB II), that clustered into two separate clades within subclade 1. The nine Pythium RB I isolates formed a distinct clade from P. iwayamai and is described here as a new species, Pythium cederbergense sp. nov. The three Pythium RB II isolates had P. canariense and P. violae as their closest relatives and were genetically diverse, suggesting the presence of several new species or a species complex that cannot be resolved with the current data, thus precluding a species description of this group. Morphological analyses showed that P. cederbergense and Pythium RB II were indistinguishable from each other but distinct from known clade G species. Clade G studies are being hampered by imprecise morphological descriptions of P. violae, P. canariense and P. iwayamai and each species being represented by only one isolate. The P. cederbergense and Pythium RB II isolates all were nonpathogenic toward rooibos, lupin and oats seedlings. One oligonucleotide was developed for each of P. cederbergense and Pythium RB II, which was able to differentiate the isolates with DNA macro-array analyses.

关键词： beta-tubulin COX1 COX2 ITS macroarray oomycete phylogeny taxonomy

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Multiplex PCR and DNA array for the detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. targeting virulence-related genes

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ANNALS OF MICROBIOLOGY 2013年第2期63卷 725-731页

作者： Thapa, Shree Prasad Han, Ah Reum Cho, Jun Mo Hur, Jang Hyun Kangwon Natl Univ Dept Environm Biol Coll Agr & Life Sci Chunchon 200701 South Korea Kangwon Natl Univ EFAP Safety Ctr Chunchon 200701 South Korea

A multiplex PCR and DNA array for quick detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. was developed using specific genetic markers derived from virulence-related genes. The genetic markers of cytK, sei, prfA, rfB, and hilA gene specifically amplified DNA fragments of 320 bp, 500 bp, 700 bp, 1.0 kb and 1.2 kb from B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., respectively. These markers are specific for the detection of the corresponding target pathogens. The sensitivity of the genetic markers was down to similar to 0.5 fg genomic DNA and similar to 10(1) CFU/ml (one bacterial cell per reaction) of bacterial culture. The combination of mPCR and DNA macroarray hybridization sensitively and specifically detected B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., in complex mixed cultures and food matrices. Thus, this mPCR and macroarray-based approach serves as rapid and reliable diagnostic tool for the detection of these five pathogens.

关键词： Genetic marker Multiplex PCR macroarray Specific detection Bacterial pathogen

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Analysis of gene expression as marker of relevant metabolisms, in three Acidithiobacillus ferrooxidans strains, in different growth conditions

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20th International Biohydrometallurgy Symposium (IBS 2013)

作者： Araya, Gonzalo Acosta, Mauricio Demergasso, Cecilia Univ Catolica Norte Ctr Biotecnol Antofagasta Chile

ISBN: (纸本)9783037858912

Acidithiobacillus ferrooxidans is a chemiolithoautotrophic Gram-negative bacterium widely spread in ambient temperature bioleaching processes. Several strains of At. ferrooxidans were isolated and studied and, some time later, questions arise about whether it was a species with a wide metabolic variation or a group of closely related species. Advances in molecular biology, phylogeny and genomics have shed some light on At. ferrooxidans strains and allows their grouping according to their relations. However, significant challenges remains to be met, such as understanding how a particular strain faces environmental challenges and how a particular kind of adaptive response affects the growth and activity of the strain. The purpose of this study was to identify differential expression signals between At. ferrooxidans strains -with different abundances and dynamics- present in the bioleaching system at Escondida mine. Culture characterization and DNA macroarrays techniques provided some answers. Analysis of growth curves showed that IESL 32 had the highest anaerobic growth rate, while aerobic growth was similar for all strains. It was shown that though the phylogenetic analysis based on 16S rRNA sequences suggested a close relation between IESL 32 and the type strain ATCC 23270, the growth curves and the expression profile showed that the type strain and strain D2 had the closest similarity. Growth experiments under different conditions, together with the comparative analysis of gene expressions in At. ferrooxidans, could be a springboard for future investigations of strain characterization to broaden our knowledge about adaptation, metabolic strategies, regulation and microbial diversity in industrial processes.

