Isolation and characterisation of P450 gene(s) in barley (Hordeum vulgare)

Linh Nguyen

In plants, P450 enzymes encoded by P450 genes play a central role in numerous biosynthetic pathways, such as the production of secondary metabolites, stress responses and disease resistance. This thesis reports upon the utilization of molecular biology techniques to study P450 gene(s) in barley (Hordeum vulgare L.). Using several combinations of degenerate primers, a large number of barley P450 gene fragments were cloned and sequenced from two commercial varieties, Chebec and Harrington. Among 247 isolated sequences, twenty six percent were homologous to genes of known function. The abundance of these sequences differed between the two cultivars. Variations in the motif sequences of the cloned genes were also found between these two cultivars. In addition to the cloned fragments, twenty-two putative barley P450 encoding genes were identified from 24,000 cereal sequences in the International Triticeae EST Cooperative (ITEC) database by homology search. Among these Expressed Sequence Tag (EST) sequences, a full-length P450 sequence was selected for further investigation in this study. This novel P450 gene, CYP72A39, was expressed at a very early vegetative stage, but no expression was detected at the reproductive stage. Comparison of expression profiles of this gene and “digital expression” databases confirmed that this gene was homologous to several cereal EST clones with tissue-specific transcripts responding to various environmental stimuli, such as stresses and disease. Among these, many transcripts in barley were obtained from stressed tissues at the vegetative stage, and two transcripts in wheat (Triticum aestivum L.) were expressed after being challenged by barley powdery mildew pathogen (Blumeria graminis f. sp. hordei L.). This suggested that CYP72A39 may play a defence role in the barley seedling. The secondary structure of CYP72A39 was investigated in a comparative analysis using a computational approach. However, attempts to express CYP72A39 in a heterologous system and functional assays of the native protein in barley did not give decisive results, due to the disordered structure of the native protein and limitations of the current method. Screening the 3’ UnTranslated Region (3’UTR) of this gene in 158 genotypes of domesticated, landrace and wild barley revealed two haplotypes, which differed by a 12 base indel positioned between two transversions. The presence of both haplotypes in wild and cultivated barley suggests this polymorphism predates the domestication of barley. This indel was mapped to the long arm of chromosome 6H, less than 10 centi-Morgans (cM) from the gene encoding resistance to powdery mildew in barley (B. graminis). A comparison between haplotype diversity and powdery mildew resistance data for over 102 genotypes showed a weak link between the ‘long’ haplotype and resistance, while the ‘short’ haplotype was associated with susceptibility. There was no evidence for a strong correlation between haplotype and quality type (malt or feed); however, more malting varieties had the long haplotype, suggesting a possible association with some attributes in modern malting barley. There was no evidence for association with other characteristics such as geographic origin, growth habit or row number. Phylogenetic relationship of the CYP72A39 to other CYP72 members was also investigated.

Isolation and characterisation of P450 gene(s) in barley (Hordeum vulgare)

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Isolation and characterisation of P450 gene(s) in barley (Hordeum vulgare)

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