RESUMEN

BACKGROUND: Chamomile (Matricaria recutita L.) has a long history of use in herbal medicine with various applications, and the flower heads contain numerous secondary metabolites which are medicinally active. In the major crop plants, next generation sequencing (NGS) approaches are intensely applied to exploit genetic resources, to develop genomic resources and to enhance breeding. Here, genotyping-by-sequencing (GBS) has been used in the non-model medicinal plant chamomile to evaluate the genetic structure of the cultivated varieties/populations, and to perform genome wide association study (GWAS) focusing on genes with large effect on flowering time and the medicinally important alpha-bisabolol content. RESULTS: GBS analysis allowed the identification of 6495 high-quality SNP-markers in our panel of 91 M. recutita plants from 33 origins (2-4 genotypes each) and 4 M. discoidea plants as outgroup, grown in the greenhouse in Gatersleben, Germany. M. recutita proved to be clearly distinct from the outgroup, as was demonstrated by different cluster and principal coordinate analyses using the SNP-markers. Chamomile genotypes from the same origin were mostly genetically similar. Model-based cluster analysis revealed one large group of tetraploid genotypes with low genetic differentiation including 39 plants from 14 origins. Tetraploids tended to display lower genetic diversity than diploids, probably reflecting their origin by artificial polyploidisation from only a limited set of genetic backgrounds. Analyses of flowering time demonstrated that diploids generally flowered earlier than tetraploids, and the analysis of alpha-bisabolol identified several tetraploid genotypes with a high content. GWAS identified highly significant (P < 0.01) SNPs for flowering time (9) and alpha-bisabolol (71). One sequence harbouring SNPs associated with flowering time was described to play a role in self-pollination in Arabidopsis thaliana, whereas four sequences harbouring SNPs associated with alpha-bisabolol were identified to be involved in plant biotic and abiotic stress response in various plants species. CONCLUSIONS: The first genomic resource for future applications to enhance breeding in chamomile was created, andanalyses of diversity will facilitate the exploitation of these genetic resources. The GWAS data pave the way for future research towards the genetics underlying important traits in chamomile, the identification of marker-trait associations, and development of reliable markers for practical breeding.

Asunto(s)

Manzanilla/genética , Flores/crecimiento & desarrollo , Sitios Genéticos/genética , Estudio de Asociación del Genoma Completo , Técnicas de Genotipaje , Polimorfismo de Nucleótido Simple/genética , Sesquiterpenos/metabolismo , Cruzamiento , Manzanilla/crecimiento & desarrollo , ADN de Plantas/genética , ADN de Plantas/aislamiento & purificación , Diploidia , Sesquiterpenos Monocíclicos , Análisis de Secuencia , Tetraploidía

RESUMEN

Plant volatiles not only have multiple defense functions against herbivores, fungi, and bacteria, but also have been implicated in signaling within the plant and toward other organisms. Elucidating the function of individual plant volatiles will require more knowledge of their biosynthesis and regulation in response to external stimuli. By exploiting the variation of herbivore-induced volatiles among 26 maize (Zea mays) inbred lines, we conducted a nested association mapping and genome-wide association study (GWAS) to identify a set of quantitative trait loci (QTLs) for investigating the pathways of volatile terpene production. The most significant identified QTL affects the emission of (E)-nerolidol, linalool, and the two homoterpenes (E)-3,8-dimethyl-1,4,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). GWAS associated a single nucleotide polymorphism in the promoter of the gene encoding the terpene synthase TPS2 with this QTL Biochemical characterization of TPS2 verified that this plastid-localized enzyme forms linalool, (E)-nerolidol, and (E,E)-geranyllinalool. The subsequent conversion of (E)-nerolidol into DMNT maps to a P450 monooxygenase, CYP92C5, which is capable of converting nerolidol into DMNT by oxidative degradation. A QTL influencing TMTT accumulation corresponds to a similar monooxygenase, CYP92C6, which is specific for the conversion of (E,E)-geranyllinalool to TMTT The DMNT biosynthetic pathway and both monooxygenases are distinct from those previously characterized for DMNT and TMTT synthesis in Arabidopsis thaliana, suggesting independent evolution of these enzymatic activities.

Asunto(s)

Arabidopsis/metabolismo , Monoterpenos Acíclicos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Estudio de Asociación del Genoma Completo , Monoterpenos/metabolismo , Sitios de Carácter Cuantitativo/genética , Sesquiterpenos/metabolismo

RESUMEN

The gut lumen is the primary site of digestion and detoxification and thus presents conditions hostile to most proteins. We used 2D-gel electrophoresis and MS/MS de novo peptide sequencing to identify the major proteins stable enough to persist in the midgut lumen of caterpillars of the cotton bollworm Helicoverpa armigera, a generalist herbivorous insect and a major crop pest worldwide. As expected, we found several enzymes responsible for digestion of carbohydrates, proteins, and lipids. In addition, we identified nondigestive proteins such as a multidomain lipocalin, a protein with pathogen recognition domains, an arginine kinase related to a class of major human allergens, and abundant proteins of unknown function. Identification of the set of proteins that are secreted into the lumen will enable us to further characterize the nutritional and defensive functions of this important intraorganismal space.

