Isolation and evaluation of the sugarcane UDP-glucose dehydrogenase gene and promoter
Date
2006-12
Authors
Van der Merwe, Jennie
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : University of Stellenbosch
Abstract
The young internodes of sugarcane are ideal targets for altering metabolism, through genetic
manipulation, to potentially control known fungal diseases such as Smut or to increase sucrose
yields in these regions that are currently being discarded. At present, no regulatory sequences
that 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 promoter
targeted for isolation in this study regulates the expression of UDP-glucose dehydrogenase (EC
1.1.1.22), an enzyme which catalyses the oxidation of UDP-glucose to UDP-glucuronic acid, a
precursor for structural polysaccharides which are incorporated into the developing cell wall. A
strong correlation between the expression of UDP-glucose dehydrogenase and a demand for
structural polysaccharides in developing tissues could therefore be expected.
The first part of this study addressed the general practicality of promoter isolation from
sugarcane, a complex polyploid. A gene encoding UDP-glucose dehydrogenase was isolated
from 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 derived
amino acid sequence showed 88 – 98% identity with UDP-glucose dehydrogenase from other
plant species, and contained highly conserved amino acid motifs required for cofactor binding
and catalytic activity. Southern blot analysis indicates a low copy number for UDP-glucose
dehydrogenase in sugarcane. The possible expression of multiple gene copies or alleles of this
gene was investigated through comparison of sequences amplified from cDNA prepared from
different tissues. Although five Single Nucleotide Polymorphisms (SNP) and one small-scale
insertion/deletion (INDEL) were identified in the aligned sequences, hundred percent identity of
the derived amino acid sequences suggested the expression of different alleles of the same gene
rather than expression of multiple copies. The finding that multiple alleles are expressed to
provide the required level of a specific enzyme, rather than the increased expression of one
dominant 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 the
isolation of a developmentally regulated promoter was investigated. The contribution of UDP glucose dehydrogenase to pentan synthesis, as well as the expression pattern and subcellular
localisation 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 relative
contributions of glycolysis, the oxidative pentose phosphate pathway and pentan synthesis to
glucose 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 significant
contribution of UDP-glucose dehydrogenase to glucose oxidation in the younger internodes. In
addition, there was significantly (P=0.05) more radiolabel in the cell wall (fiber) component
when the tissue was labelled with [1-14C]-glucose rather than [6-14C]-glucose. This also
demonstrates a selective decarboxylation of glucose in position 6 prior to incorporation into the
cell wall and is consistent with a major role for UDP-glucose dehydrogenase in cell wall
synthesis in the younger internodes.
Expression analysis showed high levels of expression of both the UDP-glucose dehydrogenase
transcript and protein in the leafroll, roots and young internodes. In situ hybridisation showed
that the UDP-glucose dehydrogenase transcript is present in virtually all cell types in the
sugarcane internode, while immunolocalisation showed that the abundance of the protein
declined in all cell types as maturity increased. Results obtained confirmed that this enzyme
plays an important role in the provision of hemicellulose precursors in most developing tissues of
the sugarcane plant, indicating that UDP-glucose dehydrogenase was indeed a suitable target for
promoter isolation.
Lastly, the promoter region and first intron, located in the 5’-untranslated region (UTR) of this
gene, were isolated and subsequently fused to the GUS reporter gene for transient expression
analysis and plant transformation. Transient expression analysis showed that the presence of the
intron was essential for strong GUS expression. Analysis of stably transformed transgenic
sugarcane plants, evaluated in a green house trial, showed that the isolated promoter is able to
drive GUS expression in a tissue specific manner under these conditions.
Description
Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2006.
Keywords
Sugarcane, Promoters, UDP-Glucose dehydrogenase, Isolation, Dissertations -- Plant biotechnology, Theses -- Plant biotechnology, Sugarcane -- Genetic engineering, Glucuronosyltransferase., Dehydrogenases, Plant genetic transformation