PCR identification of rpgip1 transgene in Pisum sativum L.

Recent efforts to increase Ascochyta blight resistance of pea have focused on the introduction of foreign genes by genetic engineering. The rpgip1 gene from Rubus idaeus was introduced by Agrobacterium-mediated transformation into Pisum sativum, cv. Baroness with the aim to increase pea resistance to fungal diseases. Notwithstanding this success, practical applications have to be preceded by the development of analytical methods for screening. Singleplex and multiplex PCR assays were employed to test primer efficiency in identifying the rpgip1 transgene in 11 pea genotypes. Five from ten primer combinations were effective in identifying transgene or insert sequences. PCR amplification using five other primer pairs revealed unspecific amplicons. According to in silico analyses, they arose from retrotransposons and pea genes including homologues of rpgip1. Two sets of primers were prepared with the aim of simultaneous amplification of different rpgip1 fragments. Fingerprints were sums of bands observed from individual pairs so the utility of multiplex assays was demonstrated. An additional advantage of multiplex PCR was clear differentiation between the transgene and endogenous pgip genes present in the donor species, R. idaeus. Sequencing of two PCR products confirms that no substantial rearrangements at the rpgip1 transgene arose during development of transgenic plants. However, a deletion occurred at 59 bp in the PGIP+VST line and a substitution at 392 bp in the PGIP line. The frequency of point mutations was not high (1.1 × 10-3) and comparable with the frequency expected for host genes based on the neutral theory of molecular evolution.European Unio

Enhancing transgenic pea (Pisum sativum L.) resistance against fungal diseases through stacking of two antifungal genes (chitinase and glucanase).

One way of enhancing and broadening resistance of plants to different biotic and abiotic stresses is to combine transgenes expressing several genes into a single line. This can be done using different strategies such as crossing, single vector with multiple genes, co-transformation, sequential transformation and IRES elements. In the present study conventional crossing method was used. Parental transgenic lines transformed via Agrobacterium tumefasciens-mediated gene transformation with pGreenII binary vector harbouring a bar gene as selectable marker in combination with the family 19 chitinase gene from Streptomyces olivaceoviridis for one line and 1,3-β-glucanase from barley (Hordeum vulgare) for the other line were used for crossing. Both chitinase and glucanase genes were cloned into pGreenII vector under the control of the constitutive double 35S-promoter from cauliflower mosaic virus. Progenies expressing the two genes were characterised at the molecular level using PCR, RT-PCR and Southern blot analysis, as well as segregation and stability studies of the respective expression levels. Leaf paint assay was used as functional test for herbicide resistant gene. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated. The synergistic effect of crossed plants was tested using in vitro assay which shows higher inhibition of spore germination

Metabolic engineering of apple by overexpression of the MdMyb10 gene

Flavonoids are low-molecular-weight phenolic compounds that are widely distributed in the plant kingdom. They have different roles in plant resistance to biotic and abiotic stresses. The transcription factor gene MdMyb10 (Gene Bank: DQ267896) was introduced into two apple (Malus domestica Borkh.) cultivars i.e. ‘Holsteiner Cox (HC)’ and ‘Gala’ via Agrobacterium-mediated transformation. The regenerated shoots were selected on kanamycin containing media. The presence of additional MdMyb10 gene in putative shoots was confirmed by PCR, RT-PCR and Southern blotting. Expression level of introduced MdMyb10 gene was analyzed by quantitative real time PCR. The results confirmed a dramatic increase in overexpression of MdMyb10 in the transgenic plants, up to 1261 and 847-folds for cultivars Holsteiner Cox and Gala, respectively compared to non-transformed negative control plants. HPLC-MS was used to determine the levels of different flavonoid compounds in both non-transgenic and transgenic plants. In MdMyb10 ‘HC’ transgenic plants, some of the polyphenols analyzed were enhanced while others were reduced in comparison to their levels in the non-transgenic plants. On the other hand, all of the analyzed polyphenol classes were induced in MdMyb10 ‘Gala’ transgenic plants in comparison to their levels in the non-transgenic plants. In the present study, the flavonoid pathway was successfully modified in apple by overexpressing the MdMyb10 transcription factor to validate the hypothesis of increased effect on plant disease resistance.Islamic Development Ban

Einsatz der dCAPS-Technologie zur Differenzierung von Trp574-Leu und Ser653- Asn in Blattproben von Raps und Clearfield®-Raps

