The Positional Cloning and Expression Analysis of the Rice Dull Gene, Dull8 (Du8)
Date Issued
2011
Date
2011
Author(s)
Pu, Chieh-han
Abstract
Rice quality improvement is an important issue in rice breeding. Starch is a major influence in rice quality because it composes 90% of rice grain. Though starch metabolism has been highly studied, so far non-sufficient evidence can be applied to explain the role of diverse starch textures in rice cultivars. Rice starch regulatory genes had been revealed and studied in the past few decades for better understanding in starch texture regulations. Dull genes are considered as amylose synthesis regulatory genes independent to Waxy. This study analyzed an EMS induced dull rice mutant line, CNY921391-du8, which exhibits low amylose content with opaque grain. Previous research had finely mapped Du8 between molecular markers CH0422 and CH0426 on chromosome 4, encompassing 12 candidate genes. Sequence analysis revealed a single point mutation of G to A in the gene encoding tetratricopeptide (TPR) repeat domain containing protein. The SNP mutation occurs on 3’ splice site of intron 11, leading to three truncated transcripts in the SNP of CNY921391-du8, compared to a single transcript of TK 8. In addition, three other du8 alleles were also found in other low amylose content mutants. The TPR domain mediates protein-protein interactions and multiprotein complexes assembly. Amino acid sequences of Du8 were highly conserved among higher plants such as Sorghum bicolor, Arabidopsis thaliana, and Ricinus communis. Real-time PCR analysis of Du8 mutant during rice grain developing stages showed that the Du8 expression pattern varied with the two dull genes, Du1 and Du3, indicating different regulation of Du8. Starch synthesis gene showing no significant variance expression patterns include OsAGPL3, OsBEIIa, OsISA2, and OsISA3. Genes with significant expression variance but not specified patterns include OsAGPL1, OsAGPL4, OsAGPS2a, OsSSI, OsSSIIa, OsSSIIb, OsSSIIc, OsSSIIIa, OsSSIIIb, OsSSIVa, OsSSIVb, and OsDPE2. Genes that were significantly reduced throughout the whole seed developing stages were OsAGPL2, OsAGPS1, OsAGPS2b, OsGBSSI, OsGBSSII, OsBEI, OsBEIIb, OsISA1, OsPUL, OsDPE1, OsPHOL, and OsPHOH. In addition, OsGBSSI, OsBEI, and OsPUL showed similar expression patterns as the isolated dull gene, Du8, and the expression levels were severely reduced in the overall rice grain developing stages of the du8 mutants. These result indicated the possibility role of Du8 in regulating these starch synthesis genes mentioned above.
Subjects
dull
tetratricopeptide repeat domain containing protein
cryptic splicing
positional cloning
real-time PCR
Type
thesis
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