Stem specific promoters from sorghum and maize for use in sugarcane
Thesis (MSc (Genetics. Institute of Plant Biotechnology))--Stellenbosch University, 2008.
Sugarcane (Saccharum spp.) is an important crop which is cultivated worldwide for the high sucrose content in its stem. Conventional plant breeding has proven to be very successful over the years with regard to the enhancement of yield characteristics but due to the exhaustion of genetic potential in the commercial sugarcane germplasm recent progress has been slow. Genetic engineering seems to be a more attractive approach to enhance sucrose content and pest resistance in the stems but requires appropriate transgenes and suitable promoter. A promoter is essential to drive the transcription of a gene and is therefore critical to the success of transgenic approaches in sugarcane crop improvement. A negligible number of strong stem-specific promoters is available for use in sugarcane and this is one of the major limitations to genetic engineering. The goal of this project was to isolate a stemspecific promoter from maize and sorghum to drive stem-specific transgene expression in sugarcane. The approach used was to source promoters from non-sugarcane grass species with less complex genomes to simplify isolation and possibly counteract silencing. A cDNA sequence (SS) (EST clone, Accession number AW746904) from sugarcane was shown by Northern and Southern analysis to be stem-specific and to have an appropriately low copy number. The SS gene sequence was not expressed in the leaves of maize, sorghum or the sugarcane cultivars and prominent expression was observed only in the stems of the sugarcane hybrids N19 and 88H0019. The SS gene sequence was used to isolate its upstream regions from a Lambda genomic library of maize (Zea mays) and a sorghum (Sorghum bicolor) Bacterial Artificial Chromosome library (BAC). Of the four sorghum and six maize clones obtained in this study, a 4500 bp maize genomic DNA fragment (λ5) was sub-cloned in three fragments into separate pBluescript vectors using the ‘forced’ cloning approach for sequence and database (BLASTN) analysis. This revealed the complete SS gene sequence (975 bp), the promoter and a 300 bp intron region. A stretch of DNA sequence from nucleotides 664-3194 from the maize clone 5 sequence was designated the maize5-pro. Following sequence alignment of the maize and sugarcane promoter regions, significant sequence identity (68%) was observed between nucleotide 1675 and 3194 in maize and nucleotide 1506 and 2947 in sugarcane. The distance between the putative TATA-box and the TSS for this promoter (30 bp) was found to fall within the expected range of 32± 7 bp. The promoter region was analysed for possible cis-acting regulatory elements and revealed several promoter elements that are common in other plant promoters. The comparisons made between the putative transcription factors in maizepro-5 and the sugarcane promoter show that both promoter sequences are very similar as they share ten of the same transcription factors. However, the transcriptional factors WBOX, SRE and SP8BFIBSP8BIB are unique to the maize5-pro and the TAAG motif to the sugarcane promoter. Primers were designed with appropriate restriction sites and the promoter and intron (2850 bp) region was amplified by PCR (Polymerase chain reaction). The amplified fragment was fused inframe to the GUS reporter gene encoding β-glucuronidase to produce a transformation test vector. This will be used in future work to assess the functionality of the promoter through the production of stable transformants in which GUS activity can be measured in a range of tissues.