Jose Antonio Garrido-Cardenas

Cloning and molecular characterization of the Δ6-desaturase from two Echium plant species: Production of GLA by heterologous expression in yeast and tobacco

The synthesis of GLA (Δ6, 9, 12-18:3) is carried out in a number of plant taxa by introducing a double bond at the Δ6 position of its precursor, linoleic acid (Δ9, 12-18:2), through a reaction catalyzed by a Δ6-desaturase enzyme. We have cloned genes encoding the Δ6-desaturase (D6DES) from two different Macaronesian Echium species, E. pitardii and E. gentianoides (Boraginaceae), which are characterized by the accumulation of high amounts of GLA in their seeds. The Echium D6DES genes encode proteins of 438 amino acids bearing the prototypical cytochrome b5 domain at the N-terminus. Cladistic analysis of desaturases from higher plants groups the Echium D6DES proteins together with other Δ6-desaturases in a different cluster from that of the highly related Δ8-desaturases. Expression analysis carried out in E. pitardii shows a positive correlation between the D6DES transcript level and GLA accumulation in different tissues of the plant. Although a ubiquitous expression in all organs is observed, the transcript is particularly abundant in developing fruits, whereas a much lower level is present in mature leaves. Functional characterization of the D6DES gene from E. gentianoides has been achieved by heterologous expression in tobacco plants and in the yeast Saccharomyces cerevisiae. In both cases, overexpression of the gene led to the synthesis of GLA. Biotechnological application of these results can be envisaged as an initial step toward the generation of transgenic oleaginous plants producing GLA.

Δ6-Desaturase sequence evidence for explosive Pliocene radiations within the adaptive radiation of Macaronesian Echium (Boraginaceae).

The oceanic islands of Macaronesia provide an ideal temporal and spatial context to test hypotheses of plant evolution using a novel set of phylogenetic markers, Delta(6)-desaturase sequences. In contrast to the limited resolution of standard molecular markers (nrDNA and plastid sequences), the Delta(6)-desaturase sequence phylogeny of Echium unequivocally reconstructs its active colonization across islands and archipelagos (Madeira, the Canary Islands, and Cape Verde), as well as its subsequent geographical and ecological speciation. Molecular-clock estimates using penalized likelihood and Bayesian inference reveal two radiation processes coincident with two dramatic climatic changes recorded in the region: the advent of the cold Canarian sea current (ca. 4 Ma) and the establishment of a strong seasonality in the Pleistocene (1.8 Ma). Though Echium had available all the diversity of present-day Macaronesian environments (xeric and mesic scrub, laurisilva, pine forest, and subalpine habitats) in the Miocene, evolutionary divergence appears to have been triggered by an extension of fluctuating xeric and mesic habitats with the advent of Pliocene conditions. These Echium radiations not only fulfill traditional predictions of adaptive radiation (i.e., common ancestry, rapid speciation, and phenotype-environment correlation), but also, uniquely among Macaronesian species, trait utility of woodiness. A Pliocene transition from annuality to a bush or tree-like condition occurred in early Echium lineages. Maintenance of woodiness in major lineages, and reversal to an herbaceous condition by three independent events, is reported for the first time in plants of oceanic islands.

Cloning and Molecular Characterization of the Acyl-CoA: Diacylglycerol Acyltransferase 1 (DGAT1) Gene from Echium

Boraginaceae species, such as those from the genus Echium, contain high levels of the Δ6-desaturated γ-linolenic (18:3n-6) and octadecatetraenoic (18:4n-3) acids. These are unusual fatty acids among the plant kingdom that are gaining interest due to their benefits to human health. The potential utility of acyltransferases aimed at an increase in oil yield and fatty acid profiling has been reported. In this work, a gene encoding an acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) was cloned from Echium pitardii. Genomic and cDNA sequences obtained revealed a gene structure composed of 16 exons, yielding a protein (EpDGAT) of 473 amino acids with high similarity to DGAT1 enzymes of plants. Protein features such as a predicted structure with a highly hydrophilic N-terminus followed by 10 transmembrane domains, as well as the presence of diverse specific signatures, also indicate that EpDGAT belongs to the DGAT1 family. indeed. DGAT activity of the protein encoded by EpDGAT was confirmed by heterologous expression of the full-length cDNA in a yeast mutant (H1246) defective in the synthesis of triacylglycerols. Fatty acid composition of the triacylglycerols synthesized by EpDGAT in H1246 yeast cultures supplemented with polyunsaturated fatty acids suggest a substrate preference for the trienoic fatty acids α-linolenic acid (18:3n-3) and γ-linolenic acid over the dienoic linoleic acid (18:2n-6). Site-directed mutagenesis has revealed the presence of a critical residue (P178 in EpDGAT) within a reported thiolase signature for binding of acyl-enzyme intermediates that might be involved in the active site of the enzyme. Transcript analysis for EpDGAT shows an ubiquitous expression of the gene which is increased in leaves during senescence.

