Molecular characterization of waxy mutants in hexaploid wheat
Matus-Cadiz, Maria Alejandra
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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 (Triticum aestivum 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.
DegreeDoctor of Philosophy (Ph.D.)
CommitteeScoles, Graham; Hucl, Pierre J.
Copyright DateDecember 1999