Isolation and evaluation of the sugarcane UDP-glucose dehydrogenase gene and promoter

[摘要] The young internodes of sugarcane are ideal targets for altering metabolism, through geneticmanipulation, to potentially control known fungal diseases such as Smut or to increase sucroseyields in these regions that are currently being discarded. At present, no regulatory sequencesthat specifically drive transgene expression in young developing sugarcane tissues are available.The objective of this study was therefore to isolate and evaluate such a sequence. The promotertargeted for isolation in this study regulates the expression of UDP-glucose dehydrogenase (EC1.1.1.22), an enzyme which catalyses the oxidation of UDP-glucose to UDP-glucuronic acid, aprecursor for structural polysaccharides which are incorporated into the developing cell wall. Astrong correlation between the expression of UDP-glucose dehydrogenase and a demand forstructural polysaccharides in developing tissues could therefore be expected.The first part of this study addressed the general practicality of promoter isolation fromsugarcane, a complex polyploid. A gene encoding UDP-glucose dehydrogenase was isolatedfrom a sugarcane genomic library. The gene contains an open reading frame (ORF) of 1443 bp,encoding 480 amino acids and one large intron (973 bp), located in the 5'-UTR. The derivedamino acid sequence showed 88 – 98% identity with UDP-glucose dehydrogenase from otherplant species, and contained highly conserved amino acid motifs required for cofactor bindingand catalytic activity. Southern blot analysis indicates a low copy number for UDP-glucosedehydrogenase in sugarcane. The possible expression of multiple gene copies or alleles of thisgene was investigated through comparison of sequences amplified from cDNA prepared fromdifferent tissues. Although five Single Nucleotide Polymorphisms (SNP) and one small-scaleinsertion/deletion (INDEL) were identified in the aligned sequences, hundred percent identity ofthe derived amino acid sequences suggested the expression of different alleles of the same generather than expression of multiple copies. The finding that multiple alleles are expressed toprovide the required level of a specific enzyme, rather than the increased expression of onedominant allele, is encouraging for sugarcane gene and promoter isolation.In the second part of the study the suitability of UDP-glucose dehydrogenase as a target for theisolation of a developmentally regulated promoter was investigated. The contribution of UDP glucose dehydrogenase to pentan synthesis, as well as the expression pattern and subcellularlocalisation of the enzyme in mature sugarcane plants was studied at the tissue and cellular level.Radiolabelling with positionally labelled glucose was used to investigate the relativecontributions of glycolysis, the oxidative pentose phosphate pathway and pentan synthesis toglucose catabolism. Significantly (P=0.05) more radiolabel was released as CO2 from [6-14C]-glucose than [1-14C]-glucose in younger internodes 3, 4 and 5, demonstrating a significantcontribution of UDP-glucose dehydrogenase to glucose oxidation in the younger internodes. Inaddition, there was significantly (P=0.05) more radiolabel in the cell wall (fiber) componentwhen the tissue was labelled with [1-14C]-glucose rather than [6-14C]-glucose. This alsodemonstrates a selective decarboxylation of glucose in position 6 prior to incorporation into thecell wall and is consistent with a major role for UDP-glucose dehydrogenase in cell wallsynthesis in the younger internodes.Expression analysis showed high levels of expression of both the UDP-glucose dehydrogenasetranscript and protein in the leafroll, roots and young internodes. In situ hybridisation showedthat the UDP-glucose dehydrogenase transcript is present in virtually all cell types in thesugarcane internode, while immunolocalisation showed that the abundance of the proteindeclined in all cell types as maturity increased. Results obtained confirmed that this enzymeplays an important role in the provision of hemicellulose precursors in most developing tissues ofthe sugarcane plant, indicating that UDP-glucose dehydrogenase was indeed a suitable target forpromoter isolation.Lastly, the promoter region and first intron, located in the 5'-untranslated region (UTR) of thisgene, were isolated and subsequently fused to the GUS reporter gene for transient expressionanalysis and plant transformation. Transient expression analysis showed that the presence of theintron was essential for strong GUS expression. Analysis of stably transformed transgenicsugarcane plants, evaluated in a green house trial, showed that the isolated promoter is able todrive GUS expression in a tissue specific manner under these conditions.

[发布机构] Stellenbosch University