A new generation of plant promoters: characterisation of two mung bean promoters with constitutive expression in tobacco

Cazzonelli, Christopher Ian. (2002). A new generation of plant promoters: characterisation of two mung bean promoters with constitutive expression in tobacco PhD Thesis, School of Integrative Biology, The University of Queensland.

       
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Author Cazzonelli, Christopher Ian.
Thesis Title A new generation of plant promoters: characterisation of two mung bean promoters with constitutive expression in tobacco
School, Centre or Institute School of Integrative Biology
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor Jimmy Botella
Wayne Graham
Total pages 189
Language eng
Subjects L
270201 Gene Expression
620499 Primary plant products not elsewhere classified
Formatted abstract I report in this thesis the cloning and characterisation of a mung bean promoter (PGEL1) in transgenic tobacco. Long distance-inverse PCR was used to amplify 2468 bp of the promoter region of a mung bean (Vigna radiata) ACC synthase gene, AIM-l. The full length PGEL1 promoter including the 5'- untranslated region was fused to the β-glucuronidase (GUS) and luciferase (LUC) reporter genes and introduced into tobacco via Agrobacterium tumefaciens-mediated transformation. Analysis of tissues from R2 single copy transformants showed that PGEL1 drives constitutive reporter gene expression throughout plant development. Quantitative analysis revealed that transgenic plants harbouring the PGEL1 promoter had on average two- to four-fold higher protein and activity levels of the reporter gene than plants transformed with the CaMV35S promoter, nevertheless reporter gene transcript levels were lower for PGEL1. My results suggest the presence of a strong translational enhancer within the 5' untranslated region of the promoter. Deletion analysis of the PGEL1 promoter in stably transformed tobacco plants, revealed that a minimal core region of 86 bp from the start of transcription (plus the 5'- untranslated region) is capable of driving strong, constitutive reporter gene expression. Comparison of the PGEL1 and CaMV35S core promoter sequences showed approximately 75 % identity between the TATA and CAAT boxes. In addition, the cloning and characterisation of a mung bean calmodulin promoter (PGEL2) in transgenic tobacco is reported. Long distance-inverse PCR was used to amplify -1807 bp of the promoter region of a mung bean calmodulin gene, MBCaM-1. A fragment of the PGEL2 promoter (-1003 bp plus the 5'-untranslated region) was fused to the β-glucuronidase (GUS) reporter gene and introduced into tobacco. Analysis of tissues from 15 R1 transformants showed that PGEL2 drives GUS expression during seed germination and seedling development. It is likely that the PGEL2 promoter is developmentally regulated. The PGEL1 and PGEL2 promoters are 61 % identical in a region containing a large inverted and several direct repeat structures. Several auxin regulatory motifs were found in both promoters. In contrast with the AIM-1 and MBCaM-1 gene expression patterns observed in mung bean, no hormonal or stress-induced expression was observed in PGEL1or PGEL2 transgenic tobacco lines. A hypothetical model is presented that describes the regulation of the PGEL1and PGEL2 promoters in mung bean and heterologous systems.
Keyword Plant propagation
Nucleic acid hybridization

 
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Created: Fri, 24 Aug 2007, 17:50:34 EST