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In this study, three members of the Bmp family were cloned and characterized in yellow catfish Pelteobagrus fulvidraco, including Bmp2a, Bmp4 and Bmp9. The predicted amino acid sequences of P. fulvidraco Bmp2a, Bmp4 and Bmp9 showed the characteristic domains of the Bmp family, including an N-terminal signal peptide, Arg-X-X-Arg site, TGF-β family signature and seven conserved cysteines, indicating that function is likely to have been conserved during evolution. mRNAs of the three Bmp genes had a variable level of expression in tissues. Compared to the control diet, a high fat diet tended to down-regulate the mRNA expression of Bmp2a, Bmp4 and Bmp9 in mesenteric fat, liver and ovary, while it tended to up-regulate their mRNA levels in muscle and kidney. The responses to dietary lipid status and the potential role in lipid metabolism have not previously been reported and reinforces the idea of their multiple functions. Our findings provide the first data about the potential role of the Bmp family in lipid metabolism in teleost.

The Black Soldier Fly (BSF) Hermetia illucens provides a promising strategy in the waste valorisation process and a sustainable alternative source of valuable nutrients, including lipids for food and feed. In the present study, the differences in growth performances and nutritional values of BSF V instar larvae and prepupae reared on vegetable waste were analyzed and compared focusing on fat content. V instar larvae showed higher capacity to bioconvert the substrate into biomass than prepupae. The nutritional composition and the fatty acid profiles were dependent on the developmental stage. The expression levels of acetyl-CoA carboxylase (acc), fatty acid synthase (fas), lipase (lip) and acyl-CoA dehydrogenase (acd) genes involved in the lipid metabolism pathway and herein characterized for the first time, were evaluated in order to understand the molecular basis underlying the observed differences in fatty acid profiles. Our results suggest that the different fatty acid profiles of BSF V instar larvae and prepupae may be related to the modulation of the lipid metabolism-related genes expression during larval development. Our study highlights substantial differences between H. illucens V instar larvae and prepupae giving important features regarding the opportunity to modulate the preferable fatty acid profile to meet the industrial requirements.

Simple Summary The increasing demand of nutrients for food and feed imposes the urgent need to implement current nutritional resources while finding valuable alternative sources of fats and proteins. The present study aims to evaluate the efficiency to bioconvert the substrate proteins of vegetable wastes into valuable larval biomass by the insect Black Soldier Fly (BSF), Hermetia illucens. Here, we report that BSF larvae and prepupae show a high protein content characterized by different profiles of valuable amino acids, including taurine, a crucial nutrient for animal feed and future fish aquaculture. Moreover, we provide insights into the genetic basis of taurine biosynthesis in BSF for the first time and we show that the regulation of the genes associated with taurine synthesis influences the taurine content in BSF larvae and prepupae. These findings on peculiar BSF phenotypes encourage the utilization of larvae and/or prepupae to meet different nutritional requirements of fish species as alternative source of relevant amino acids including taurine. Notably, the bioconversion process by BSF represents a sustainable and economically interesting joint solution to meet the protein demand for animal and aquafeed in the next decades as well as a sustainable biotechnological tool for vegetable waste valorization. Abstract Insects have been recognized as sustainable alternative sources of nutrients for food and feed. The Black Soldier Fly (BSF), Hermetia illucens, is a particularly promising species for its great potential in the waste valorization to produce, during the bioconversion process, high-value fat and proteins that currently represent a valuable source for fish feed. The present study aims to evaluate the efficiency to use substrate proteins in two different BSF developmental stages as sustainable biotechnological tools for vegetable waste management. We provide insights into the nutritional values of both V instar larvae and prepupae in terms of valuable amino acids with special focus on taurine, a crucial nutrient for fish. Moreover, we cloned four key genes from BSF involved in the taurine biosynthesis pathway, 2-aminoethanethiol dioxygenase (Hiado), cysteine dioxygenase (Hicdo), cysteine sulfonate decarboxylase (Hicsad), and glutamate decarboxylase (Higad). The gene expression analysis in larvae and prepupae by qPCR showed development-specific profiles suggesting they influence the taurine content during BSF development. These findings showed peculiar phenotypes in larvae and prepupae that can be selected for different biotechnological applications as sustainable source of relevant amino acids and taurine to support the increasing demand for animal feed and aquafeed in the next decades.

Insects have been recognized as sustainable alternative sources of nutrients for food and feed. The Black Soldier Fly (BSF), Hermetia illucens, is a particularly promising species for its great potential in the waste valorization to produce, during the bioconversion process, high-value fat and proteins that currently represent a valuable source for fish feed. The present study aims to evaluate the efficiency to use substrate proteins in two different BSF developmental stages as sustainable biotechnological tools for vegetable waste management. We provide insights into the nutritional values of both V instar larvae and prepupae in terms of valuable amino acids with special focus on taurine, a crucial nutrient for fish. Moreover, we cloned four key genes from BSF involved in the taurine biosynthesis pathway, 2-aminoethanethiol dioxygenase (Hiado), cysteine dioxygenase (Hicdo), cysteine sulfonate decarboxylase (Hicsad), and glutamate decarboxylase (Higad). The gene expression analysis in larvae and prepupae by qPCR showed development-specific profiles suggesting they influence the taurine content during BSF development. These findings showed peculiar phenotypes in larvae and prepupae that can be selected for different biotechnological applications as sustainable source of relevant amino acids and taurine to support the increasing demand for animal feed and aquafeed in the next decades.

