Isolation, characterisation and molecular evolution of the actin gene family of the New Zealand black-footed abalone, Haliotis iris.
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Molluscs are biologically important; they form a diverse taxon and the most well known members of the Lophotrochozoa, the most understudied Bilaterian group. Despite this importance, few studies have characterised molluscs at the genetic level. The New Zealand black-footed abalone Haliotis iris, an economically and culturally valued species, was chosen as a model for genetic characterisation of a molluscan actin gene family. In H. iris, actin is essential for the production of a large muscular foot, which forms the bulk of the body mass. The structure, expression and evolution of actin genes were investigated to elucidate the function of the actin gene family in H. iris. H. iris actin genes were isolated by PCR using gene subtype-specific primers designed from previously characterised partial H. iris actin sequences and generic primers derived from H. rufescens (Californian red abalone) and Cyprinus carpio (common carp). Three full length genes, H.irisA1, H.irisA2 and H.irisA3, and three partial genes, H.irisA1a, H.irisA1b and H.irisA1c, were isolated. The full length genes showed 82-95% sequence similarity to mollusc actin gene sequences deposited in GenBank. Sequence conservation confirmed the identity of the putative actin genes. The six genes contained a single variable length intron between codons 41 and 42. Intron lengths were: 174 nt, H.irisA1; 1,078 nt, H.irisA2; 581 nt, H.irisA3; 301 nt, H.irisA1a; 282 nt, H.iris1l and 229 nt, H.irisA1c. The predicted proteins of the full length genes contained 375 aa and lacked the second amino acid usually found in invertebrate actin proteins. Southern hybridisation of genomic DNA suggested there was a large gene family composed of at least eight members. The expression of H.irisA1, H.irisA2 and H.irisA3 in developmental stages and adult tissues was investigated by RT-PCR. RT-PCR demonstrated differential expression of H. iris actin genes during development and in adult tissues. H.irisA1 and H.irisA2 were expressed at low levels in fertilised eggs and blastula, with expression increasing in trochophore and veliger larvae. H.irisA3 was not expressed in eggs, but was faintly detected in blastula and highly expressed in trochophore and veliger larvae. H.irisA1 was ubiquitously expressed in adult gill, gonad, hepatopancreas, foot and mantle tissue, suggesting it may be a cytoplasmic-type actin. H.irisA2 was expressed in all tissues except the hepatopancreas, although low expression may not have been detectable by electrophoresis of RT -PCR products. Further characterisation is required to confirm whether H.irisA2 encodes a cytoplasmic-type actin. H.irisA3 was expressed at high levels in the muscular foot and mantle, and was faintly detected in gonad, suggesting it may be a muscle type actin. Phylogenetic analyses of H. iris actin genes and other molluscan actin genes available on GenBank were performed using maximum parsimony and maximum likelihood methods. Analyses suggested that haliotid actins can be divided into two orthologous clades, the first clade containing H.irisA1, H.irisA1a, H.irisA1b, H.irisA1c, H.virgA1a, H.virgA1b, H.virgA1c and H. Rufescens actin, the second clade containing H.irisA2, H.irisA3 and H. discus hannai actin. Orthology indicated that the last common ancestor of haliotids had at least two actin genes. Clustering of actin genes from individual haliotid species within orthologous actin gene clades suggests paralogy resulting from duplication of actin genes within species. Evidence for gene orthology between mollusc actin genes was found, but further characterisation of actin genes from other mollusc species is required to infer the evolutionary significance of orthology.