Quantification of individual low-molecular-weight glutenin subunit transcripts in developing wheat grains by competitive RT-PCR

Oligonucleotide primers and competitive templates were developed so that competitive reverse transcriptase-polymerase chain reaction (RT-PCR) techniques could be used to measure the accumulation of transcripts corresponding to individual low-molecular-weight glutenin subunit (LMW-GS) genes in developing wheat (Triticum aestivum) grains. Primers were based on LMW-GS genes previously cloned and sequenced from the cultivar 'Cheyenne' or on the N-terminal amino acid sequences of the major LMW-GSs accumulated in the cultivar 'Yecoro Rojo'. Competitive templates varied from the LMW-GS target genes by either a restriction site or a small deletion between the primer binding sites. Amounts of individual LMW-GS transcripts were quantified by titrating the amplification products produced from RT-PCR of RNA against amplification products produced from known amounts of the corresponding competitive template. Competitive RT-PCR analysis revealed that transcripts for the different LMW-GS genes varied only 4.5-fold in their levels of accumulation in developing 'Cheyenne' grains 15 days post-anthesis (DPA). Nucleotide sequencing of two of the amplification products revealed LMW-GS genes not previously described in 'Cheyenne'. Both of these genes encode proteins with a single cysteine residue in the repetitive region. Clones corresponding to these new sequences were not represented in a cDNA library prepared from 'Cheyenne' endosperm RNA even though the sequences were abundant in developing seeds and present in the RNA population from which the library was prepared. Competitive RT-PCR was shown to be a sensitive method for quantifying the expression of closely related members of complex gene families that cannot be readily distinguished by hybridization analysis. Such techniques should provide insight into the regulation of LMW-GS genes that are critical for wheat flour quality.