The metagenomics worldwide research

Metagenomics is the technique, or set of techniques, whose main objective is to determine the microbial population that can be found in a determined environment, studied in the context of its community. For this, it uses the techniques of massive sequencing, or next generation sequencing, due to the difficulties presented by traditional techniques when trying to transfer all the microorganisms present in a given environment to the laboratory. Metagenomics is a newly created technique, which was born at the beginning of the twenty-first century, and since then the interest of the world scientific community in fields as diverse as medicine, biotechnology, agriculture or genetics has not left to grow. In this article, the authors make a historical review of the metagenomics, analyze and evaluate the different massive sequencing platforms used for metagenomic assays, review the current literature on this subject and advance future problems with which researchers who decide to go deeper in this field could find. In this way, the prior knowledge of the researcher will facilitate the approach of his research.

A distinct subfamily of papain-like cystein proteinases regulated by senescence and stresses in Glycine max

GMCP3 encodes a cystein proteinase of Glycine max belonging to the papain-like family (C1A in MEROPS database) that was previously found to be involved in the mobilization of protein reserves during seed germination. Here, we report that GMCP3 is induced by senescence and diverse stresses in non-seed tissues, thus indicating a more general function in plants. Cladistic analysis of papain-like proteins of plants indicated that GMCP3, along with related proteases of other species, belongs to a distinct new group within the C1A family, which can also be distinguished by the four-exon structure of the gene. We also describe the genomic organization of GMCP3 revealing the presence of two closely related copies that are transcriptionally regulated in a similar way, although only one appears to be functional.

Cloning and molecular characterisation of a Δ8-sphingolipid-desaturase from Nicotiana tabacum closely related to Δ6-acyl-desaturases

Investigation on the absence of Δ6-desaturase activity in Nicotiana tabacum has led to the cloning of a new desaturase gene from this organism (NTDXDES) that exhibited unexpected biochemical activity. Cladistic analysis shows clustering of NTDXDES together with functional Δ6-acyl-desaturases of near Solanales plants, such as Borago and Echium. This group lies apart from that of previously characterised Δ8-sphingolipid-desaturases, which also includes two putative tobacco members identified in this study. Moreover, strong expression of NTDXDES is found in leaves, flowers, fruits and developing seeds of tobacco plants that is highly dependent on the development phase, with transcriptional activity being higher at stages of active tissue growth. This pattern is similar to that showed by Δ6-acyl-desaturases characterised in Boraginaceae species. However, functional assays using a yeast expression system revealed that the protein encoded by NTDXDES lacks Δ6-desaturase activity, but instead it is able to desaturate sphingolipid substrates by introducing a double bond on the Δ8-position. These data indicate that NTDXDES represent a novel desaturase gene placed in a different evolutionary lineage to that of previously characterised Δ8-desaturases.

DNA Sequencing Sensors: An Overview

The first sequencing of a complete genome was published forty years ago by the double Nobel Prize in Chemistry winner Frederick Sanger. That corresponded to the small sized genome of a bacteriophage, but since then there have been many complex organisms whose DNA have been sequenced. This was possible thanks to continuous advances in the fields of biochemistry and molecular genetics, but also in other areas such as nanotechnology and computing. Nowadays, sequencing sensors based on genetic material have little to do with those used by Sanger. The emergence of mass sequencing sensors, or new generation sequencing (NGS) meant a quantitative leap both in the volume of genetic material that was able to be sequenced in each trial, as well as in the time per run and its cost. One can envisage that incoming technologies, already known as fourth generation sequencing, will continue to cheapen the trials by increasing DNA reading lengths in each run. All of this would be impossible without sensors and detection systems becoming smaller and more precise. This article provides a comprehensive overview on sensors for DNA sequencing developed within the last 40 years.

