T-DNA tagging In Brassica carinata with a promoterless gus : NPTII gene fusion vector
Date
1998-04-01
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Degree Level
Doctoral
Abstract
An efficient system for or 'Agrobacterium'-mediated transformation of Brassica carinata was used together with a promoterless gus::nptII gene fusion to isolate putative promoter sequences. Cotyledonary petioles were transformed using the promoterless gene fusion construct. Only transformation events in which the promoterless gene fusion had integrated downstream from plant regulatory sequences were expected to produce viable tissue under kanamycin selection. Forty-two transgenic plants were recovered. Transformation efficiency was approximately 0.6%. Regenerated plants were screened for GUS expression in different tissues and organs by histological and fluorometric assays. Tissue-specific GUS expression was detected (stigmas, seed coat, leaf edges and vascular tissue) in some plants, while strong constitutive GUS expression was detected in others (based on GUS histological assays). Using subgenomic libraries, putative promoter fragments were isolated from the plants which exhibited GUS expression in stigmas, leaf edges and constitutively. A putative promoter fragment from a plant which exhibited GUS expression only in the stigma was fused with the gus gene and reintroduced by Agrobacterium -mediated transformation into B. napus, B. carinata, Arabidopsis' and tobacco . GUS expression was observed in the stigma of B. napus but not in ' B. carinata'. In Arabidopsis and tobacco GUS expression. was not tissue specific (weakly constitutive or restricted to two or more tissues). The 3' DNA sequence (15 kb) flanking the gus::nptII insert in the plant with GUS expression in the stigma was also isolated using a subgenomic library. A gene for a cytochrome P450 like protein was discovered on the minus DNA strand of the 3' sequence with a start codon approximately 6.5 kb from the T-DNA left border.
Description
Keywords
plant science, T-DNA tagging, brassica carinata, marker genes, promoterless GUS::NPTII gene fusion, GUS expression, transgenic plants
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Plant Sciences
Program
Plant Sciences