关键词： At. ferrooxidans gene expression anaerobic growth macroarray

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低密度cDNAmacroarray对干扰素α抗病毒蛋白的筛选

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中华肝脏病杂志 2011年第10期19卷 774-778页

作者：管世鹤杨凯王琴程中乐潘颖吴园园杨东亮安徽医科大学第二附属医院检验科合肥230601 华中科技大学同济医学院附属同济医院临床免疫研究室

目的基于低密度cDNAmacroarray技术筛选出差异表达的干扰素（IFN）仅抗病毒基因，以探讨IFNα抗病毒蛋白的表达与HBV复制的关系。方法以一定浓度的IFNa处理肝胚瘤细胞株HepG2和HepG2．2．15细胞6h，用cDNAmacroarray分析比较两细胞株IF... 详细信息

目的基于低密度cDNAmacroarray技术筛选出差异表达的干扰素（IFN）仅抗病毒基因，以探讨IFNα抗病毒蛋白的表达与HBV复制的关系。方法以一定浓度的IFNa处理肝胚瘤细胞株HepG2和HepG2．2．15细胞6h，用cDNAmacroarray分析比较两细胞株IFN仅抗病毒基因表达谱，并筛选出差异表达的IFNa抗病毒基因。将表达HBV核心蛋白（HBc）的质粒DHBc-EGFP转染HepG2细胞，RT-PCR法分析HBc对IFN仅抗病毒基因表达的影响。将表达抗黏病毒A蛋白（MxA）的表达质粒pcDNA3．1Flag-MxA转染HepG2．2．15，以酶联免疫吸附试验、Dotblot、Southernblot等方法分别检测HepG2．2．15细胞表达释放的HBsAg与HBeAg、细胞外HBVDNA和细胞内HBVDNA复制中间体（松弛环状DNA、双股线性DNA），以判断HBV复制情况。两组间数据比较采用t检验，组间不同时间点数据比较采用单因素方差分析。结果cDNAmacroarray分析显示HepG2和HepG2．2．15细胞的抗病毒基因表达谱具有差异性：IFNa抗病毒基因中干扰素诱导跨膜蛋白（IFITM）1、IFITM2、IFITM3、RING4等在HepG2．2．15细胞的表达被部分抑制，而重要的抗病毒蛋白MxA表达被完全抑制。HBc转染组细胞中MxAmRNA表达的相对水平为0．31±0．05，低于空白对照组的0．74±0．04，差异有统计学意义，P〈0．05。MxA蛋白转染HepG2．2．15细胞48、72h后，MxA转染组细胞上清液中HBsAg的S／CO值分别为1．42±0．21和1．58±0．18，HBeAg的s／co值为1．44±0．14和2．28±0．24，而空白对照组细胞上清液中HBsAg的S／CO值为1．92±0．19和2．79±0．25，HBeAg的S／CO值为2．31±0．46和3．37±0．29，两组细胞上清液中HBV抗原的s／CO值差异均有统计学意义，P值均〈0．05。细胞外HBVDNA、胞内HBV复制中间体DNA均无明显变化。结论HBV及其抗原成分的复制和表达影响着IFNa抗病毒蛋白的表达；HBV通过抑制IFNd抗病毒蛋白的表达而发挥拮抗IFNa的抗病毒活性。

关键词：肝炎病毒,乙型干扰素抗病毒蛋白 cDNA macroarray

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Identification and expression analysis of genes induced by phosphate starvation in leaves and roots of Brassica napus

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PLANT GROWTH REGULATION 2011年第1期65卷 65-81页

作者： Ren, Feng Chen, Liang Guo, Qian-Qian Zhong, Hui Wu, Yan Chang, Li-Li Li, Xue-Bao Huazhong Normal Univ Coll Life Sci Hubei Key Lab Genet Regulat & Integrat Biol Wuhan 430079 Peoples R China