Scutellaria lateriflora is well known for its medical applications because of the presence of flavanoids and alkaloids. The present study aimed to explore the molecular aspects and regulations of flavanoids. Five partial cDNAs encoding genes that are involved in the flavonoid biosynthetic pathway: phenylalanine ammonia lyase (SlPAL), cinnamate 4-hydroxylase (SlC4H), 4-coumaroyl CoA ligase (Sl4CL), chalcone synthase (SlCHS), and chalcone isomerase (SlCHI) were isolated from S. lateriflora. Organ expression analysis showed that these genes were expressed in all organs analyzed with the highest levels correlating with the richest accumulation of wogonin in the roots. Baicalin and baicalein differentially accumulated in S. lateriflora plants, with the highest concentration of baicalin and baicalein detected in the leaves and stems, respectively. Exogenous methyl jasmonate (MeJA) significantly enhanced the expression of SlCHS and SlCHI, and accumulation of baicalin (22.54 mg/g), baicalein (1.24 mg/g), and wogonin (5.39 mg/g) in S. lateriflora hairy roots. In addition, maximum production of baicalin, baicalein, and wogonin in hairy roots treated with MeJA was approximately 7.44-, 2.38-, and 2.12-fold, respectively. Light condition increased the expression level of SlCHS, the first committed step in flavonoid biosynthesis in hairy roots of S. lateriflora after 3 and 4 weeks of development compared to the dark condition. Dark-grown hairy roots contained a higher content of baicalin and baicalein than light-grown hairy roots, while light-grown hairy roots accumulated more wogonin than dark-grown hairy roots. These results may helpful for the metabolic engineering of flavonoids biosynthesis in S. lateriflora. Keywords: Flavonoids, Gene characterization, Light/dark regulation, Methyl jasmonate, Scutellaria lateriflora

Background Genetically modified cotton accounts for 64% of the world’s cotton growing area (22.3 million hectares). The genome sequencing of the diploid cotton progenitors Gossypium raimondii and Gossypium arboreum as well as the cultivated Gossypium hirsutum has provided a wealth of genetic information that could be exploited for crop improvement. Unfortunately, gene functional characterization in cotton is lagging behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods. We present here a simple, fast and efficient method for the transient transformation of G. hirsutum that can be used for gene characterization studies. Results We developed a transient transformation system for gene characterization in upland cotton. Using β-glucuronidase as a reporter for Agrobacterium-mediated transformation assays, we evaluated multiple transformation parameters such as Agrobacterium strain, bacterial density, length of co-cultivation, chemicals and surfactants, which can affect transformation efficiency. After the initial characterization, the Agrobacterium EHA105 strain was selected and a number of binary constructs used to perform gene characterization studies. 7-days-old cotton seedlings were co-cultivated with Agrobacterium and transient gene expression was observed 5 days after infection of the plants. Transcript levels of two different transgenes under the control of the cauliflower mosaic virus (CaMV) 35S promoter were quantified by real-time reverse transcription PCR (qRT-PCR) showing a 3–10 times increase over the levels observed in non-infected controls. The expression patterns driven by the promoters of two G. hirsutum genes as well as the subcellular localization of their corresponding proteins were studied using the new transient expression system and our observations were consistent with previously published results using Arabidopsis as a heterologous system. Conclusions The Agrobacterium-mediated transient transformation method is a fast and easy transient expression system enabling high transient expression and transformation efficiency in upland cotton seedlings. Our method can be used for gene functional studies such as promoter characterization and protein subcellular localization in cotton, obviating the need to perform such studies in a heterologous system such as Arabidopsis. Electronic supplementary material The online version of this article (10.1186/s13007-018-0319-2) contains supplementary material, which is available to authorized users.

The hypothesis of the present study is that apoptosis through an intrinsic mitochondrial pathway may mediate high fat diet (HFD)-induced changes in the metabolism of Pelteobagrus fulvidraco. To this end, we cloned the full-length cDNA sequences of Cycs, Apaf1, Casp9, Casp3a, and Casp3b involved in the mitochondria apoptotic pathway, and explored their mRNA tissue expressions and transcriptional responses to HFD. All of these members shared similar domains to their orthologous vertebrate genes. They were constitutively expressed in all analyzed tissues but varied from tissue to tissue. Compared to the control, HFD up-regulated the mRNA expression of partial genes among these five key genes (Cycs, Apaf1, Casp9, Casp3a, and Casp3b) in mesenteric fat, intestine, ovary and the kidney, indicating the induction of apoptosis in these tissues; in contrast, HFD down-regulated mRNA levels of partial genes among the five key genes (Cycs, Apaf1, Casp9, Casp3a, and Casp3b) in the heart, spleen and gill tissues, indicating the inhibition of apoptosis in these tissues. The present study will facilitate further exploration into the functions of these genes at the molecular level and disclose the critical involvement of these genes against nutrient changes, indicating that processes of apoptosis in various tissues may differentially be modified by HFD.

Meiotic recombination promotes genetic variation by reciprocal exchange of genetic material producing novel allelic combinations that influence important agronomic traits in crop plants. Therefore, harnessing meiotic recombination has the potential to accelerate crop improvement via classical breeding. Numerous genes involved in crossover formation have been identified in model systems. For example, SPO11 mediates generation of meiotic DNA double-strand breaks (DSBs) across all eukaryotes, which may be repaired as crossovers. However, downstream regulators of recombination remain to be identified, including those with species-specific roles. To isolate crossover frequency modifiers I performed a high-throughput forward genetic screen using EMS mutagenesis of Arabidopsis carrying a fluorescent crossover reporter line called 420. The primary screen isolated nine mutants from ~3,000 scored individuals that showed significantly higher (high crossover rate, hcr) or lower (low crossover rate, lcr) crossover frequency, including a new fancm allele. Four mutants (hcr1, hcr2, hcr3 and lcr1) were mapped by sequencing and candidate genes identified. The hcr1 mutation was confirmed as being located within the PROTEIN PHOSPHATASE X-1 (PPX-1) gene, using isolation of an independent allele and complementation studies. Similarly, the lcr1 mutation was confirmed to be within the gene TBP-ASSOCIATED FACTOR 4B (TAF4B). Using immunocytological staining I observed that hcr1 did not show changes in DSB-associated foci (RAD51), but it did show a significant increase in crossover-associated MLH1 foci. The hcr1 mutation increases crossovers mainly in the sub-telomeric chromosome regions, which remain sensitive to crossover interference. Also the genetic interaction between the hcr1 and fancm mutations is additive. These results support a model where PPX- 1 acts to limit recombination via the Class I interfering CO pathway, downstream of DSB formation. In summary, this genetic screen has led to discovery of novel genes that regulate meiotic recombination and their functional characterization may find utility in crop breeding programs., Marie-Curie “COMREC” network FP7 ITN-606956

