J. Bříza

Cloning and characterisation of chs-specific DNA and cDNA sequences from hop (Humulus lupulus L.)

A complete sequence of chalcone synthase (CHS) gene from hop was cloned. The gene designated chs _H1 consists of two exons and one 187 bp intron. CHS protein predicted from chs_H1 cDNA has 42.5 kDa and retains conserved domains and residues including 26 amino acids at positions identical to those identified by crystallography as characteristic for catalytic domains of alfalfa CHS (EC 2.3.1.74). Cloned CHS_H1 protein shows specific CHS activity with 4-coumaroyl-CoA. Structure modelling revealed clear differences between CHS_H1 and phlorisovalerophenone synthase, the only published CHS-like homologue from hop. Conserved motifs like H, and G boxes characteristic for the light regulated and stress inducible genes were identified within promoter region of chs _H1 gene. Highly specific expression of chs _H1 mRNA was detected by quantitative RT PCR in glandular trichomes during cone maturation. Much lower, but significant levels of chs _H1 mRNA were detected at the stage of hop flowering in petioles (100%), developed flowers (96%), and in stem apexes (78%), while the lowest levels of mRNA were found in the roots (31%) and leaf blades (9%). Southern blot analyses predicted at least five additional chs -like genes related to chs _H1. A genomic arrangement different from phlorisovalerophenone synthase sequences was found for these genes. RFLP analyses using DNA from 15 genotypes revealed several distinct dendrogram clusters, suggesting specific re-arrangements of hop chs -like genes during evolution and/or during the breeding and selection processes. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Use of phosphomannose isomerase-based selection system for Agrobacterium -mediated transformation of tomato and potato

Two selection systems for Agrobacterium tumefaciens mediated transformation of tomato and potato were compared. In the tomato (Lycopersicon esculentum cv. Moneymaker), the highest transformation rate, 4.2 %, of cotyledon explants on mannose-selection medium was obtained when mannose/sucrose concentration in the regeneration medium was 5/15 g dm−3. The best transformation efficacy with the commonly used concentration of 100 mg dm−3 kanamycin as a selection agent was 9 %. In the potato (Solanum tuberosum cv. Bintje), the highest transformation frequency was 53.3 % when mannose concentration in the regeneration medium was 5 g dm−3 during the first 3 weeks after transformation and 10 g dm−3 afterwards. The optimum concentration of sucrose was 20 g dm−3. The transformation efficiency using kanamycin as a selection agent at a concentration 100 mg dm−3 was 33.3 % with potato. Our results demonstrate that the transformation efficiency using mannose selection is 1.6-fold higher for potato and about 2 times lower for tomato comparing with the ordinary protocol using kanamycin.

Valerophenone synthase-like chalcone synthase homologues in Humulus lupulus

Valerophenone synthase homologue of chalcone synthase (CHS) is the first key enzyme described to be involved in the biosynthesis of bitter acids, the compounds produced in hop lupulin glands valuable for the taste of beer. The complete sequence of a novel homologue of CHS chs 4 was isolated from hop. Protein predicted from chs 4 cDNA has 43.45 kDa and length 395 amino acids. It was found by the analysis of chs 4 flanking sequences that this gene is in the cluster with other CHS homologues — chs 3 and vps. The intron identified in chs 4 has been found to be homological to vps and chs 3 introns. Expression of chs 4 was partially characterized using reverse transcription polymerase chain reaction and it was found that chs 4 is specifically expressed in the glandular tissue of hop cones likewise vps. The predicted protein sequence CHS 4 was compared with other CHS-like proteins.

The "putative" role of transcription factors from HlWRKY family in the regulation of the final steps of prenylflavonid and bitter acids biosynthesis in hop (Humulus lupulus L.).

Lupulin glands localized in female hop (Humulus lupulus L.) cones are valuable source of bitter acids, essential oils and polyphenols. These compounds are used in brewing industry and are important for biomedical applications. In this study we describe the potential effect of transcription factors from WRKY family in the activation of the final steps of lupulin biosynthesis. In particular, lupulin gland-specific transcription factor HlWRKY1 that shows significant similarity to AtWRKY75, has ability to activate the set of promoters driving key genes of xanthohumol and bitter acids biosynthesis such as chalcone synthase H1, valerophenone synthase, prenyltransferase 1, 1L and 2 and O-methyltransferase-1. When combined with co-factor HlWDR1 and silencing suppressor p19, HlWRKY1 is able to enhance transient expression of gus gene driven by Omt1 and Chs_H1 promoters to significant level as compared to 35S promoter of CaMV in Nicotiana. benthamiana. Transformation of hop with dual Agrobacterium vector bearing HlWRKY1/HlWDR1 led to ectopic overexpression of these transgenes and further activation of lupulin-specific genes expression in hop leaves. It was further showed that (1) HlWRKY1 is endowed with promoter autoactivation; (2) It is regulated by post-transcriptional gene silencing (PTGS) mechanism; (3) It is stimulated by kinase co-expression. Since HlWRKY1 promotes expression of lupulin-specific HlMyb3 gene therefore it can constitute a significant component in hop lupulin regulation network. Putative involvement of HlWRKY1 in the regulation of lupulin biosynthesis may suggest the original physiological function of lupulin components in hop as flower and seed protective compounds.