Phosphorus (P) is one of the essential nutrient elements for plant growth and development because of its low availability and mobility in soil. To investigate genes which are involved in Pi starvation response in leaves and roots of Brassica napus, a survey of genes induced in Pi starvation was done by macroarray analysis. Among 3,000 cDNA clones, 94 genes were identified to be up-regulated, while 46 genes were detected to be down-regulated by Pi starvation in B. napus. The Pi starvation-induced genes represent a large range of functional categories including cell defense, cellular signal transduction, cellular transport, biogenesis of cellular components, protein synthesis, proteins with binding function, regulation of metabolism and so on. By comparative analysis, over 40% genes were overlapped with Pi starvation-induced genes in Arabidopsis, but the remaining was not identified in Arabidopsis. From the genes identified by macroarray analysis, 22 genes were selected for further quantitative reverse transcription (RT)-PCR assay and confirmed as Pi starvation-induced genes convincingly. The temporal expression patterns of 22 genes in roots and leaves were different during Pi starvation. Most of the 22 genes were expressed at relatively low level in leaves and roots, and at relatively high level in other tissues. The data presented in this study will facilitate the understanding of molecular mechanism of Pi starvation response in B. napus.

关键词： Brassica napus macroarray Phosphate starvation Gene identification

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In silico comparison of Yersinia pestis and Yersinia pseudotuberculosis transcriptomes reveals a higher expression level of crucial virulence determinants in the plague bacillus

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INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY 2011年第2期301卷 105-116页

作者： Chauvaux, Sylvie Dillies, Marie-Agnes Marceau, Michael Rosso, Marie-Laure Rousseau, Sandrine Moszer, Ivan Simonet, Michel Carniel, Elisabeth Inst Pasteur Yersinia Res Unit F-75724 Paris 15 France Inst Pasteur Plate Forme Puces ADN 2 F-75724 Paris 15 France Univ Lille Nord France Inserm U1019 CNRS UMR8204 Ctr Infect & Immun Lille F-59019 Lille France Inst Pasteur F-75724 Paris 15 France

Although Yersinia pestis and Yersinia pseudotuberculosis are genetically very similar (97% nucleotide sequence identity for most of the chromosomal genes), they exhibit very different patterns of infection. Y. pestis causes plague which is usually fatal in the absence of treatment, whereas Y. pseudotuberculosis generally triggers non-life-threatening intestinal symptoms. This drastic difference in pathogenicity may result from the acquisition of a few species-specific genes, but also from differences in their transcriptional regulation networks. In this study, we performed an in silico comparative whole-genome transcriptome analysis of Y. pestis and Y. pseudotuberculosis grown in parallel under 8 distinct conditions to determine whether they exhibit differences in their regulatory networks. In this analysis, 304 genes common to both species were found to display significant inter-species differences in transcriptional levels, with 91% of them being more expressed in Y. pestis. Remarkably, 3 major virulence determinants conserved in the 2 species (the pYV virulence plasmid, the High Pathogenicity Island, and the ail locus) were among the genes more expressed in Y. pestis. Furthermore, the induction at 37 degrees C of pYV-borne genes was considerably greater in Y. pestis than in Y. pseudotuberculosis. Conversely, the rovA transcriptional regulator gene was more transcribed in Y. pseudotuberculosis. We also performed a clustering analysis of the transcriptome data of both Y. pestis and Y. pseudotuberculosis, which allowed to group genes according to their expression profiles. This analysis identified groups of genes with unknown functions which, based on regulation patterns similar to those of known virulence genes, are potential new virulence determinants in Y. pestis. In conclusion, this is the first comparative analysis at the whole-genome level of the transcription profiles of Y. pestis and Y. pseudotuberculosis. Our results suggest that the higher pathogenicity of

关键词： Comparative transcriptomics Analysis of variance Clustering analysis Transcriptional regulation macroarray

