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procedures previously applied successfully by Walter et al. (1999) and Clapham et al. (2000) on Norway spruce were used in combination. After regeneration of transgenic plantlets these can be tested for pinosylvin synthase activity and resistance towards fungal pathogens. Methods and materials Vectors and enzymes and media All enzymes were obtained from Gibco BRL (Life technologies, USA) except calf intestinal alkaline phosphatase which was obtained from Roche, USA. The pKK233-2 plasmid vector containing the PSS1gene (Fliegman et al. 1992) was a gift from Prof. Joachim Schröder (Institut für Biologie II, Freiburg, Germany). The pCW122 plasmid vector was a gift from Dr. Christian Walter (Forest Research, New Zeeland). The vector was designed by Walter et al. (1994) for biolistic transfer of genes into cultured embryogenic cells of Pinus radiata. pCW122 contains the uidA reporter gene (GUS), derived from Escherichia coli under the control of a cauliflower mosaic virus (CaMV) tandem 35S promoter, and the nptII (neomycin phosphotransferase) antibiotic resistance gene, derived from E. coli, controlled by a CaMV 35S promoter. Both genes have a Kozak consensus sequence (Kozak 1986) around the ATG start codon, which improves the intensity of translation. NptII acts on antibiotics of the aminoglycoside group (including neomycin, kanamycin and geneticin) which are inactivated by phosphorylation. pCW122 contains two Bam HI sites framing GUS, and a single Hind III site proximal to GUS. pKK233-2 and pCW122 were maintained in E. coli strain DH5α grown in Luria-Bertani medium (LB) (Sambrook et al. 1989) containing 100 µg/ml ampicillin. 4 The PSS1-gene. The pinosylvin synthase gene, PSS1, accession no. X60753 was cloned by Fliegman et al. (1992) from stress-induced Scots pine hypocotyls and the complete coding region without intron was inserted into the plasmid pKK233-2 (Fliegman et al. 1992, Schanz et al. 1992). Since the environment around the PSS1 start codon contained no suitable cloning site, an Nco I site covering the PSS1 start ATG was created by site directed mutagenesis (Fliegman et al. 1992). Construction of plasmids for expression of PSS1 in Norway spruce PSS1 was excised from pKK233-2 and inserted into a modified pCW122 vector in both the sense and antisense directions. The cloning steps are shown in Figure 2. pKK233-2 was opened with Nco I and dephosphorylated using calf intestinal alkaline phosphatase (CIAP) (Roche, USA). An Nco I-Hind III adaptor was produced from the primers 5’ TCGAAGCTTAGCTGT ‘3 and 5’ CATGGACAG- CTAAGCTTCGA ‘3 and ligated on to the open ends. A 40:1 adaptor to vector ratio was used for the ligation. The ligation mix was digested with Hind III. As Nco I-Hind III adaptor dimers also cut by Hind III would be present and compete for insertion into Hind III sites, the Hind III restriction fragments were separated by gel electrophoresis and the PSS1 fragment was retrieved from the gel. pCW122 was digested with Bam HI to cut out GUS. The restriction fragments were separated and the fragment containing pCW122 without GUS was religated using T4 ligase and transformed into One shot® TOP 10 chemically competent E. coli (Invitrogen, USA) according to the manufactures instructions. The cells were cultured and plasmid DNA was obtained using the QIAprep spin miniprep kit (Qiagen Ltd., USA) and checked by restriction analysis. Re-ligated pCW122 without GUS was used
