Recent research has focused on the molecular characterization of null waxy (Wx), 'Wx-A1b, Wx-B1b', and 'Wx-D1b', alleles that produce no detectable Wx proteins in the endosperm starch of allohexaploid wheat (<i>Triticum aestivum</i> L.; 2n = 6x = 42; AABBDD). The major objectives of this thesis were to (1) isolate and characterize a Wx wheat cDNA and (2) to identify aberrant 'Wx' transcripts encoded by the null 'Wx-A1b' allele of CDC Wx2, a waxy hexaploid wheat line, which result in an absent Wx-A1 protein (~59 kD). In the first study, a cDNA library prepared from developing wheat kernels (cv. Fielder) was screened using a homologous PCR-digoxigenin labeled wheat cDNA probe. A 2.2 kb cDNA clone denoted GBSSIMMI (Accession no. Y16340) was sequenced and identified as encoding a Wx-D1 protein. The deduced amino acid sequence showed 94% similarity with a wheat Wx-A1 peptide, 96% similarity with a wheat Wx-B1 peptide, and 100% identity with two wheat Wx-D1 peptides. A 33-nucleotide deletion, encoding 11 amino acids (AMLCRAVPRRA), was detected within the GBSSIMMI cDNA relative to a previously isolated wheat cDNA (accession no. X57233). Complementation analysis using a glycogen synthase deficient 'E. coli' strain and an 'in vitro' starch synthase assay did not indicate that GBSSIMMI encoded a functional Wx-D1 protein. In the second study, two sister lines CDC Wx2 and CDC Wx6 were obtained by crossing lines Bai-Huo (carries null 'Wx-D1b' allele; lacks Wx-D1 protein) and Kanto 107 (carries null 'Wx-A1b' and -'B1b' alleles; lacks Wx-A1 and -B1 proteins). Waxy protein profiling, amylose concentration determinations, Northern blot analysis, and reverse transcriptase PCR (RT-PCR) analysis were conducted. Ten RT-PCR derived cDNA clones were selected from each genotype and characterized by DNA sequencing analyses. The waxy phenotype of CDC Wx2, lacking Wx-A1, -B1, and -D1 proteins and possessing a reduced amylose concentration ~4%), was associated with dramatically reduced levels of a 2.4 kb 'Wx' transcript when compared to the higher levels in a wildtype control line. DNA sequencing of clones from Kanto 107 and CDC Wx2 characterized two types of aberrant 'Wx' transcripts, one containing intron 1 and another containing introns 1 and 4. Intron 1 in both types of aberrant 'Wx' transcripts contained a premature stop codon which resulted in the translation of a truncated Wx protein ~4 or 11 kD). Analysis of CDC Wx6, lacking Wx-B1 and -D1 proteins and possessing a reduced amylose concentration (~14%) failed to reveal aberrant ' Wx' transcripts, suggesting that the RNA defects in this study were not responsible for the absence of the Wx-B1 or -D1 proteins. Thus, the aberrant Wx transcripts were encoded by the null 'Wx-A1b' allele. The presence of a premature stop codon in the 'Wx' transcripts encoded by the null 'Wx-A1b' allele explained the absence of the ~59 kD Wx-A1 protein in CDC Wx2 and its parental line Kanto 107.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-10212004-002025 |
| Date | 01 January 2000 |
| Creators | Matus-Cadiz, Maria Alejandra |
| Contributors | Scoles, Graham, Hucl, Pierre J. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-10212004-002025 |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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