Modifying the Sulfur Amino Acid Content of Seed Protein via Genetic Engineering

A. Titel und Inhaltsverzeichnis I-VII 1. Einleitung 1 2. Material und Methoden 15 3. Ergebnisse 31 4. Diskussion 69 5. Zusammenfassung/Summary 89 6. Literatur 91 7. Anhang 111In dieser Arbeit wurde ein Transformationssystem für den Agrobacterium tumefaciens -vermittelten Gentransfer in die Körnerleguminose Vicia faba etabliert. Epicotylsegmente von steril angezogenen Keimlingen wurden mit den Agrobakterienstämmen EHA101 bzw. EHA105 inokuliert, die die codierenden Sequenzen für (a) uidA, (b) ein mutiertes lysC-Gen einer feedback-insensitiven bakteriellen Aspartatkinase aus E. coli, (c) für ein methioninreiches 2S Albumin aus Helianthus annuus (sfa8) transferierten. Die Gene lysC und sfa8 standen unter Kontrolle samenspezifischer Promotoren. Auf Kallus-induzierendem Kulturmedium wurden 10-30% Kanamycin- bzw. Phosphinotricin-resistente Kalli selektiert, deren transgener Status mittels GUS-Assay bzw. PAT-Assay bestätigt werden konnte. Zwischen 1% und 6% der selektierten Kalli waren in der Lage, in der zweiten Kulturphase auf thidiazuronhaltigem Medium Sprosse zu bilden. Diese wurden in einer dritten Kulturphase auf BAP-haltiges Medium transferiert, vermehrt und nach Bewurzelung bzw. in vitro-Pfropfung zu vollständigen Pflanzen regeneriert. Mit verschiedenen Parametern der Kulturbedingungen wie Hormontyp und -konzentration, Kohlenstoffart und -gehalt, Temperatur u.a. wurden Optimierungsversuche mit begrenztem Erfolg durchgeführt. Verbesserungen konnten durch Massnahmen wie die Temperaturabsenkung von 20°C auf 15°C in Phase III oder die Verwendung von Caseinhydrolysat als Zusatz im Kulturmedium erreicht werden. Alle transgenen Pflanzen wurden mittels Southern Blot Analyse charakterisiert und die Integration des Transgens in das Pflanzengenom bestätigt. Die cytologischen Untersuchungen ergaben überwiegend diploide Pflanzen, aber auch tetraploide Formen und Aneuploidien traten auf, die größtenteils mit einem aberranten Habitus verbunden waren. Von 12 ausführlich untersuchten Linien wiesen sieben Samenbildung auf. Die Nachkommenschaft dieser sieben Linien wurde mit PCR-Analysen auf die genetische Stabilität der eingeführten Merkmale über mind. zwei Generationen getestet. Es konnte gezeigt werden, dass die integrierten Gene nach Selbstung der Linien entsprechend den Mendel´schen Gesetzen weitervererbt wurden. Die Expression der übertragenen Genkonstrukte wurde mit enzymatischen Assays (GUS, PAT, AK), durch Transkriptionsanalyse und mit Hilfe der Immunodetektion nachgewiesen. Aminosäureanalysen der transgenen, mit lysC-transformierten Linien 12-2 und 12-40 ergaben Veränderungen im Profil der freien Aminosäuren, u.a. eine relative Erhöhung von Threonin (8-10x) und Methionin (2-3x) in den Samen gegenüber dem WT. In Linien, die mit dem Gen für das methioninreiche SFA8-Albumin transformiert worden waren, liessen sich keine Veränderungen im Gehalt der schwefelhaltigen proteingebundenen Aminosäuren nachweisen. Trotz der relativ geringen Transformationsfrequenz und der langen Kulturdauer bietet das vorgestellte Transformationssystem einen geeigneten Ansatz zur stabilen Übertragung erwünschter Merkmale in die Körnerleguminose Vicia faba. Damit wird es erstmalig möglich, auch bei Vicia faba gentechnische Methoden in der Pflanzenzüchtung nutzen zu können.Among the major grain legume crops, Vicia faba belongs to those where the production of transgenic plants has not yet been convincingly reported. We have produced stably transformed lines of faba bean with an Agrobacterium tumefaciens-mediated gene transfer system. Stem segments from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA101 or EHA105, carrying binary vectors conferring (1) uidA, (2) a mutant lysC gene, coding for a bacterial aspartate kinase insensitive to feedback control by threonine or lysine from E.coli, (3) the coding sequence for a methionine-rich sunflower 2S-albumin. Kanamycin or Phosphinotricin-resistant calli were obtained on callus initiation medium at a frequency of 10-30%. Shoot regeneration was achieved on thidiazuron containing medium in a second culture step. A subsequent transfer of shoots to BA-containing medium was necessary for stem elongation and leaf development. Shoots were rooted or grafted onto young seedlings in vitro and mature plants were recovered and transferred to the greenhouse. Investigation on factors influencing the regeneration efficiency, e.g. hormone type and concentration or sugar type and concentration, were conducted with lesser results. A decrease in temperature from 20°C to 15°C was found to be benefical for shoot development in phase III. Molecular analysis confirmed the integration of the transgenes into the plant genome by Southern blotting. Cytological studies showed chromosomal aberration in 25% of all transgenic lines, mostly correlated with a differing phenotype. From 12 well-characterized lines seven were found to be fertil. The introduced DNA is physically transmitted as a dominant Mendelian trait and the inheritance of the transgene up to the -at least- third generation was confirmed by PCR in seed-bearing lines. Expression of the foreign genes was demonstrated by RT-PCR, Western analysis and enzyme activity assays (GUS, PAT, AK). HPLC analyses of seed amino acids in lysC-containing lines showed changes in the free amino acid composition, above all in threonine content (up to 10-fold increase compared to wildtype), and to a lesser extent in methionine content (2-3-fold). No change in proteinbound amino acid composition was found in the two SFA-lines 8-5 and 8-66 expressing the methionin-rich 2S-albumin from sunflower. Although at present the system is time-consuming and of relatively low efficiency, it represents a feasible approach for the production of genetically engineered faba beans. Future prospects were discussed

