Pesquisa de atividade antibacteriana de Paenibacillus ssp isolados de amostras de solo e água

14

Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data

<p>Abstract</p> <p>Background</p> <p>Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes.</p> <p>Results</p> <p>By the use of two distinct algorithms, implemented by <it>geNorm </it>and <it>NormFinder</it>, we have assessed the gene expression of nine candidate reference genes in cotton: <it>GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 </it>and <it>GhUBQ14</it>. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of <it>GhPP2A1 </it>and <it>GhUBQ14 </it>genes were the most stable across all samples and also when distinct plants organs are examined. <it>GhACT4 </it>and <it>GhUBQ14 </it>present more stable expression during flower development, <it>GhACT4 </it>and <it>GhFBX6 </it>in the floral verticils and <it>GhMZA </it>and <it>GhPTB </it>during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development.</p> <p>Conclusion</p> <p>We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of <it>GhUBQ14 </it>and <it>GhPP2A1 </it>housekeeping genes as superior references for normalization of gene expression measures in different cotton plant organs; <it>GhACT4 </it>and <it>GhUBQ14 </it>for flower development, <it>GhACT4 </it>and <it>GhFBX6 </it>for the floral organs and <it>GhMZA </it>and <it>GhPTB </it>for fruit development. We also provide the primer sequences whose performance in qPCR experiments is demonstrated. These genes will enable more accurate and reliable normalization of qPCR results for gene expression studies in this important crop, the major source of natural fiber and also an important source of edible oil. The use of bona fide reference genes allowed a detailed and accurate characterization of the temporal and spatial expression pattern of two MADS-box genes in cotton.</p

Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues

<p>Abstract</p> <p>Background</p> <p>Cotton (<it>Gossypium </it>spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization.</p> <p>Results</p> <p>Nucleotide analysis revealed high identity with cotton <it>E2 </it>homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the <it>E2 </it>active site, and an intron that is spliced in <it>E2 </it>homologues, but not in <it>GhGDRP85</it>. The <it>GhGDRP85 </it>gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in <it>Arabidopsis thaliana</it>. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2.</p> <p>Conclusions</p> <p>uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.</p

Transcriptome analysis of Gossypium hirsutum flower buds infested by cotton boll weevil (Anthonomus grandis) larvae

Background\ud Cotton is a major fibre crop grown worldwide that suffers extensive damage from chewing insects, including the cotton boll weevil larvae (Anthonomus grandis). Transcriptome analysis was performed to understand the molecular interactions between Gossypium hirsutum L. and cotton boll weevil larvae. The Illumina HiSeq 2000 platform was used to sequence the transcriptome of cotton flower buds infested with boll weevil larvae.\ud \ud \ud Results\ud The analysis generated a total of 327,489,418 sequence reads that were aligned to the G. hirsutum reference transcriptome. The total number of expressed genes was over 21,697 per sample with an average length of 1,063 bp. The DEGseq analysis identified 443 differentially expressed genes (DEG) in cotton flower buds infected with boll weevil larvae. Among them, 402 (90.7%) were up-regulated, 41 (9.3%) were down-regulated and 432 (97.5%) were identified as orthologues of A. thaliana genes using Blastx. Mapman analysis of DEG indicated that many genes were involved in the biotic stress response spanning a range of functions, from a gene encoding a receptor-like kinase to genes involved in triggering defensive responses such as MAPK, transcription factors (WRKY and ERF) and signalling by ethylene (ET) and jasmonic acid (JA) hormones. Furthermore, the spatial expression pattern of 32 of the genes responsive to boll weevil larvae feeding was determined by “in situ” qPCR analysis from RNA isolated from two flower structures, the stamen and the carpel, by laser microdissection (LMD).\ud \ud \ud Conclusion\ud A large number of cotton transcripts were significantly altered upon infestation by larvae. Among the changes in gene expression, we highlighted the transcription of receptors/sensors that recognise chitin or insect oral secretions; the altered regulation of transcripts encoding enzymes related to kinase cascades, transcription factors, Ca2+ influxes, and reactive oxygen species; and the modulation of transcripts encoding enzymes from phytohormone signalling pathways. These data will aid in the selection of target genes to genetically engineer cotton to control the cotton boll weevil.FAPESP [2009/53998-3]CNPq [310.612/2011-0, 306025/2010-8]Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ

Avaliação da atividade in vitro e in vivo de Paenibacillus provenientes de amostras de ambiente

35

Avaliação da atividade in vitro e in vivo de Paenibacillus provenientes de amostras de ambiente

35

Pesquisa de atividade antibacteriana de Paenibacillus ssp isolados de amostras de solo e água

14

Clonagem molecular e caracterização funcional de genes envolvidos na biossíntese de óleos essenciais (Monoterpenos) em Eucalyptus grandis

45

Construção e análise de uma biblioteca de cDNA visando a identificação de genes envolvidos na síntese de alcalóides em Psychotria brachyceras (Rubiaceae)

15

Construção e análise de uma biblioteca de cDNA visando a identificação de genes envolvidos na síntese de alcalóides em Psychotria brachyceras (Rubiaceae)

15