Cloning, characterization, and expression analysis of starch branching enzyme cDNA from wheat (Triticum aestivum cv. Fielder)
Nair, Ramesh Bhaskaran
The major objectives of this study were to analyze the spatial and temporal distribution of starch branching enzyme (SBE) activity in wheat (Triticum aestivum L. cv. Fielder), to clone and characterize Sbe cDNA and to analyze the expression of genes encoding the SBE isoforms. An assay based on SBE-mediated unprimed synthesis of á-1,4 glucan by phosphorylase a was standardized to detect SBE activity in the protein extracts of wheat tissues. Analysis of SBE activity in the various parts of a wheat plant revealed highest activity in developing kernels. Within a kernel, the embryo axis exhibited higher SBE activity than either the endosperm or scutellum. During kernel development, SBE activity was highest in kernels 10 days post-anthesis (DPA). As the kernels matured beyond 10 DPA SBE activity decreased. Kernel dry weight, starch and amylose concentration increased rapidly until 20 DPA whereas the moisture concentration decreased during this period. Quantitative image analysis of starch granules suggested two distinct granule types (type A and type B that varied in size and shape) were present in kernels after 15 DPA. Image analysis of starch granules isolated at the different stages of kernel development suggested that granules formed immediately after anthesis developed as type A granules while the type B granules were initiated at 12 to 15 DPA. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to isolate partial cDNA that corresponded to Sbe1 and Sbe2 transcripts expressed in wheat kernels. Northern blot analysis, using the isolated Sbe1 and Sbe2 cDNA detected two transcripts of 4.9 and 2.8 kb in kernels and a single transcript of 2.8 kb in leaves and roots. The 2.8 kb Sbe transcript detected in kernels corresponded in size to that of Sbe transcripts reported from other plants. However, the 4.9 kb transcript detected in wheat kernels seems to represent a unique Sbe transcript. During kernel development, Sbe1 and Sbe2 genes were differentially expressed. Northern blot analysis also revealed that 4.9 and 2.8 kb Sbe1 transcripts were expressed in kernels of three hexaploid wheat cultivars, two tetraploid wheat cultivars and in a barley cultivar. A cDNA library constructed from 12 day old kernels was screened to isolate full length Sbe1 and Sbe2 cDNA clones. The longest clone isolated using the Sbe1 cDNA probe contained a truncated cDNA that represented the 4.9 kb transcript expressed in wheat kernels. RT-PCR analysis suggested that the 4.9 kb transcript was formed as a result of duplication of Sbe1 mRNA sequences. A full-length cDNA (2970 bp) that corresponded to the 2.8 kb Sbe2 transcript expressed in wheat kernels was also isolated. The translated region of the cDNA predicted a 823 amino acid primary product with a molecular mass of 91.4 kDa. The isolated cDNA clones were able to restore BE activity in BE deficient E. coli indicating that the isolated cDNA coded for a functional BE. Southern blot analysis suggested that the Sbe1 and Sbe2 transcripts were encoded by more than one gene.
DegreeDoctor of Philosophy (Ph.D.)
CommitteeScoles, Graham; Devine, Malcolm
Copyright DateApril 1997
wheat -- physiology