Cloning, Site-specific Mutation And Functional Analysis Of Chemical-inducible Promoter

Chemically inducible systems that control expression of native gene or transgene in plant have potential applications in gene function determination and genetic engineering. Most of plants, when attacked by avirulent pathogens, usually produce a hypersensitive response (HR) at the locus attacked and develop a systemic acquired resistance (SAR) throughout entire plant by expressing a set of defense genes including pathogenesis-related (PR) genes. One of these PR genes, PR-la, responds not only to avirulent pathogen, but also to some chemicals, such as SA (salicylic acid) and BTH (benzothiadiazole) etc., and the responding ability to the inducers is controlled by its promoter.In order to use the responding ability to the inducers of PR-la promoter, two fragments, IP1 (900 bp) and IPs (603 bp) were cloned from tobacco genomic DNA by Polymerase Chain Reaction (PCR) with primers designed according to the sequence reported in Genbank. Sequence analysis of the amplified 900 bp fragment indicated that the cloned sequence had 99% of homology to the known sequence. Its transcription start site, TATA box and consensus sequence "TGAC" conserved in PR genes were identical to those of PR-la promoter. By artificially changing A to C at -137 bp site upstream from transcription start point of cloned promoter, two site-mutation promoters, IPMs (603 bp) and IPM1 (900 bp) were created. Furthermore, by inserting "anther box" element to the mutated area of two site-mutation promoters, another two promoters, IPMas and IPMal, were created. In order to study the chemical-inducible capacity of wild and modified PR-la promoters, a coding sequence of GUS (|3-glucuronidase) gene was fused to their downstream, and the chimeric genes were cloned into pBin!9-based plant expression vector. These vectors were introduced into tobacco cells by Agrobacterium twnefaciens mediation and transgenic plants were obtained and verified by Southern Blot.GUS expression in the transgenic tobacco was induced by exogenous applied SA and BTH, and the expression pattern showed that:1. 900 bp promoter-directed GUS expression was highly induced by SA and BTH, while the 603 bp promoter, whether mutated or not, did not respond to SA and BTH induction, which indicated that the element in response to SA and BTH lied among 575~872 bp from transcription start site.2. Specific mutation from A to C at -137 bp site upstream from transcription start point had no effect on the inducibility of the promoter in response to SA and BTH.3. Inserting the anther box into a given area of the chemical-inducible promoter resulted an anther-specific expression of GUS upon the induction of chemical inducers, such as BTH.4. BTH had a higher efficiency to induce GUS expression than SA indicated by GUS staining strength and had no phytotoxicity to plant within effective concentration.5. GUS expression induced by BTH was systemic and had been detected in leaf, sepal, anther and mature pollen of transgenic tobacco plants, while induction effects of SA was spatial-temporally limited.6. Systemic GUS expression was induced 15 days after BTH treatment and the GUS activity was still detectable 25 days later.7. Induced GUS expression was BTH dose-dependent under 1.2 mM.