Asunto(s)

Sistema Digestivo/metabolismo , Proteínas de Insectos/análisis , Lepidópteros/metabolismo , Proteoma/análisis , Secuencia de Aminoácidos , Animales , Arginina Quinasa/análisis , Arginina Quinasa/genética , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Electroforesis en Gel Bidimensional/métodos , Hidrolasas/análisis , Hidrolasas/genética , Proteínas de Insectos/genética , Larva/genética , Larva/metabolismo , Lepidópteros/genética , Lipocalinas/análisis , Lipocalinas/genética , Datos de Secuencia Molecular , Mapeo Peptídico/métodos , Proteoma/genética , Proteómica/métodos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos

RESUMEN

Tumor-associated antibodies are frequently detected in cancer patients. To ask whether the recognized antigens are rejection antigens, we screened a cDNA expression library of the mouse TS/A tumor with TS/A-immune serum and isolated 8 IgG-reactive clones, representing self-antigens that were expressed in normal tissues and other tumor lines. Three of the antigens had previously been identified in the human system by this cloning strategy. None of the antigens revealed to be a rejection antigen in normal mice demonstrated by an otherwise effective plasmid immunization. For one of the identified antigens, alpha-catenin, it is shown that the induction of IgG antibodies by protein immunization does not correlate with tumor rejection. For another antigen, vimentin, it is shown that vimentin-deficient but not vimentin-competent mice reject vimentin-expressing tumors indicating T -cell tolerance despite the fact that tumor cell immunization induces antivimentin IgG antibodies. Tissue damage induced by adenovirus infection induced an antibody response similar to tumor cell immunization, exemplified with 2 of the antigens. We conclude that the tumor-induced antibodies mirror tissue damage and that the antibody-inducing antigens can serve as rejection antigens if they are recognized as foreign.

Asunto(s)

Antígenos de Neoplasias/inmunología , Proteínas del Citoesqueleto/metabolismo , Inmunoglobulina G/metabolismo , Hígado/inmunología , Neoplasias/inmunología , Vimentina/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Animales , Formación de Anticuerpos/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Autoantígenos/inmunología , Autoinmunidad , Proteínas del Citoesqueleto/inmunología , Biblioteca de Genes , Humanos , Tolerancia Inmunológica , Inmunoglobulina G/inmunología , Hígado/lesiones , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/genética , Neoplasias/metabolismo , Linfocitos T/inmunología , Vimentina/genética , Vimentina/fisiología , alfa Catenina

RESUMEN

HER-2/neu (HER-2) is a cell surface proto-oncogene that is often overexpressed in carcinomas. Passive administration of anti-HER-2 antibodies in breast cancer patients has achieved promising results, but less is known about the role of antibodies in active immunization. We asked whether B cells/antibodies are needed for tumor immunity induced by plasmid (HER-2 and GM-CSF) immunization. HER-2 specific tumor immunity relied completely on both CD4+ and CD8+ T cells. IFN-gamma, and to a lesser extent IL-4, seemed to be crucial cytokines during tumor rejection. Protection was associated with production of anti-HER-2 IgG antibodies in B cell competent mice. After immunization, however, B cell-deficient mice rejected HER-2-expressing tumors as efficiently as control littermates. We conclude that T cells are the main effector cells in DNA vaccine induced immunity against HER-2 and that anti HER-2 antibodies are not necessary to elicit a protective anti tumor immune response in this model.

Asunto(s)

Linfocitos B/fisiología , Linfocitos T CD4-Positivos/inmunología , Neoplasias Mamarias Experimentales/prevención & control , Receptor ErbB-2/genética , Vacunación , Vacunas de ADN/administración & dosificación , Animales , Anticuerpos Monoclonales/metabolismo , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Humanos , Inmunidad Celular , Interferón gamma/metabolismo , Depleción Linfocítica , Masculino , Neoplasias Mamarias Experimentales/inmunología , Ratones , Ratones Endogámicos BALB C , Plásmidos , Proto-Oncogenes Mas , Receptor ErbB-2/inmunología , Tasa de Supervivencia

RESUMEN

The Del1 mutant of the green alga Chlamydomonas reinhardtii with a defined deletion in the chloroplast encoded psbA gene is unable to grow photoautotrophically. We show here that this mutant can be transformed with PCR-generated psbA fragments of varying length to yield photosynthetically growing colonies. PCR fragments need not be purified but can be directly precipitated from the amplification reaction onto tungsten particles, allowing fast and efficient mutagenesis experiments. Flanking regions bordering the deletion breakpoints have been systematically shortened from both sides. The shortest fragment giving rise to significant numbers of transformants contains about 50 bp upstream and 120 bp downstream of the deletion breakpoint. This technique greatly simplifies comprehensive structure-function analyses of the D1 protein in Chlamydomonas, but could perhaps be adapted to other chloroplast genes in this or other organisms as well.