Submitted to: Plant Science
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/12/1999
Publication Date: N/A
Citation: Interpretive Summary: Molecular biology offers the promise of novel ways to improve fiber properties of cotton. We have previously isolated and characterized a lipid transfer gene Ltp6 that is specifically expressed in cotton fiber cells. Plant genes are directed or controlled by DNA sequences called promoters. In the present research the DNA sequence of the promoter for the Ltp6 gene and a series of five modifications (deletions) to the promoter were made and linked with a reporter gene, GUS, which provides a blue color in the cells where it is active. These were then transformed into tobacco leaf disks and in plants with some of the modifications the trichomes (fiber cells on the leaf) were blue which indicated that the promoter was active in trichomes. One construction Ltp-447-GUS was only active in the trichome cells. The unmodified promoter was at least 1,000 times weaker than the 35S general promoter from the cauliflower mosaic virus. Modifications of the promoter were made in a sequential manner until no promoter activity was detectable. Thus, we identified those parts of the promoter necessary for activity in trichome cells. Tobacco trichomes and cotton fiber cells may thus share some common regulatory elements for tissue-specific expression.
Technical Abstract: A cotton (Gossypium hirsutum) genomic clone (1.7-kb DNA insert) harboring the lipid transfer gene Ltp6 specifically expressed in fiber cells had been previously isolated and characterized. By using PCR amplification, the 447 bp Ltp6 promoter and a series of 5' deletions of the promoter were generated and cloned into a pBI101 plasmid upstream of the GUS (B-glucuronidase) reporter gene. These constructs were introduced into Agrobacterium tumefaciens LBA4404, and leaf disks of tobacco (Nicotiana tabacum L.) were transformed with Agrobacterium tumefaciens LBA4404 carrying various promoter-GUS pBI101 plasmids. Histochemical analyses of the transgenic tobacco seedlings indicated that the Ltp6 promoter (nt - 447 to -1, undeleted) directed GUS expression only in trichomes (hair cells). Fluorometric GUS assays showed that the promoter activity of the undeleted Ltp6 promoter was at least 1,000 times weaker than that of the 35S promoter of califlower mosaic virus (CaMV). Sequential deletions of the promoter gradually decreased the expression level of the GUS gene. No GUS activity was observed when the 5' deletion of the Ltp6 promoter reached to nt -86, which removed the putative CAAT and TATA promoter elements.