ABSTRACT
A wheat gene, denoted Sbe1, encoding a type I starch-branching enzyme (SBEI) was isolated from a genomic library and shown to comprise 14 exons distributed over a 5.7 kb DNA region. Analyses of kernel RNA by 5' rapid amplification of cDNA ends (5'-RACE) and reverse transcription-polymerase chain reaction (RT-PCR) demonstrated a considerable sequence variation at the 5' ends of SBEI gene transcripts. DNA sequence alignments between the 5'-RACE products and the Sbe1 genomic DNA indicated that the first two exons and first intron were differentially processed to generate three classes of the mature transcript. One form of the SBEI gene transcript in 12-day old kernels contained the exon I+II+III combination at the 5' end, whereas other forms differed by inclusion of intron 1 or exclusion of exon II sequences. RT-PCR analysis of Sbe1-uidA::nptII chimeric mRNA produced in transgenic wheat cultured cells confirmed that the isolated Sbe1 was able to produce all three forms of SBEI gene transcripts by alternative splicing of the primary mRNA. The variants of processed Sbe1 mRNA were potentially translated into N-terminal variants of the SBEI precursor with different transit peptide sequences.
Subject(s)
1,4-alpha-Glucan Branching Enzyme/genetics , Alternative Splicing , Triticum/genetics , Amino Acid Sequence , Base Sequence , DNA, Plant/chemistry , DNA, Plant/genetics , DNA, Plant/isolation & purification , Exons , Gene Expression , Genes, Plant/genetics , Genetic Variation , Introns , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Plant/genetics , RNA, Plant/metabolism , Recombinant Fusion Proteins/genetics , Seeds/genetics , Sequence Analysis, DNA , Tissue Distribution , Transcription, Genetic , Transformation, Genetic , Triticum/cytology , Triticum/enzymologyABSTRACT
The cauliflower mosaic virus 35S (35S) and the enhanced 35S (E35S) promoters fused with maize alcohol dehydrogenase (Adh1) intron1 or maize shrunken locus (sh1) intronl along with maize Adh1 and rice actin (Act1) promoters fused to their respective first introns were tested for transient expression of the E.coli ß-glucuronidase (gus) reporter gene in cultured barley (Hordeum vulgare L) cells. The plasmids, carrying the respective promoterintron combinations to drive the gus fused to nopaline synthase (nos) terminator, were introduced into cultured barley cells using a particle gun. The rice Act1 promoter with its first intron gave the highest expression of all promoter intron combinations studied. This was followed by the E35S promoter and no significant differences were observed between the other two promoters tested. The rice actin promoter is now being used to drive selectable marker genes to obtain stably transformed cereal cells.