Full-length cDNAs encoding ξ-carotene desaturase (CmZDS), lycopene ε-cyclase (CmLCYE), β-ring carotene hydroxylase (CmCHXB), and zeaxanthin epoxidase (CmZEP), and partial-length cDNA encoding ε-ring carotene hydroxylase (CmCHXE) were isolated in Chamoe (Cucumis melo L. var. makuwa), an important commercial fruit. Sequence analyses revealed that these proteins share high identity and common features with other orthologous genes. Expression levels of entire genes involved in the carotenoid biosynthetic pathway were investigated in the peel, pulp, and stalk of chamoe cultivars Ohbokggul and Gotgam. Most of the carotenoid biosynthetic genes were expressed at their highest levels in the stalk, whereas carotenoids were highly distributed in the peel. The expression levels of all carotenoid biosynthetic genes in fruits of the native cultivar Gotgam chamoe were higher than those in the cultivar Ohbokggul chamoe, consistent with the abundant carotenoid accumulation in Gotgam chamoe fruits and trace carotenoid content of Ohbokggul chamoe fruit. Lutein and β-carotene were the dominant carotenoids; high levels (278.05 μg g−1 and 112.02 μg g−1 dry weight, respectively) were found in the peel of Gotgam chamoe. Our findings may provide a foundation for elucidating the carotenoid biosynthetic mechanism in C. melo and inform strategies for developing new chamoe cultivars with improved characteristics.

The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a foodborne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on triclabendazole (TCBZ), and overuse has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty-acid-binding protein (FABP) superfamily has proposed multifunctional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterized FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome, and EST data mining with proteomics and phylogenetics to reveal a liver fluke FABP superfamily of seven clades: previously identified clades I-III and newly identified clades IV-VII. All new clade FABPs were analyzed using bioinformatics and cloned from both liver flukes. The extended FABP data set will provide new study tools to research the role of FABPs in parasite biology and as therapy targets.

Valsa mali, a parasitic fungus, is a destructive pathogen of apple tree that causes heavy economic losses in China. The pathogen secretes various cell wall-degrading enzymes (CWDEs) that degrade plant cell-wall components, and thus facilitate its entry into host cells. Therefore, functional analysis of the genes encoding CWDEs is necessary to understand virulence of V. mali toward apple tree. Here, we identified and cloned an endo-β-1,4-xylanase gene, VmXyl1 in V. mali. The full-length cDNA of VmXyl1 is 1626 bp containing 5′- and 3′-non-coding regions, as well an open reading frame of 1320 bp that encodes a protein with a calculated molecular mass and an isoelectric point of 43.8 kDa and 4.4, respectively. The predicted amino acid sequences showed significant homology to a family GH10 of glycosyl hydrolases. The apple branch extract and beechwood xylan, but not glucose, induced the expression of VmXyl1. Furthermore, VmXyl1 had high expression levels in the apple tree bark during the pathogen infection. The deletion of VmXyl1 did not affect mycelia growth; however, it significantly reduced pycnidia formation in V. mali. The deletion strains showed a reduced virulence toward apple leaves and twigs. Moreover, the mutant strains had reduced endo-β-1,4-xylanase activity and growth when cultured using beechwood xylan as the only carbon source. Reintroducing wild-type VmXyl1 into the mutant strains rescued the defect phenotype. We conclude that VmXyl1 determines the virulence of V. mali toward apple tree. These results provide valuable insight into the plant–pathogen molecular interactions.

Valsa mali, a parasitic fungus, is a destructive pathogen of apple tree that causes heavy economic losses in China. The pathogen secretes various cell wall-degrading enzymes (CWDEs) that degrade plant cell-wall components, and thus facilitate its entry into host cells. Therefore, functional analysis of the genes encoding CWDEs is necessary to understand virulence of V. mali toward apple tree. Here, we identified and cloned an endo-β-1,4-xylanase gene, VmXyl1 in V. mali. The full-length cDNA of VmXyl1 is 1626 bp containing 5′- and 3′-non-coding regions, as well an open reading frame of 1320 bp that encodes a protein with a calculated molecular mass and an isoelectric point of 43.8 kDa and 4.4, respectively. The predicted amino acid sequences showed significant homology to a family GH10 of glycosyl hydrolases. The apple branch extract and beechwood xylan, but not glucose, induced the expression of VmXyl1. Furthermore, VmXyl1 had high expression levels in the apple tree bark during the pathogen infection. The deletion of VmXyl1 did not affect mycelia growth; however, it significantly reduced pycnidia formation in V. mali. The deletion strains showed a reduced virulence toward apple leaves and twigs. Moreover, the mutant strains had reduced endo-β-1,4-xylanase activity and growth when cultured using beechwood xylan as the only carbon source. Reintroducing wild-type VmXyl1 into the mutant strains rescued the defect phenotype. We conclude that VmXyl1 determines the virulence of V. mali toward apple tree. These results provide valuable insight into the plant–pathogen molecular interactions.