as vector for transformation into Norway spruce, it was termed pCW122*. pCW122* was re-opened by digestion with Hind III, the ends were dephosphorylated by CIAP, and PSS1 was ligated into the Hind III sites. The construct was transformed into E. coli TOP10 cells and ten clones were obtained. Of these, one proved to have PSS1 inserted in the antisense orientation, the others contained re-ligated plasmid without insert. pCW 122 pKK 233-2 Met ………. .......... B.... uid A ....B..H............ ..........CCATGG.... PSS 1 ....H……...... .............. Nco I digest with Bam H I digest with Nco I reli gate, clone in ligate Hin d III - Nco I adaptor E. coli , miniprep onto Nco I site ......................B..H.................... digest with Hin d III ligate Met TCGAAGCTTAGCTGTCCATGG....PSS1…H Hind III Nco I digest with Hin d III Met AGCTTAGCTGTCCATGG.... PSS1....H Hin d III Nco I Met ......................B.. AGCTTAGCTGTCCATGG.... PSS 1 .... H.................... Figure 2. Transfer of the pinosylvin synthase gene (PSS1) from the vector pKK233-2 into the vector pCW122. The distances are not drawn to scale. B=Bam HI site, H=Hind III site. pCW122 was cut with Bam H1 to remove uidA. After religation, the pCW122 (minus uidA) (now denoted pCW122*) was opened by digestion with Hind III. The pKK233-2 vector was opened with Nco I and a Hind III- Nco I adaptor was ligated onto the open ends. By digesting pKK233-2 with Hind III, PSS1 plus the adaptor was excised as a Hind III fragment and finally ligated into pCW122* . The resulting PSS1- antisense clone was sequenced (at MWG Biotech, Germany) throughout PSS1 and through both sites of insertion and used subsequently for transformation of Norway spruce. It was termed pCW122*PSS1-AS. Finally pCW122*PSS1-AS was cut by Hind III. The digestion mix was heated to 70 ºC to inactivate Hind III, ethanol-precipitated, then ligated directly. The ligation mix was used for transformation into E. coli TOP10 cells. Two of the clones obtained were verified by restriction analysis to contain the plasmid pCW122* carrying the PSS1gene in sense direction. This plasmid was used as vector for transformation into Norway spruce and termed pCW122*PSS1-S. The plasmid WRG 1515 (Hilliou et al. 1999) was used as control vector for measurement of transient GUS expression. Capture and maintenance of embryogenic cell lines. The embryogenic cell lines 11703-B3 and 186-3C from Picea abies (L.) Karst. were initiated from single mature zygotic embryos and maintained by weekly subcultures as described in Walter et al. (1999). The BMI-S1 medium (Krogstrup 1986) was used for subculturing, supplemented with 2,4dichlorophenoxyacetic acid (5 µM), 6-benzylaminopurine (2 µM), kinetin (2 µM), sucrose 30 g/l, casein hydrolysate 500 mg/l (Sigma C-9386), Lglutamine (450 mg/l), m-inositol (1000 mg/l). The medium was solidified with gellan gum (3 g/l) (Sigma P8169) which was mixed with the macro nutrient solution plus half of the water and autoclaved at 121 °C for 20 minutes. All other medium constituents were mixed with the remaining half of the water and filter sterilized. Before sterilization pH was adjusted to 5.7 with NaOH and HCl (Table 1). The autoclaved and the filter sterilized components were mixed and 25 ml medium poured into 90 mm Petri dishes. Transformation Embryogenic cell lines in supplemented BMI-S1 medium, taken 7 days after subculture, were filtered through a sterile sieve to retain aggregates larger than 0.5 mm. The medium, containing small and medium sized aggregates, were allowed to become sediment in 50 ml falcon tubes, producing 15 ml of sedimented cells to which 30 ml fresh supplemented BMI-S1 + 25 mM sorbitol was added. After mixing, aliquots of culture (2 ml) were pumped through filter paper discs (Whatman no. 1001042), retaining the cell aggregates 5
Page 1 and 2: Transformation of Norway spruce wit
Page 3: Figure 1. Pathways for stilbenoid a
Page 7 and 8: g/1) added to solidify the gel. The
Page 9 and 10: Measurement of neomycin-phosphotran
Page 11 and 12: Bombardment Cell line B63 K8 11703-
Page 13 and 14: current study we removed the GUS re
Page 15 and 16: Krogstrup, P. 1986. Embryo-like str

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