The 5’-region of the chitinase gene cabch29, derived from Brassica oleracea var. capitata, has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants...The 5’-region of the chitinase gene cabch29, derived from Brassica oleracea var. capitata, has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants. Different 5’-deletion fragments were linked to reporter geneβ-glucuronidase (GUS) as translational fusions, and the expression of these chimeric genes was analyzed in vegetative organs and tissues. Sequences up to -651 showed some basal GUS activity with nearly equal levels in wounded and intact tissues. The addition of further upstream sequences (-651 to -1284) enhanced expression level, and the expression driven by this fragment was inducible by a factor of two to three-fold by wounding. Histochemical analysis of different tissue from transgenic plants that contain cabch29 promoter-GUS fusion gene demonstrated woundinducible and tissue-specific cabch29 promoter activity in plants containing the 1308 base pair fragment. The location of GUS activity appears to be cell-specific, being highest in vascular cells and epidermal cells of stem, leaf and roots. Meanwhile, the temporal and spatial expression of cabch29-GUS fusion gene has been investigated. Among the different vegetative organs, a high level of GUS activity was observed in stem and a moderate one in roots;whereas, wounding stress led to a high level of GUS in stem and moderate one in leaf.展开更多
Chitinase, which catalyzes the hydrolysis of the β-1,4-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin, is involved in inducible plants defense system. By construction of cabbage (Brassica olerac...Chitinase, which catalyzes the hydrolysis of the β-1,4-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin, is involved in inducible plants defense system. By construction of cabbage (Brassica oleracea var.capitata) genomic library and screening the library with pRCH8, a probe of rice chitinase gene fragment, a chitinase genomic sequence was isolated. The complete nucleotide sequence of the putative cabbage chitinase gene (cabch29) was determined, with its longest open reading frame (ORF)encoding a polypeptide of 413 aa. This polypeptide consists of a 21 aa N-terminal signal peptide,two chitin-binding domains different from those of other classes of plant chitinases, and a catalytic domain. Homology analysis illustrated that this cabch29 gene has 58.8% identity at the nucleotide level with the pRCH8 ORF probe and has 50% identity at the amino acid level with the catalytic domains of chitinase from bean,maize and sugar beet. Meanwhile,several kinds of cis- elements,such as TAT.A box, CAAT box, GATA motif, ASF-1 binding site, wound-response elements and AATAAA, have also been discovered in the flanking region of cabch29 gene.展开更多
文摘The 5’-region of the chitinase gene cabch29, derived from Brassica oleracea var. capitata, has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants. Different 5’-deletion fragments were linked to reporter geneβ-glucuronidase (GUS) as translational fusions, and the expression of these chimeric genes was analyzed in vegetative organs and tissues. Sequences up to -651 showed some basal GUS activity with nearly equal levels in wounded and intact tissues. The addition of further upstream sequences (-651 to -1284) enhanced expression level, and the expression driven by this fragment was inducible by a factor of two to three-fold by wounding. Histochemical analysis of different tissue from transgenic plants that contain cabch29 promoter-GUS fusion gene demonstrated woundinducible and tissue-specific cabch29 promoter activity in plants containing the 1308 base pair fragment. The location of GUS activity appears to be cell-specific, being highest in vascular cells and epidermal cells of stem, leaf and roots. Meanwhile, the temporal and spatial expression of cabch29-GUS fusion gene has been investigated. Among the different vegetative organs, a high level of GUS activity was observed in stem and a moderate one in roots;whereas, wounding stress led to a high level of GUS in stem and moderate one in leaf.
文摘Chitinase, which catalyzes the hydrolysis of the β-1,4-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin, is involved in inducible plants defense system. By construction of cabbage (Brassica oleracea var.capitata) genomic library and screening the library with pRCH8, a probe of rice chitinase gene fragment, a chitinase genomic sequence was isolated. The complete nucleotide sequence of the putative cabbage chitinase gene (cabch29) was determined, with its longest open reading frame (ORF)encoding a polypeptide of 413 aa. This polypeptide consists of a 21 aa N-terminal signal peptide,two chitin-binding domains different from those of other classes of plant chitinases, and a catalytic domain. Homology analysis illustrated that this cabch29 gene has 58.8% identity at the nucleotide level with the pRCH8 ORF probe and has 50% identity at the amino acid level with the catalytic domains of chitinase from bean,maize and sugar beet. Meanwhile,several kinds of cis- elements,such as TAT.A box, CAAT box, GATA motif, ASF-1 binding site, wound-response elements and AATAAA, have also been discovered in the flanking region of cabch29 gene.