Farm questionnaires for monitoring genetically modified crops: a case study using GM maize

Monitoring is a statutory requirement for the cultivation of genetically modified (GM) crops in the European Community. Questionnaires for farmers to report on observations of effects linked with the cultivation of GM crops can form a useful part of a monitoring regime. A questionnaire for GM maize (Zea mays L.) was designed, with questions focusing on potential effects related to the GM maize grown, as well as on background information about cultivation methods and on individual field situations. In this paper we present the methodological approach of the monitoring regime, the structuring of the data, and the contents and structure of the questionnaire. The statistical requirements and background for an appropriate evaluation and interpretation of the data are described. Results of interviews made from 2001 to 2005 are also presented. It is envisaged that this approach will be developed for monitoring other cultivated GM plants and traits, and may be applicable in monitoring certain non-farmed environments

LinBi: Linking biodiversity and culture information

The LinBi project aims to enhance the discoverability of digitized objects from natural history collections hosted by institutes all over Europe. This enhancement is achieved by publishing new and enriched content to the Europeana collections platform. The use of simple vocabularies and machine-readable metadata encourages reuse and has the additional advantage of facilitating the clustering of interesting content for user groups beyond biodiversity and natural history researchers. Linking the collections of Europe together in an openly-available platform and sharing our common cultural and natural heritage with a broad audience will increase the public’s awareness of biodiversity collections. Furthermore, it will help us reach out to new user groups such as teachers, journalists and artists, who were previously unaware of, or distant from, our collections. Suitable content was selected and harmonized for interlinking with Europeana. Contributions include a large quantity of herbarium sheets, digitized glass plate negatives taken between 1880 and 1930 and a portrait collection dating from the late 19th and early 20th century. With the help of the DoeDat crowdsourcing platform, existing metadata were enriched and mobilized to allow for publication in the form of open linked data. The integration of geographical data and common names allows the Europeana platform to link scientific specimens with literature and fine art from different collections and to guide users to interesting and inspiring content via themed virtual exhibitions. One such theme is composed of content from the "Wild Roses of Crépin" collection, which will be enriched by pictures of living plants, herbarium specimens and illustrations old and modern. A second content cluster consists of an enriched and curated collection of botanical illustrations originating from a corpus of special and rare books ranging from the 15th to 19th centuries. Careful curation increases the potential for re-use and provides additional oppertunities for the general public to interact with this collection. The LinBi platform has the long-term ambition of forming a sustainable and open source solution integrated into the Europeana Core Service. This will further improve cooperation between institutes by building international infrastructure and networks, thus contributing to a more open cross-border society

BMP7, a Putative Regulator of Epithelial Homeostasis in the Human Prostate, Is a Potent Inhibitor of Prostate Cancer Bone Metastasis in Vivo

Bone morphogenic protein 7 (BMP7) counteracts physiological epithelial-to-mesenchymal transition, a process that is indicative of epithelial plasticity. Because epithelial-to-mesenchymal transition is involved in cancer, we investigated whether BMP7 plays a role in prostate cancer growth and metastasis. BMP7 expression in laser-microdissected primary human prostate cancer tissue was strongly down-regulated compared with normal prostate luminal epithelium. Furthermore, BMP7 expression in prostate cancer cell lines was inversely related to tumorigenic and metastatic potential in vivo and significantly correlated to E-cadherin/vimentin ratios. Exogenous addition of BMP7 to human prostate cancer cells dose-dependently inhibited transforming growth factor β-induced activation of nuclear Smad3/4 complexes via ALK5 and induced E-cadherin expression. Moreover, BMP7-induced activation of nuclear Smad1/4/5 signaling transduced via BMP type I receptors was synergistically stimulated in the presence of transforming growth factor β, a growth factor that is enriched in the bone microenvironment. Daily BMP7 administration to nude mice inhibited the growth of cancer cells in bone. In contrast, no significant growth inhibitory effect of BMP7 was observed in intraprostatic xenografts. Collectively, our observations suggest that BMP7 controls and preserves the epithelial phenotype in the human prostate and underscore a decisive role of the tumor microenvironment in mediating the therapeutic response of BMP7. Thus, BMP7 can still counteract the epithelial-to-mesenchymal transition process in the metastatic tumor, positioning BMP7 as a novel therapeutic molecule for treatment of metastatic bone disease