Differential expression of the ornithine decarboxylase gene during carposporogenesis in the thallus of the red seaweed Grateloupia imbricata (Halymeniaceae).

This paper describes the cloning of the ornithine decarboxylase gene from a red seaweed, Grateloupia imbricata (Rhodophyta), the characterization of its expression throughout the reproductive process, and demonstrates how polyamines are involved in seaweed reproduction. In addition, the data indicate that the basal perennial and non-spore-forming thalli behave physiologically and genetically differently from the distal reproductive tissue. The common polyamines putrescine, spermidine and spermine have been associated with carposporogenesis in red seaweeds. Ornithine decarboxylase (ODC, EC 4.1.1.17) produces the diamine putrescine from the non-protein amino acid, ornithine. ODC is predominant in the synthesis of polyamines in G. imbricata. The gene encoding the ornithine decarboxylase in G. imbricata was cloned by genomic polymerase chain reaction (PCR) using degenerate primers against conserved motives, followed by chromosome walking using inverse PCR (iPCR). The encoded protein (GiODC, accession # FJ223132) was very similar to other ODCs, bearing the characteristic conserved domain of pyridoxal-dependent decarboxylases. The expression of the GiODC gene was investigated by real-time PCR and in situ hybridization (ISH), and was observed to vary according to cystocarp differentiation. It was weakly transcribed in apical parts of fertile tissue where the cystocarps are located, while the transcript levels were comparatively high in the basal part. This expression pattern correlated with the levels of free polyamines, which were higher at the basal part.

Trends in plant research using molecular markers

AbstractMain conclusionA deep bibliometric analysis has been carried out, obtaining valuable parameters that facilitate the understanding around the research in plant using molecular markers. The evolution of the improvement in the field of agronomy is fundamental for its adaptation to the new exigencies that the current world context raises. In addition, within these improvements, this article focuses on those related to the biotechnology sector. More specifically, the use of DNA markers that allow the researcher to know the set of genes associated with a particular quantitative trait or QTL. The use of molecular markers is widely extended, including: restriction fragment length polymorphism, random-amplified polymorphic DNA, amplified fragment length polymorphism, microsatellites, and single-nucleotide polymorphisms. In addition to classical methodology, new approaches based on the next generation sequencing are proving to be fundamental. In this article, a historical review of the molecular markers traditionally used in plants, since its birth and how the new molecular tools facilitate the work of plant breeders is carried out. The evolution of the most studied cultures from the point of view of molecular markers is also reviewed and other parameters whose prior knowledge can facilitate the approach of researchers to this field of research are analyzed. The bibliometric analysis of molecular markers in plants shows that top five countries in this research are: US, China, India, France, and Germany, and from 2013, this research is led by China. On the other hand, the basic research using Arabidopsis is deeper in France and Germany, while other countries focused its efforts in their main crops as the US for wheat or maize, while China and India for wheat and rice.

Advanced microbial analysis for wastewater quality monitoring: metagenomics trend

Urban Wastewater treatment plants (UWWTPs) have played an important and fundamental role in society for water purification of contaminated human wastewaters over the last decades. Microorganisms are very important in UWWTP as their metabolic activity significantly reduces the organic load of the UWW, although there is an uncertain gap in our knowledge regarding microbial consortium structure and their activity in UWWTP operation on a large scale. On the other hand, effluents of UWWTPs have come to be a new source of fresh water to ease water scarcity in many regions of the world, especially in intensive irrigation practices. Many concerns over health risks relating to the direct reuse of this water are very well known. However, if a proper disinfection treatment is applied, these are strongly reduced as conventional methodologies have demonstrated over the last decades. In line with this, the continuous development of new devices for analytical measurement that increase the sensitivities (limit of detection) are showing that other potential risks for both environmental and human health may be associated with UWW reuse. In this work, the most important aspects related to microorganisms in UWWTPs and UWW effluents are presented. Moreover, the new developments on genetic tools for detection of microorganisms are presented, with special emphasis on metagenomic methodology. A bibliometric analysis of what has been published so far is also carried out.