Bei Clearfield®-Raps (Brassica napus L.) handelt es sich um Rapssorten, in denen eine Unkrautbekämfung mit Herbiziden aus der Gruppe der ALS-Inhibitoren möglich ist. Zwei Allele der ALS (Acetolactat-Synthase), die eine Target-Site Resistance (wirkortspezifische Resistenz, bzw. Toleranz) bewirken, sind in zwei der fünf ALS-Gene des Raps eingekreuzt. Der Clearfield®-Raps differenziert sich vom konventionellen Raps durch die Aminosäure Asparagin an Stelle von Serin im Kodon 653 des ALS I Gens und bewirkt eine erhöhte Toleranz speziell gegen Herbizide aus der Gruppe der Imidazolinone. Das Allel von ALS III differenziert sich durch die Aminosäure Leucin an Stelle von Trypthophan im Kodon 574 und bewirkt eine Toleranz hauptsächlich gegen Imidazolinone, Sulfonylharnstoffe und Triazolopyrimidine (gruppenübergreifende Toleranz). Letzteres ist für eine erschwerte Bekämpfung von Clearfield®-Raps als Ausfallraps mit Sulfonylharnstoffen und Triazolopyrimidinen z.B. in Getreide verantwortlich. In dieser Arbeit wird ein Test vorgestellt, der eine Allel-Diskriminierung mittels der dCAPS-Technologie (derived cleaved amplified polymorphic sequence) ermöglicht. So lässt sich ein Clearfield®- Raps von einem konventionellen Raps an Blättern und anderen Pflanzenteilen mittel Molekulargenetik differenzieren. Durch eine Kombination von PCR und der Behandlung der PCR-Produkte durch spezifische Restriktionsendonukleasen werden die Resistenz-Allele auf dem ALS I (nach Behandlung mit MnlI) und ALS III (nach Behandlung mit NcoI) mittels Gelelektrophorese nachgewiesen. Für den Nachweis einer Minderwirkung von Sulfonylharnstoffen gegen Ausfallraps reicht oft die Identifizierung von Leu574 aus. Um sicher zu gehen und einen Clearfield®-Raps von natürlich auftretenden Raps-Varianten mit einer Resistenz zu unterscheiden sollte immer auch ein Nachweis von Asn653 durchgeführt werden. Die hier dargestellte Technik setzt keine spezielle Laboreinrichtung voraus und lässt sich mit molekulargenetischen Grundkenntnissen etablieren. So können landwirtschaftliche Einrichtungen flexibel auf einen Nachweisbedarf von Clearfield®-Raps in der Praxis reagieren.Stichwörter: Ausfallraps, Auskreuzung, Clearfield®, dCAPS, Herbizidtoleranz, Imidazolinone, Leu574, Ser, SNPs, Sulfonylharnstoffe, Triazolopyrimidine, UnkrautbekämpfungThe utilization of the dCAPS technology to discriminate Trp574-Leu and Ser653-Asn in leaf samples of Clearfield® oilseed rape and conventional oilseed rapeClearfield® rape (Brassica napus L.) derives from classical breeding methods and makes weed control with ALS-inhibiting herbicides in oilseed rape possible. Two alleles of the ALS (acetolactate synthase), which are responsible for target site resistance (or tolerance, respectively), were crossed into two of the five ALS genes of oilseed rape. The allele of Clearfield® oilseed rape in ALS I is different from other ALS alleles by an exchange of the amino acid serine by asparagine in the codon 653 and is responsible for tolerance to imidazolinones. The allele in ALS III differs by an exchange of tryptophan to leucine in the codon 574 and causes a broad tolerance to imidazolinones, sulfonylureas, and triazolopyrimidines. The latter allele hinders mainly the control of volunteer Clearfield® oilseed rape with sulfonylureas and triazolopyrimidines e.g. in cereals. To decipher the alleles in Clearfield® oilseed rape and conventional oilseed rape a test based on the dCAPS (derived cleaved amplified polymorphic sequence) technology was developed and is presented here. With this test a Clearfield® oilseed rape can be discriminated in leaf samples or other parts of plants from conventional oilseed rape.A combination of PCR and digestion with restriction endonucleases is used to discriminate the alleles of ALS I (after incubation of PCR-products with MnlI) and of ALS III (after incubation of PCR-products with NcoI). The identification of Leu574 is in most cases probably sufficient for proving reduced efficacy of sulfonylureas and triazolopyrimidines. To come to a clear identification and to discriminate Clearfield® oilseed rape from natural occurring target-site resistant oilseed rape both alleles should be analysed in samples. The dCAPS technology does not require special lab equipment and can be performed with the basic equipment of every molecular biological working lab. With the herein presented protocol the agricultural consulting and research facilities can react flexible on the need to prove Clearfield® oilseed rape variations from conventional oilseed rape in practice. Keywords: dCAPS, herbicide tolerance, imidazolinone, outcrossing, single nucleotide polymorphism, sulfonylureas, triazolopyrimidines, volunteer rape, weed contro