Production of human papillomavirus type 16 E7 oncoprotein fused with β-glucuronidase in transgenic tomato and potato plants

The human papillomavirus type 16 (HPV 16) oncogene E7 fused with the gene for β-glucuronidase (gus) was used in plant transformation experiments. The E7 gene modified for lower cancerogenicity and fused with the 5′ end of the gus in cassettes with cauliflower mosaic virus 35S promoter and transcription terminator produced high contents of fusion proteins in potato protoplasts. Expression vectors harbouring E7 fusion cassettes were used for Agrobacterium tumefaciens LBA4404 mediated transformation of either potato (Solanum tuberosum L. cv. Bintje) or tomato (Lycopersicon esculentum Mill. cv. Moneymaker). A fusion gene was found in all rooted regenerants using polymerase chain reaction with primers providing amplified fragments from E7 and gus genes. GUS activity was revealed in all regenerants obtained. Nevertheless, the level of GUS expression in different constructs varied much more than in transient expression experiments with potato protoplasts. Especially, expression level in plants carrying vectors with the whole E7 gene fused with gus was lowered by 2–3 orders of magnitude comparing with fusion of the first 41 codons of E7 and gus. Southern hybridisation of 18 tomato and 23 potato regenerants revealed mostly multiple tandem integration of T-DNA into the plant genome and Western blot proved the presence of the fusion protein in 9 tomato and 11 potato plants out of 41 tested individuals.

Cry3A δ-endotoxin gene mutagenized for enhanced toxicity to spruce bark beetle in a receptor binding loop

Bacillus thuringiensis Cry3A gene was redesigned for high expression in Norwegian spruce and the sequence was slightly modified to allow for simple N- and C- terminal deletions and domain II loop 1 exchange for synthetic oligos. Modified Cry3A toxins from 13 variants of the synthetic gene were expressed in Escherichia coli BL21 and their toxicity on spruce bark beetle larvae was tested using spruce bark sandwiches. Mutant toxins with N-terminal deletion and loop 1 duplication showed increased toxicity. Key words : Bacillus thuringiensis , Ips typographus, Picea abies, resistance.

Germinal Excision and Reinsertion Frequencies of the Mobile Element Ds Transposed from Two Unlinked T-DNA Loci in Tomato

Acceptor sites of unlinked transposed Ds element from two T-DNA loci in tomato were mapped. Experimental data obtained from TC1 progeny testing were employed for estimation of germinal excision frequency (GEF) of Ds element and frequency of its reinsertion (FR). The donor T-DNAs 1481J and 1601D, containing a 35S:NPT transformation marker, a 35S:BAR or nos:BAR excision marker conferring phosphinothricine resistance and a Ds element in the 5′ untranslated leader of the nos (or 35S): BAR gene, were located on chromosome 7 and 8, respectively. Ds transposition was induced by 105121 T-DNA carrying stabilized Ac (sAc) which provides a source of transposase and 2′:GUS marker conferring β-glucuronidase activity. Tomato plants harbouring the Ds in 1481J or 1601D locus and sAc were crossed and F1D, were crossed individually as seed parents to wild-type plants to generate TC1 progenies. TC1 seed was germinated on phosphinothricine (Basta)-containing medium, and individual seedlings carrying a transposed Ds and lacking sAc were identified by PCR (to detect the Ds) on phosphinothricine resistant individuals that lacked β-glucuronidase activity. From segregation ratio in TC1 the germinal excision and reinsertion frequencies of the Ds element were estimated for individual F1 plants. A total of 14560 TC1 seedlings of 1481J and 16195 TC1 seedlings of 1601D was analyzed. We observed high variation between individual plants as regards both GEF and FR despite of donor locus (1481J or 1601D), however, the average germinal excision frequencies as well as average frequencies of reinsertion were very similar for both donor loci: GEF1481J = 24 %, GEF1501D = 25 %, FR1481J = 42 %, FR1601D = 46 %.