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Molecular techniques for pathogen identification and fungus detection in the environment

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IMA FUNGUS 2011年第2期2卷 177-189页

作者： Tsui, Clement K. M. Woodhall, James Chen, Wen Levesque, C. Andre Lau, Anna Schoen, Cor D. Baschien, Christiane Najafzadeh, Mohammad J. de Hoog, G. Sybren Univ British Columbia Dept Forest Sci Vancouver BC V6T 1Z4 Canada Food & Environm Res Agcy York YO41 1LZ N Yorkshire England Agr & Agri Food Canada Cent Expt Farm Ottawa ON Canada Ctr Infect Dis & Microbiol Westmead NSW 2145 Australia Univ Sydney Westmead Hosp Westmead NSW 2145 Australia Plant Res Int Business Unit Biointeract & Plant Hlth NL-6700 AA Wageningen Netherlands Tech Univ Berlin Environm Microbiol D-10587 Berlin Germany CBS KNAW Fungal Biodivers Ctr NL-3584 CT Utrecht Netherlands Mashhad Univ Med Sci Sch Med Dept Parasitol & Mycol Mashhad Iran

Many species of fungi can cause disease in plants, animals and humans. Accurate and robust detection and quantification of fungi is essential for diagnosis, modeling and surveillance. Also direct detection of fungi enables a deeper understanding of natural microbial communities, particularly as a great many fungi are difficult or impossible to cultivate. In the last decade, effective amplification platforms, probe development and various quantitative PCR technologies have revolutionized research on fungal detection and identification. Examples of the latest technology in fungal detection and differentiation are discussed here.

关键词： FISH LAMP macroarray medical mycology molecular diagnostics molecular ecology padlock probe pathogenic fungi plant pathology rolling circle amplification

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Polyketide synthase pathways identified from a metagenomic library are derived from soil Acidobacteria

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FEMS MICROBIOLOGY ECOLOGY 2011年第1期78卷 176-187页

作者： Parsley, Larissa C. Linneman, Jonathan Goode, Ann M. Becklund, Kristen George, Isabelle Goodman, Robert M. Lopanik, Nicole B. Liles, Mark R. Auburn Univ Dept Biol Sci Auburn AL 36849 USA Georgia State Univ Dept Biol Atlanta GA USA Univ Wisconsin Dept Plant Pathol Madison WI 53706 USA Catholic Univ Louvain Unite Genie Biol B-1348 Louvain Belgium Rutgers State Univ Sch Environm & Biol Sci New Brunswick NJ 08903 USA

Polyketides are structurally diverse secondary metabolites, many of which have antibiotic or anticancer activity. Type I modular polyketide synthase (PKS) genes are typically large and encode repeating enzymatic domains that elongate and modify the nascent polyketide chain. A fosmid metagenomic library constructed from an agricultural soil was arrayed and the macroarray was screened for the presence of conserved ketosynthase [beta-ketoacyl synthase (KS)] domains, enzymatic domains present in PKSs. Thirty-four clones containing KS domains were identified by Southern hybridization. Many of the KS domains contained within metagenomic clones shared significant similarity to PKS or nonribosomal peptide synthesis genes from members of the Cyanobacteria or the Proteobacteria phyla. However, analysis of complete clone insert sequences indicated that the BLAST analysis for KS domains did not reflect the true phylogenetic origin of many of these metagenomic clones that had a %G+C content and significant sequence similarity to genes from members of the phylum Acidobacteria. This conclusion of an Acidobacteria origin for several clones was further supported by evidence that cultured soil Acidobacteria from different subdivisions have genetic loci closely related to PKS domains contained within metagenomic clones, suggesting that Acidobacteria may be a source of novel polyketides. This study also demonstrates the utility of combining data from culture-dependent and -independent investigations in expanding our collective knowledge of microbial genomic diversity.

关键词： polyketide synthase soil macroarray ketosynthase metagenomics Acidobacteria

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