Genes in the ZIP family encode transcripts to store and transport bivalent metal micronutrient, particularly iron (Fe) and or zinc (Zn). These transcripts are important for a variety of functions involved in the developmental and physiological processes in many plant species, including most, if not all, Poaceae plant species and the model species Arabidopsis. Here, we present the report of a genome wide investigation of orthologous ZIP genes in Setaria italica and the identification of 7 single copy genes. RT-PCR shows 4 of them could be used to increase the bio-availability of zinc and iron content in grains. Of 36 ZIP members, 25 genes have traces of signal peptide based sub-cellular localization, as compared to those of plant species studied previously, yet translocation of ions remains unclear. In silico analysis of gene structure and protein nature suggests that these two were preeminent in shaping the functional diversity of the ZIP gene family in S. italica. NAC, bZIP and bHLH are the predominant Fe and Zn responsive transcription factors present in SiZIP genes. Together, our results provide new insights into the signal peptide based/independent iron and zinc translocation in the plant system and allowed identification of ZIP genes that may be involved in the zinc and iron absorption from the soil, and thus transporting it to the cereal grain underlying high micronutrient accumulation.

Salinity is a severe abiotic stress limiting agricultural yield and productivity. Plants have evolved various strategies to cope with salt stress. Chloroplasts are important photosynthesis organelles, which are sensitive to salinity. An understanding of molecular mechanisms in chloroplast tolerance to salinity is of great importance for genetic modification and plant breeding. Previous studies have characterized more than 53 salt-responsive genes encoding important chloroplast-localized proteins, which imply multiple vital pathways in chloroplasts in response to salt stress, such as thylakoid membrane organization, the modulation of photosystem II (PS II) activity, carbon dioxide (CO2) assimilation, photorespiration, reactive oxygen species (ROS) scavenging, osmotic and ion homeostasis, abscisic acid (ABA) biosynthesis and signaling, and gene expression regulation, as well as protein synthesis and turnover. This review presents an overview of salt response in chloroplasts revealed by gene characterization efforts.

Scutellaria baicalensis has a wide range of biological activities and has been considered as an important traditional drug in Asia and North America for centuries. A partial-length cDNA clone encoding phytoene synthase (SbPSY) and full-length cDNA clones encoding phytoene desaturase (SbPDS), ξ-carotene desaturase (SbZDS), β-ring carotene hydroxylase (SbCHXB), and zeaxanthin epoxidase (SbZEP) were identified in S. baicalensis. Sequence analyses revealed that these proteins share high identity and conserved domains with their orthologous genes. SbPSY, SbPDS, SbZDS, SbCHXB, and SbZEP were constitutively expressed in the roots, stems, leaves, and flowers of S. baicalensis. SbPSY, SbPDS, and SbZDS were highly expressed in the stems, leaves, and flowers and showed low expression in the roots, where only trace amounts of carotenoids were detected. SbCHXB and SbZEP transcripts were expressed at relatively high levels in the roots, stems, and flowers and were expressed at low levels in the leaves, where carotenoids were mostly distributed. The predominant carotenoids in S. baicalensis were lutein and β-carotene, with abundant amounts found in the leaves (517.19 and 228.37 μg g-1 dry weight, respectively). Our study on the biosynthesis of carotenoids in S. baicalensis will provide basic data for elucidating the contribution of carotenoids to the considerable medicinal properties of S. baicalensis., EXCLI Journal ; Vol. 14, 2015

Cynomolgus macaques (Macaca fascicularis) are an established animal model of asthma, which exhibit different responses to allergen exposure that are clinically relevant. The chemokine ligand gene (CCL7) encodes Monocyte Chemotactic Protein-3, which has an important role in asthma pathogenesis. While CCL7 polymorphism in humans is associated with asthma phenotype, very little is known about CCL7 in nonhuman primate models of respiratory disease. The objective of this study was to isolate and characterize CCL7 gene in cynomolgus macaques of Indonesian origin. In this study, we used sequencing and bioinformatics technique for gene isolation, characterization, and protein 3D structure prediction. We isolated a 2253 base-pair (bp) sequence of CCL7 in cynomolgus macaques, which exhibited 95% similarity in coding sequence to human CCL7. The amino acid sequence was more closely clustered with human CCL7 than with that of rodents. Importantly, the predictive protein structure of CCL7 was similar to that in humans. These similarities in CCL7 suggests the potential of cynomolgus macaque as a translational model to study asthma, particularly in the context of genetics and role of chemokines such as CCL7.

Full-length cDNAs encoding &xi, carotene desaturase (CmZDS), lycopene &epsilon, cyclase (CmLCYE), &beta, ring carotene hydroxylase (CmCHXB), and zeaxanthin epoxidase (CmZEP), and partial-length cDNA encoding &epsilon, ring carotene hydroxylase (CmCHXE) were isolated in Chamoe (Cucumis melo L. var. makuwa), an important commercial fruit. Sequence analyses revealed that these proteins share high identity and common features with other orthologous genes. Expression levels of entire genes involved in the carotenoid biosynthetic pathway were investigated in the peel, pulp, and stalk of chamoe cultivars Ohbokggul and Gotgam. Most of the carotenoid biosynthetic genes were expressed at their highest levels in the stalk, whereas carotenoids were highly distributed in the peel. The expression levels of all carotenoid biosynthetic genes in fruits of the native cultivar Gotgam chamoe were higher than those in the cultivar Ohbokggul chamoe, consistent with the abundant carotenoid accumulation in Gotgam chamoe fruits and trace carotenoid content of Ohbokggul chamoe fruit. Lutein and &beta, carotene were the dominant carotenoids, high levels (278.05 &mu, g g&minus, 1 and 112.02 &mu, 1 dry weight, respectively) were found in the peel of Gotgam chamoe. Our findings may provide a foundation for elucidating the carotenoid biosynthetic mechanism in C. melo and inform strategies for developing new chamoe cultivars with improved characteristics.

