Sucrose phosphate synthase gene family isolation and characterisation in sugarcane

The sucrose phosphate synthase (SPS) genes in sugarcane are encoded by a large multigenic family. Previous work on sugarcane and other grasses suggests the presence of five SPS gene families and includes evidence of multiple sub-families within the Poaceae. In sugarcane (Saccharum officinarum), the source of 70% of the world’s sugar, sucrose accumulation in the stem is dependent on SPS activity. Accumulating evidence of the role of SPS indicates that SPS is a valuable marker for the accumulation and the biosynthesis of sucrose. Therefore it would be desirable to increase the knowledge and have a better understanding of the genetic structure of SPS. In order to unravel the complexity of this multigene family, isolation and characterisation of these genes in sugarcane is crucial. Probes were designed independently for each gene family, through a series of bioinformatics assisted procedures and were confirmed by PCR amplification, cloning and sequencing as being family specific. These probes were DIG labelled and hybridised to leaf and stem cDNA libraries to detect the SPS genes. Screening of the cDNA libraries yielded the full-length gene of SPS gene family member II. These same probes were also utilised in the screening of a BAC library, which yielded over 30 corresponding positive BAC clones. In order to isolate the remaining expressed SPS genes, a 3’ and 5’ Rapid Amplification of cDNA Ends (RACE) was performed on stem tissue cDNA from an elite sugarcane variety Q117.