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  • 1
    ISSN: 1432-203X
    Keywords: Key words Chlorophyll fluorescence ; Kanamycin resistance screening ; Tobacco ; Arabidopsis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The applicability of a chlorophyll fluorescence assay for kanamycin (Km) resistance screening in transgenic tobacco (Nicotiana tabacum) and Arabidopsis thaliana plants was investigated. In wild-type leaves incubated in the presence of 200 mg/l Km, a decrease in maximum variable fluorescence ((Fv)m) and a significant increase in constant fluorescence (Fo) were observed. Using (Fv)m/Fo as a screening parameter, we were able to distinguish Km-treated samples from untreated samples within 4 days. This parameter was applied to Km resistance screening using tobacco plants transformed with the nptII gene via Agrobacterium. Among 74 shoots selected on medium containing 200 mg/l Km, 37 plants were scored as Km sensitive by the chlorophyll fluorescence assay. These 74 scorings proved to be accurate, as reconfirmed by (1) polymerase chain reaction amplification of the transgene, (2) enzymatic assay of neomycin phosphotransferase and (3) leaf disc assay. Using the chlorophyll fluorescence assay, we could also screen 3-week old Arabidopsis plants carrying the nptII gene. These results clearly demonstrate the reliability and efficiency of this nondestructive assay for Km resistance screening of transgenic plants.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1617-4623
    Keywords: Key words Protein kinase ; Wounding ; Abscisic acid ; Methyl jasmonate ; Signal transduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA encoding a protein kinase, which may be involved in the wound signal transduction pathway, was isolated from Nicotiana tabacum. The cDNA, named WAPK, is 1227 bp in length and contains an ORF of 1017 bp. The ORF encodes a polypeptide of 339 amino acids, with a calculated molecular mass of 38234 Da. Analysis of the deduced amino acid sequence shows that the N-terminal region of WAPK contains a catalytic region composed of eleven subdomains which are typically found in Ser/Thr protein kinases. This region shows 78–84% sequence identity with similar regions of abscisic acid (ABA)-induced and external-stimuli-responsive protein kinases. However, the C-terminal region of WAPK shows little homology with similar regions of Ser/Thr protein kinases, except for a 16-amino acid stretch near the end of the catalytic domain. Kinase assays using a WAPK fusion protein expressed in E. coli revealed that WAPK autophosphorylates on serine residue(s). The WAPK gene is predominantly expressed in flowers, moderately in roots, and poorly in leaves. Transcripts were not detected in stems. The WAPK gene was induced by wounding (within 1.5 h), by abscisic acid (within 0.5 h), and by methyl jasmonate (within 2 h). The induction pattern of WAPK mRNA upon wounding was not affected by treatment with diethyldithiocarbamic acid, a reagent which inhibits jasmonic acid biosynthesis. These results suggest that the WAPK gene is regulated by ABA in the wound signal transduction pathway.
    Type of Medium: Electronic Resource
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