Nicotine is the major alkaloid compound in cultivated tobacco (Nicotiana tabacum) that could potentially be converted into carcinogenic compound (nor-nicotine). The PMT gene encoding putrescine N-methyltransferase (PMT) is one of the two key genes that play a prominent role in nicotine biosynthesis. The aimed of this study was to isolate and characterize the cDNA sequence originated from Indonesian local tobacco cv. Sindoro1 (Ntpmt_Sindoro1). The results showed that the Ntpmt_Sindoro1 was 1124 bp in length. This cDNA fragment encodes for 374 amino acid residues. The predicted polypeptide from the cDNA is a hidrophilic protein, and has a predicted molecular weight of 40.95 kDa. The predicted amino acids sequence also showed high similarity to the PMT gene product Nicotiana sp. available in the GenBank data base. The amino acid sequences also exert conserved residues specifically exhibited only by PMT gene originated from N. tabacum. Clustering analysis revealed that Ntpmt_Sindoro1 belongs to the same clade as the PMT3 gene, a member of the N. tabacum PMT gene family. The Ntpmt_Sindoro1 cDNA sequence covering exon1-exon8 of the PMT gene fragment has been registered in the GenBank data base, under the accession number JX978277.

Genes in the ZIP family encode transcripts to store and transport bivalent metal micronutrient, particularly iron (Fe) and or zinc (Zn). These transcripts are important for a variety of functions involved in the developmental and physiological processes in many plant species, including most, if not all, Poaceae plant species and the model species Arabidopsis. Here, we present the report of a genome wide investigation of orthologous ZIP genes in Setaria italica and the identification of 7 single copy genes. RT-PCR shows 4 of them could be used to increase the bio-availability of zinc and iron content in grains. Of 36 ZIP members, 25 genes have traces of signal peptide based sub-cellular localization, as compared to those of plant species studied previously, yet translocation of ions remains unclear. In silico analysis of gene structure and protein nature suggests that these two were preeminent in shaping the functional diversity of the ZIP gene family in S. italica. NAC, bZIP and bHLH are the predominant Fe and Zn responsive transcription factors present in SiZIP genes. Together, our results provide new insights into the signal peptide based/independent iron and zinc translocation in the plant system and allowed identification of ZIP genes that may be involved in the zinc and iron absorption from the soil, and thus transporting it to the cereal grain underlying high micronutrient accumulation.

Dystrophinopathies are X-linked recessive diseases caused by mutations in the DMD gene. Our objective was to identify mutations in this gene by Multiplex Ligation Probe Amplification (MLPA), to confirm the clinical diagnosis and determine the carrier status of at-risk relatives. Also, we aimed to characterize the Dystrophinopathies argentine population and the DMD gene. We analyzed a cohort of 121 individuals (70 affected boys, 11 symptomatic women, 37 at-risk women and 3 male villus samples). The MLPA technique identified 56 mutations (45 deletions, 9 duplications and 2 point mutations). These results allowed confirming the clinical diagnosis in 63% (51/81) of patients and symptomatic females. We established the carrier status of 54% (20/37) of females at-risk and 3 male villus samples. We could establish an association between the most frequent deletion intron breakpoints and the abundance of dinucleotide microsatellites loci, despite the underlying mutational molecular mechanism remains to be elucidated. The MLPA demonstrate, again, to be the appropriate first mutation screening methodology for molecular diagnosis of Dystrophinopathies. The reported results permitted to characterize the Dystrophinopathies argentine population and lead to better understanding of the genetic and molecular basis of rearrangements in the DMD gene, useful information for the gene therapies being developed. Fil: Luce, Leonela Natalia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Dalamon, Viviana Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Ferrer, Marcela Maria. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Parma, Diana Lidia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Szijan, Irena. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Giliberto, Florencia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

This study reports the characterization of a novel human gene, chromosome 3 open reading frame 6 (C3orf6), mapped to chromosome 3q28, within the critical region of hereditary spastic paraplegia SPG14 locus. Based on computational "spliced" EST alignment and RT-PCR, two C3orf6 transcript variants were identified. The longer C3orf6 transcript contains a 1449-nt ORF, encoding a protein of 482 aa, while the shorter variant contains a 921-nt ORF, encoding for a protein of 306 aa. C3orf6 gene is organised on 12 exons and the shorter transcript comes from an alternative splicing event skipping exon 6. The two mRNA are differentially expressed in brain and in several other human tissues with a predominant level for the shorter transcript. By database analysis, EST assembling and RT-PCR, we identified the transcripts of mouse and rat C3orf6 orthologous genes. The involvement of C3orf6 in the spastic paraplegia was investigated by sequencing all coding exons and flanking sequences in the SPG14 family, excluding the presence of causative mutations.

Lycium chinense is a shrub that has health benefits and is used as a source of medicines in Asia. In this study, a full-length cDNA clone encoding β-ring carotene hydroxylase (LcCHXB) and partial-length cDNA clones encoding phytoene synthase (LcPSY), phytoene desaturase (LcPDS), ξ-carotene desaturase (LcZDS), lycopene β-cyclase (LcLCYB), lycopene ε-cyclase (LcLCYE), ε-ring carotene hydroxylase (LcCHXE), zeaxanthin epoxidase (LcZEP), carotenoid cleavage dioxygenase (LcCCD1), and 9-cis epoxycarotenoid dioxygenase (LcNCED) were identified in L. chinense. The transcripts were constitutively expressed at high levels in leaves, flowers and red fruits, where the carotenoids are mostly distributed. In contrast, most of the carotenoid biosynthetic genes were weakly expressed in the roots and stems, which contained only small amounts of carotenoids. The level of LcLCYE transcripts was very high in leaves and correlated with the abundance of lutein in this plant tissue. During maturation, the levels of lutein and zeaxanthin in L. chinense fruits dramatically increased, concomitant with a rise in the level of β-cryptoxanthin. LcPSY, LcPDS, LcZDS, LcLCYB, and LcCHXE were highly expressed in red fruits, leading to their substantially higher total carotenoid content compared to that in green fruits. Total carotenoid content was high in both the leaves and red fruits of L. chinense. Our findings on the biosynthesis of carotenoids in L. chinense provide insights into the molecular mechanisms involved in carotenoid biosynthesis and may facilitate the optimization of carotenoid production in L. chinense.