Resistance to ACNU induced toxicity in transgenic tobacco suspension cultures with ada gene transferred from Escherichia coli

The protein coding region of theE. coli DNA repair geneada combined with the CaMV 35S promoter has been transferred to tobacco by means ofAgrobacterium tumefaciens Ti plasmid. In transgenic plants having theada gene in a sense orientation, detectable amounts of O6-alkylguanine-DNA-alkyltransferase has been found whereas in non-transformed plants this activity is absent. Cell suspension cultures derived from the former plants showed lower sensitivity to the toxic (growth inhibiting) effects of the bifunctional alkylating agent 1-(2-chloroethyl)-1-nitroso-3-(aminomethyl-1,3-diazinylo)-methylurea compared with cell cultures derived from a control non-transformed plant or from transgenic plants harbouring theada gene in an opposite, non-sense orientation.

Genome-wide transcriptome profiling of transgenic hop (Humulus lupulus L.) constitutively overexpressing Hl WRKY1 and Hl WDR1 transcription factors

BackgroundThe hop plant (Humulus lupulus L.) is a valuable source of several secondary metabolites, such as flavonoids, bitter acids, and essential oils. These compounds are widely implicated in the beer brewing industry and are having potential biomedical applications. Several independent breeding programs around the world have been initiated to develop new cultivars with enriched lupulin and secondary metabolite contents but met with limited success due to several constraints. In the present work, a pioneering attempt has been made to overexpress master regulator binary transcription factor complex formed by HlWRKY1 and HlWDR1 using a plant expression vector to enhance the level of prenylflavonoid and bitter acid content in the hop. Subsequently, we performed transcriptional profiling using high-throughput RNA-Seq technology in leaves of resultant transformants and wild-type hop to gain in-depth information about the genome-wide functional changes induced by HlWRKY1 and HlWDR1 overexpression.ResultsThe transgenic WW-lines exhibited an elevated expression of structural and regulatory genes involved in prenylflavonoid and bitter acid biosynthesis pathways. In addition, the comparative transcriptome analysis revealed a total of 522 transcripts involved in 30 pathways, including lipids and amino acids biosynthesis, primary carbon metabolism, phytohormone signaling and stress responses were differentially expressed in WW-transformants. It was apparent from the whole transcriptome sequencing that modulation of primary carbon metabolism and other pathways by HlWRKY1 and HlWDR1 overexpression resulted in enhanced substrate flux towards secondary metabolites pathway. The detailed analyses suggested that none of the pathways or genes, which have a detrimental effect on physiology, growth and development processes, were induced on a genome-wide scale in WW-transgenic lines.ConclusionsTaken together, our results suggest that HlWRKY1 and HlWDR1 simultaneous overexpression positively regulates the prenylflavonoid and bitter acid biosynthesis pathways in the hop and thus these transgenes are presented as prospective candidates for achieving enhanced secondary metabolite content in the hop.

Transformation of Tobacco cpDNA with Fusion E7GGG/GUS Gene and Homologous Recombination Mediated Elimination of the Marker Gene

ABSTRACT A transformation vector harboring the marker gene aadA conferring spectinomycin resistance and the fusion E7GGG/gus gene of interest was employed for the transformation of tobacco cpDNA via biolistics. The gene of interest consisted of the oncogenic E7GGG gene from human papillomavirus strain 16 fused with the reporter gus gene. Both transgenes were equipped with the same promoter and termination sequences arranged as direct repeats. Biolistics with circular vector yielded 20 shoots rooted in spectinomycin containing medium; with linear vector no rooted plants were obtained. After 4 months of in vitro selection the plants were burst into flower in a greenhouse and pollinated with non-transgenic plant pollen. Seeds were harvested individually from each plant capsule and planted onto selection medium. Rare white seedlings were recovered by transfer onto medium without spectinomycin and self-pollinated. GUS activity of 15 seedlings from each self-pollinated plant was measured and 26 plants with higher activity (and one without any GUS activity) were PCR analyzed for the presence of both the marker gene and the gene of interest. Finally, 8 plants were analyzed in detail by Southern hybridization, RT-PCR, and Western blot. We identified 6 plants harboring the E7GGG/gus gene only, where the marker gene was eliminated by homologous recombination; one plant with both transgenes; and one with the aadA gene only. RT-PCR showed the presence of the respective mRNAs in all 8 plants analyzed but Western blot proved that only the GUS part of the fusion E7GGG/GUS protein was present in all plants harboring the fusion gene. We discuss why E7GGG protein could not be detected.