Characterisation of genes expressed in various tissues of PEA (Pisum sativum L.); correlation of genotype and phenotype

Abstract

Genes encoding representatives of two subfamilies from the vicilin storage protein family in pea (Pisum sativum L.) have been sequenced and characterised. One, encoding convicilin, shows that this protein differs from vicilin by the insertion of a hydrophilic region near the N-terminus. The transcription start point has been determinedand the pattern of expression in developing seeds elucidated. By theexpression of this gene in tobacco, the specific polypeptide product ofthe gene was identified as a minor component of convicilin , with a lowerMr than the major species . The other gene subfamily investigated wasThat encoding the vicilin 47,000 Mr polypeptide. A gene and a cDNA weresequenced, and the gene found to diverge from the cDNA in the 3" regionof the coding sequence. No product from this divergent gene could beidentified.A member of the legumin gene family (legK) was sequenced and found to be inactive due to a mutation of the start codon. The region of DNAencoding the start codon of this gene was amplified by polymerase chainreaction from a pea line in which the gene was known to be active . Thesequence of this revealed the presence of a normal start codon. Two dimensional protein gels were run with seed extracts from these twolines , and the product of (legK) demonstrated by its occurrence in theline with the functional gene. A method for the extraction and purification of the major pea root protein was established. The protein was shown to have a Mr of 16,000 and not to be susceptible to cleavage by cyanogen bromide. Partial amino acid sequence data was obtained from the purified protein .A differential screen of cDNA from purple and green poddedvarieties of pea was conducted, and differentially expressed cDNAsisolated . The nature of the expression of these cDNAs was studied in thetwo lines and the cause of instability in the purple podded phenotypeinvestigated . A genomic library was constructed from the purple poddedline . Two genes were selected by the differentially expressed cDNAs andtheir DNA sequences determined. A gene encoding a pectinestera- likesequence, and the pod expressed cDNA used to select it , were found to betwo members of a small multigene family in pea. The second gene selectedproved to encode a protein containing two distinct domains; the N-terminal region being of a repetitive proline-rich nature and the C-terminal region being hydrophobic and cysteine rich . This gene waspresent as a single copy in the pea genome and its expression appearedto be linked to pigmentation.