School of Botany - Theses
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ItemCharacterization of pistil-specific genes encoding proline-rich proteins from Nicotiana alata(University of Melbourne, 1990)This thesis describes investigations into the structure and expression of a group of pistil-specific genes encoding proline-rich proteins in Nicotiana alata. Two cDNA libraries prepared from pistil mRNA of N. alata were screened with a probe encoding a carrot extensin. Low stringency hybridization and washing conditions were used to enhance the chance of detecting DNA sequences encoding high proportions of proline residues but which might be distinct from extensin. Three classes of cDNA clones, corresponding to three proline-rich protein genes (PRP1, PRP2 and PRP3), were isolated from the pistil cDNA libraries. None of these cDNA clones represented a full-length transcript Two genomic clones (PRP3g5 and PRP3g12) corresponding to the PRP3 gene were also characterized. The PRP3g5 clone probably represents a pseudogene of the PRP3 gene family, whereas the clone PRP3g12 is likely to correspond to a functional PRP3 gene. A typical TATA box and several putative polyadenylation signal are present in the sequence of the clone PRP3g12, and the ATG initiation codon of this gene is found to be located in a context which is probably optimum for translation initiation. The clone PRP3g12 does not contain any introns. The three proline-rich proteins predicted from the partial cDNA clones and the genomic clone are all rich in proline residues (29% for PRP1,49% for PRP2 and 30% for PRP3). In addition, PRP1 is also rich in asparagine (14%), and PRP3 is rich in serine (19%). These three genes have a similar codon usage preference for praline: more than 60% of all proline residues are coded by CCA. The 114 amino acid sequence of PRP2 can be divided into two domains; one (nucleotides 1-210) contains 5 repeats of (Pro)4-Ala interspersed with 4 repeats of the pentapeptide Gln-Leu-Pro-Ile-Arg, and the other is composed mainly of tandemly reiterated (Pro)2-5-Gly-Tyr repeats. The first 23 of the 151 amino acid residues in the PRP3 protein predicted from the genomic clone PRP3g12 are hydrophobic and resemble to a signal peptide. The PRP3 resembles extensin in containing six Ser-(Pro)4 repeats which are separated, in most cases, by a Ser-Pro dipeptide. Southern analysis under stringent conditions showed each of the three PRP cDNAs hybridized to a number of restriction fragments for each of the four restriction enzymes used. This indicates that each of these PRP genes belongs to a small multigene family. The carrot extensin genomic clone (pDCSA1) hybridized to a set of restriction fragments different from any of the three PRP genes, suggesting that another gene more homologous to the carrot extensin than any of the three PRP genes is present in N. alata. The hybridization pattern of the clone pDC5A1 is relatively simple, indicating a low copy number of the extensin gene in the genome. Northern analysis indicated that the three PRP genes are different from each other and from the extensin gene. All the three PRP genes are effectively pistil- specific, in contrast to the extensin gene which is expressed in all tissues tested although the level, number and size of the extensin transcripts differs in different tissues of N. alata. The PRP3 gene is developmentally regulated and its maximum expression correlates with the maturity of the pistil. After wounding, the expression of the extensin gene is increased in all tissues tested, whereas, the mRNA levels of both PRP2 and PRP3 decreased in wounded pistils. Wounding had no detectable effect on the expression of the PRP3 gene in stem and leaf tissues, while a small PRP2 transcript (smaller than the major transcript expressed in wounded pistils) is induced in the same tissues after wounding.