Карнозинска киселина, фенолни дитерпен, поседује бројне биолошке активности због чега је потенцијално веома значајна за фармацеутску индустрију. Грчка жалфија (Salvia fruticosa Mill.) и рузмарин (Rosmarinus officinalis L.) богати су природни извори овог једињења. Иако је много тога познато у вези структуре и биолошких активности карнозинске киселине, веома мало се не зна о њеном биосинтетском путу у биљкама. Основни циљ дисертације био је изучавање биосинтезе карнозинске киселине на молекуларном нивоу, изолацијом и функционалном карактеризацијом гена укључених у овај процес. У ту сврху, извршена је анализа постојеће цДНК библиотеке жлезданих длака грчке жалфије, што је за резултат имало идентификацију два гена, названих SfCPS и SfKSL, који потенцијално кодирају дитерпен синтазе. Гени кандидати су изоловани и функционално окарактерисани у бактерији Escherichia coli Mig., квасцима (Saccharomyces cerevisiae Meyen ex E. C. Hansen) и дувану (Nicotiana benthamiana Domin). Хетеролога екпресија гена SfCPS и SfKSL резултирала је синтезом милтирадиена, што је утврђено GC-MS анализом и 1D и 2D NMR спектроскопијом (1H, 13C, DEPT, COSY H-H, HMQC и HMBC). За проналажење преосталих гена одговорних за биосинтезу карнозинске киселине, обављено је секвенционирање транскриптома грчке жалфије и рузмарина, уз коришћење 454 GS FLX Titanium платформе. На основу резултата фитохемијске анализе LC-PDA-LTQ-Orbitrap FTMS методом, жлездане длаке младих листова благо стресираних биљака генотипа “Кавуси” грчке жалфије и жлездане длаке млађих стадијума развоја комерцијалног генотипа рузмарина (B & T World Seeds) изабране су као биљни материјал за анализу транскриптома... Carnosic acid is a phenolic diterpene potentially highly significant for the pharmaceutical industry, due to its numerous biological activities. Cretan sage (Salvia fruticosa Mill.) and Rosemary (Rosmarinus officinalis L.) are rich natural sources of this compound. Although the structure and biological activities of carnosic acid are widely known, its biosynthetic pathway in plants remains unexplored. The main aim of the dissertation was to investigate the carnosic acid biosynthesis at the molecular level, by isolating and functionally characterizing the genes involved in this process. For that purpose, the existing Cretan sage trichome cDNA library has been analyzed, which has resulted in the identification of two putative diterpene synthase genes, named SfCPS and SfKSL. The candidate genes were isolated and functionally characterized in Escherichia coli Mig., yeast (Saccharomyces cerevisiae Meyen ex E. C. Hansen) and Nicotiana benthamiana Domin. Heterologous expression of SfCPS and SfKSL genes has resulted in the synthesis of miltiradiene, which has been confirmed by GC-MS analysis and 1D and 2D NMR spectroscopy (1H, 13C, DEPT, COSY H-H, HMQC и HMBC). In order to retrieve the remaining genes responsible for the biosynthesis of carnosic acid, the glandular trichomes of Cretan sage and Rosemary have been sequenced using 454 GS FLX Titanium platform. Based on the results of the LC-PDA-LTQ-Orbitrap FTMS phytochemical analysis, glandular trichomes of Cretan sage young leaves (genotype Kavoussi) exposed to mild stress, and glandular trichomes on the Rosemary leaves of the younger developmental stage (commercial genotype B&T World Seeds), were chosen for the transcriptome analysis...

European eel (Anguilla anguilla, L., 1758) is suffering a strong population decrease and at the same time it is a very appreciated species and by now it has not been possible closing its cycle life. In fact, this species does not mature in captivity unless hormonally induced. So all the production is up to the natural population. All these factors together make urgent achieving the closing of the productive cycle and for this aim it is important to understand the reproductive physiology and the reasons of this development blockage. The present thesis wants to be a new contribution to the knowledge of reproductive physiology in female European eel submitted at hormonal treatment. To achieve this goal, expression of genes not previously studied in this species (cyp19a1, ara, arb, gnrhr1a, gnrhr1b, gnrhr2, zpb and zpc) was analyzed in eels reared under a constant thermal regime, accordingly to the usual rearing conditions. Also, the effect of rearing temperature on gene expression and steroid profile (T, 11-KT and E2) was studied. In fact, eels migrate to Sargasso Sea to reproduce and during the travel experiment temperature changes, while traditionally they are reared at a constant high temperature which could affect vitellogenesis progression and final oocyte quality. For the study it was necessary cloning and characterizing some genes which have not still been sequenced in European eel. Gene expression was studied by qPCR after designing primer and optimizing the qPCR race. Steroid profiles were analyzed by immunoassays and the gonadal development stages were established by histology. The first result obtained at the end of the study were six new genes characterized in European eel. The analysis of gene expression allowed to understand the involvement of specific genes during vitellogenesis (arb, gnrhr1b and gnrhr2) in different brain regions. The temperature was conformed as a crucial environmental factor affecting vitellogenesis. On one hand, eels matured at lower starting temperatures showed better reproductive parameters which could have an influence in the final oocyte quality. On the other hand higher temperatures are necessary to achieve further vitellogenetic stages, Mazzeo, I. (2014). Effect of thermal regime on the expression of key reproductive genes during hormonally-induced vitellogenesis in female European eels [Tesis doctoral no publicada]. Universitat Politècnica de València. doi:10.4995/Thesis/10251/48490., TESIS