Cloning of a Gene for an Acyl-CoA Dehydrogenase from Pisum sativum L. and Purification and Characterization of Its Product as an Isovaleryl-CoA Dehydrogenase

Isovaleryl-CoA dehydrogenase (IVD, EC 1.3.99.10) catalyzes the third step in the catabolism of leucine in mammals. Deficiency of this enzyme leads to the clinical disorder isovaleric acidemia. IVD has been purified and characterized from human and rat liver, and the x-ray crystallographic structure of purified recombinant human IVD has been reported. Nothing is known about IVD activity in plants, although cDNA clones from Arabidopsis thaliana and partial sequences from Gossypium hirsutum and Oryza sativa have been identified as putative IVDs based on sequence homology and immuno cross-reactivity. In this report we describe the identification and characterization of an IVD from pea, purification of the enzyme using a novel and rapid auxin affinity chromatography matrix, and cloning of the corresponding gene. At the amino acid level, pea IVD is 60% similar to human and rat IVD. The specific activity and abundance of plant IVD was found to be significantly lower than for its human counterpart and exhibits developmental regulation. Substrate specificity of the plant enzyme is similar to the human IVD, and it cross-reacts to anti-human IVD antibodies. Molecular modeling of the pea enzyme based on the structure of human IVD indicates a high degree of structural similarity among these enzymes. Glu-244, shown to function as the catalytic base in human IVD along with most of the amino acids that make up the acyl CoA binding pocket, is conserved in pea IVD. The genomic structure of the plant IVD gene consists of 13 exons and 12 introns, spanning approximately 4 kilobases, and the predicted RNA splicing sites exhibit the extended consensus sequence described for other plant genes

Enhanced Abiotic Stress Tolerance of Vicia faba L. Plants Heterologously Expressing the PR10a Gene from Potato

Pathogenesis-related (PR) proteins are known to play relevant roles in plant defense against biotic and abiotic stresses. In the present study, we characterize the response of transgenic faba bean (Vicia faba L.) plants encoding a PR10a gene from potato (Solanum tuberosum L.) to salinity and drought. The transgene was under the mannopine synthetase (pMAS) promoter. PR10a-overexpressing faba bean plants showed better growth than the wild-type plants after 14 days of drought stress and 30 days of salt stress under hydroponic growth conditions. After re-moving the stress, the PR10a-plants returned to a normal state, while the wild-type plants could not be restored. Most importantly, there was no phenotypic difference between transgenic and non-transgenic faba bean plants under well-watered conditions. Evaluation of physiological parameters during salt stress showed lower Na+-content in the leaves of the transgenic plants, which would reduce the toxic effect. In addition, PR10a-plants were able to maintain vegetative growth and experienced fewer photosystem changes under both stresses and a lower level of osmotic stress injury under salt stress compared to wild-type plants. Taken together, our findings suggest that the PR10a gene from potato plays an important role in abiotic stress tolerance, probably by activation of stress-related physiological processes

Energy Informatics - Current and Future Research Directions

Due to the increasing importance of producing and consuming energy more sustainably, Energy Informatics (EI) has evolved into a thriving research area within the CS/IS community. The arti- cle attempts to characterize this young and dynamic field of research by de- scribing current EI research topics and methods and provides an outlook of how the field might evolve in the fu- ture. It is shown that two general re- search questions have received the most attention so far and are likely to dominate the EI research agenda in the coming years: How to leverage infor- mation and communication technol- ogy (ICT) to (1) improve energy effi- ciency, and (2) to integrate decentral- ized renewable energy sources into the power grid. Selected EI streams are reviewed, highlighting how the re- spective research questions are broken down into specific research projects and how EI researchers have made con- tributions based on their individual academic background