Project 4 - Genes and enzymes responsible for cell wall synthesis in cereals

1. Planned Outcomes/Aims

The broad aim of this project is to identify and characterize genes encoding the major enzymes involved in synthesizing individual components of cell walls.  An important component of the Project will be to develop robust procedures for the rapid analysis of gene function.

2. Contracted Outputs

The specific output from the Project will be to:

• Identify major genes involved in cell wall synthesis, in particular genes encoding polysaccharide synthases, hydrolases and structural proteins
  
  
• Functional analysis of major genes, using ‘loss-of-function’ and ‘gain-of function’ procedures.

3. Research Methodology

3.1 Identification of candidate genes and proteins

Bioinformatics: Available databases from Arabidopsis, barley and rice will be searched to identify sequences related to known plant polysaccharide synthases (eg CelA) and glycosyl hydrolases, or to glycosyl transferases from yeast and mammals.  We will use both linear alignments and hydrophobic cluster analyses to identify similar sequences in the databases.

3.2 Gene functional analysis

An important component of the Project will be to develop a range of systems for functional analysis of genes.  These will include gain-of-function systems for analysing cereal genes by transgenesis into Arabidopsis, tobacco and yeast.  Alteration in phenotype will be used in screening assays prior to subsequent more detailed assays of gene function.  Loss-of-function, or gene knockout, experiments will be performed by transgenesis into barley, pasture grasses (Lolium multiflorum), yeast and Arabidopsis, depending on the gene to be analysed, and by virus-induced gene silencing.

3.3 Proteomics:

Separation and identification of proteins by two-dimensional (2D) gel electrophoresis can be used for gene expression analysis at the protein level.  Between 1,000 and 2,000 proteins can be routinely separated on 2D gels.  There are a number of approaches but in summary, individual proteins are identified based upon a combination of amino acid composition, peptide mass spectrometry fingerprinting, N-terminal sequence, Mr by MS and pI data, which are then analysed through appropriate protein databases (eg SWISS-PROT) and OWL) and can also be correlated with nucleotide databases.

3.4 Protein expression systems:

Heterologous expression of cDNAs in bacterial, yeast, Pichia pastorius and baculovirus systems have been successfully employed to express mammalian Golgi-derived glycosyl transferases involved in glycoprotein and polysaccharide synthesis.  These proteins have been used for biochemical analysis of activity and specificity.  A similar approach will be taken to produce cereal polysaccharide synthases for analysis.

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