Karnozinska kiselina, fenolni diterpen, poseduje brojne biološke aktivnosti zbog čega je potencijalno veoma značajna za farmaceutsku industriju. Grčka žalfija (Salvia fruticosa Mill.) i ruzmarin (Rosmarinus officinalis L.) bogati su prirodni izvori ovog jedinjenja. Iako je mnogo toga poznato u vezi strukture i bioloških aktivnosti karnozinske kiseline, veoma malo se ne zna o njenom biosintetskom putu u biljkama. Osnovni cilj disertacije bio je izučavanje biosinteze karnozinske kiseline na molekularnom nivou, izolacijom i funkcionalnom karakterizacijom gena uključenih u ovaj proces. U tu svrhu, izvršena je analiza postojeće cDNK biblioteke žlezdanih dlaka grčke žalfije, što je za rezultat imalo identifikaciju dva gena, nazvanih SfCPS i SfKSL, koji potencijalno kodiraju diterpen sintaze. Geni kandidati su izolovani i funkcionalno okarakterisani u bakteriji Escherichia coli Mig., kvascima (Saccharomyces cerevisiae Meyen ex E. C. Hansen) i duvanu (Nicotiana benthamiana Domin). Heterologa ekpresija gena SfCPS i SfKSL rezultirala je sintezom miltiradiena, što je utvrđeno GC-MS analizom i 1D i 2D NMR spektroskopijom (1H, 13C, DEPT, COSY H-H, HMQC i HMBC). Za pronalaženje preostalih gena odgovornih za biosintezu karnozinske kiseline, obavljeno je sekvencioniranje transkriptoma grčke žalfije i ruzmarina, uz korišćenje 454 GS FLX Titanium platforme. Na osnovu rezultata fitohemijske analize LC-PDA-LTQ-Orbitrap FTMS metodom, žlezdane dlake mladih listova blago stresiranih biljaka genotipa “Kavusi” grčke žalfije i žlezdane dlake mlađih stadijuma razvoja komercijalnog genotipa ruzmarina (B & T World Seeds) izabrane su kao biljni materijal za analizu transkriptoma. Rezultat sekvencioniranja bio je anotacija velikog broja novih sekvenci, a posebno značajna bila je identifikacija gena metaboličkih puteva povezanih sa sekundarnim metabolizmom. Izolovano je ukupno 24 gena koji su kodirali za enzime familije citohrom P450 (CYP) monooksigenaze. Funkcija enzima CYP76Sf2 grčke žalfije, i CYP76Ro3 i CYP76Ro4 ruzmarina, kao feruginol sintaza, utvrđena je esejima funkcionalne karakterizacije u kvascima i duvanu. Kloniranje i funkcionalna karakterizacija gena koji svojom zajedničkom aktivnošću dovode do sinteze feruginola kod grčke žalfije i ruzmarina, pruža osnovu budućim istraživanjima biosintetskog puta karnozinske kiseline. Uz to, izvršena je lokalizacija biosintetskog puta karnozinske kiseline u žlezdanim dlakama grčke žalfije i ruzmarina. Priloženi rezultati doprinose boljem razumevanju metabolizma karnozinske kiseline, i time omogućavaju primenu najsavremenijih biotehnoloških metoda za proizvodnju ovog farmaceutski značajnog jedinjenja. Carnosic acid is a phenolic diterpene potentially highly significant for the pharmaceutical industry, due to its numerous biological activities. Cretan sage (Salvia fruticosa Mill.) and Rosemary (Rosmarinus officinalis L.) are rich natural sources of this compound. Although the structure and biological activities of carnosic acid are widely known, its biosynthetic pathway in plants remains unexplored. The main aim of the dissertation was to investigate the carnosic acid biosynthesis at the molecular level, by isolating and functionally characterizing the genes involved in this process. For that purpose, the existing Cretan sage trichome cDNA library has been analyzed, which has resulted in the identification of two putative diterpene synthase genes, named SfCPS and SfKSL. The candidate genes were isolated and functionally characterized in Escherichia coli Mig., yeast (Saccharomyces cerevisiae Meyen ex E. C. Hansen) and Nicotiana benthamiana Domin. Heterologous expression of SfCPS and SfKSL genes has resulted in the synthesis of miltiradiene, which has been confirmed by GC-MS analysis and 1D and 2D NMR spectroscopy (1H, 13C, DEPT, COSY H-H, HMQC и HMBC). In order to retrieve the remaining genes responsible for the biosynthesis of carnosic acid, the glandular trichomes of Cretan sage and Rosemary have been sequenced using 454 GS FLX Titanium platform. Based on the results of the LC-PDA-LTQ-Orbitrap FTMS phytochemical analysis, glandular trichomes of Cretan sage young leaves (genotype Kavoussi) exposed to mild stress, and glandular trichomes on the Rosemary leaves of the younger developmental stage (commercial genotype B&T World Seeds), were chosen for the transcriptome analysis...

9 páginas, 6 figuras, 1 tabla, In this study, we present the characterization of a newly identified gene, MgC1q, revealed in suppression subtractive hybridization and cDNA libraries from immunostimulated mussels. Based on sequence homology, molecular architecture and domain similarity, this new C1q-domain-containing gene may be classified as a member of the C1q family and, therefore, part of the C1q–TNF superfamily. The expression of MgC1q was detected all along the mussel ontogeny, being detectable within 2 h post-fertilization, with a notable increase after 1 month and continuing to increase until 3 months. Measurable transcript levels were also evident in all analyzed tissues of naïve adult mussels, and the hemocytes showed the highest expression levels. Experimental infection of adult mussels with Gram positive or Gram negative bacteria significantly modulated the MgC1q expression, and confirmed it as an immune-related gene. Intra- and inter-individual sequence analyses revealed extraordinary diversity of MgC1q at both the DNA and cDNA levels. While further research is needed to define its function, our data indicate that MgC1q is a pattern recognition molecule able to recognize pathogens during innate immune responses in Myitilus galloprovincialis. The high sequence variability suggests that somatic diversification of these nonself recognition molecules could have occurred., This work has been funded by the EU Integrated Project FOOD-CT-2005-007103 and AGL2008-05111/ACU from the Spanish Ministerio de Ciencia e Innovación. Camino Gestal wishes to acknowledge additional funding from the Spanish Ministerio de Educación y Ciencia through the “Ramón y Cajal” Contract.

NR 20140805

Genes coding for proteins involved in generegulation and/or development are of great inter-est in the study of the biology of Schistosomamansoni. This trematode is the etiologic agent ofschistosomiasis and presents a complex life cyclewith drastic morphologic changes between stages.Recently, some strains have become resistant tothe drugs currently in use to eradicate the disease(D Cioli et al. 1995 Pharmac Ther 68: 35-85). Thestrategy of gene discovery program in S. mansoniby using the EST (expressed sequence tag) ap-proach (GR Franco et al. 1995 Gene 152 : 141-147)has been very efficient in the discovery of new S.mansoni genes, which were unlikely to be identi-fied using classical procedures based on pheno-type. A class of genes that interested us particu-larly were those that in other organisms wereknown to be involved in the regulation of embryo-genesis. Among these, we selected one that pre-sented a high homology to a Drosophila genenamed mago nashi. In diptera this gene is involvedin the process of germ plasm assembly and itsmutation results in sterility of F1 progeny and alsoin the formation of the perpendicular axes (REBoswell et al. 1991 Development 113: 373-384).We reasoned that this gene might conceivably playa role in the morphogenetic changes seen in thelife cycle of the parasite . We thus decided to char-acterize S. mansoni mago nashi further by obtain-ing its full length cDNA and genomic sequences,as well as studying its expression pattern at thedifferent life stages of the worm.The whole cDNA was sequenced in both di-rections yielding 485 nucleotides (nt) that codedfor a protein of 146 amino acids with 84% of ho-mology to the Drosophila homologue (Fig. 1). Pro-cedures for plasmidial DNA preparation, sequenc-ing and analysis of sequences have been previouslydescribed (Franco loc. cit.). From the cDNA se-quence, primers were designed and the genomicgene amplified from S. mansoni total DNA. Thegenomic amplified product was bigger than thecorrespondent cDNA amplified product when twodifferent pairs of primers were used (Fig. 2). ThePCR amplification products were cloned in a plas-mid vector (pUC18) by using the Pharmacia Sureclone kit and sequenced in both directions usingfluorescent primers in an automated DNA se-quencer. Consistent with the results of Fig. 2, threeintrons were identified in the genomic sequence,two of 34 nt and one with 33 nt producing a totalof a 101 nt of intron sequences (Fig. 3).Our next step was to investigate the gene ex-pression pattern at the different life cycle stages.Through the RT-PCR technique using cDNA ob-tained from different stages and specific primers,it was shown that mago nashi is expressed in allstages studied i.e. egg, schistosomula and adultworm (data not shown). This was not unexpectedalthough this gene was first identified in droso-phila embryos, it was also identified in adult flies(PA Newmark & RE Boswell 1994 Development120: 1303-1313). The gene had also been shownto be expressed in a large variety of human adulttissues such as lung, kidney, liver, heart, pancreas,brain and placenta (X- Zhao et al. 1998 Genomics47: 319-322).Since this work was started, the mago nashigene has been identified in a variety of differentorganisms. These include mouse, human,Caenorhabditis elegans, Brugia malayi,Arabidopsis thaliana and Oryza sativa . Alignmentof the conceptual translations of these mago genes

Musa acuminata cultivar pisang ambon lumut is a native climacteric fruit from Indonesia. Climacteric fruit ripening process is triggered by the gaseous plant hormone ethylene. The rate limiting enzyme involved in ethylene biosynthesis is ACC synthase (ACS) which is encoded by ACS gene family. The objective of this study is to identify MA-ACS gene family in M. acuminata cultivar pisang ambon lumut and to study the MA-ACS1 gene expression. The result showed that there were nine M. acuminata ACS gene family members called MA-ACS1–9. Two of them (MA-ACS1 and MA-ACS2) were assessed using reverse transcriptase PCR (RT-PCR) for gene expression study and it was only MA-ACS1 correlated with fruit ripening. The MA-ACS1 gene fragment has been successfully isolated and characterized and it has three introns, four exons, and one stop codon. It also shows highest homology with MACS1 gene from M. acuminata cultivar Hsian Jien Chiao (GenBank accession number AF056164). Expression analysis of MA-ACS1 using quantitative PCR (qPCR) showed that MA-ACS1 gene expression increased significantly in the third day, reached maximum at the fifth day, and then decreased in the seventh day after harvesting. The qPCR expression analysis result correlated with the result of physical analysis during fruit ripening. Key words: pisang ambon lumut, MA-ACS gene family, gene characterization, gene expression, quantitative PCR (qPCR)

Earlier we detected a novel enzymatic activity in salivary gland secretion of the medicinal leech, splitting isopeptide bonds between the glutamine gamma-carboxamide and lysine epsilon-amino group. This activity is due to destabilase. We described its partial amino acid sequence and sequences to two closely related cDNAs, but none of them perfectly matched the protein isolated. Here we report the isolation and sequence peculiarities of the third cDNA of the family as well as the complete sequence of the destabilase protein. The inferred mature protein product of this cDNA matches the independently determined destabilase protein sequence. It contains 115 amino acid residues including 14 highly conserved Cys residues and is formed from a precursor containing specific leader peptide.

Oil palm ((Elaeis guineensis Jacq) is one of the most productive oil crops in the world. It can produce oil up to 90% of its dry weight and has the highest oil content reported among other plant tissues. WRI1 gene is one of many contributing genes that control oil content in oil palm fruit. Despite its obvious scientific and economic interest, literature and molecular resources about WRI1 gene in oil palm are still limited. The objective of this research was to isolate sequence and characterize the WRI1 gene from oil palm genome. WRI1 gene was successfully amplified from oil palm cDNA library. The amplification produced a single DNA fragment of 1 000 bp and which was cloned into pJET cloning vector for sequencing. The sequencing result showed two clones that were identified as WRI1 transcription factor.