Annual Progress Report on Malting Barley Research March, 2002

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Tissue-specific gene products from the above differential display PCRs were used by Dr.
Andris Kleinhofs (Washington State U.) as probes to find the corresponding Morex
genomic clones in a BAC library. We have also adopted additional options, the inverse
PCR procedure and PCR-based gene walking. Before the promoter region can be
subcloned from a BAC genomic clone, it is necessary to determine the 5’ end of the
mRNA. Determination of the 5' (and, where needed, 3') end sequences of mRNAs are
performed by Rapid Amplification of cDNA ends (RACE) using the GeneRacer TM kit
(InVitrogen), as described by the supplier. These techniques were described in the 2001
AMBA progress report.
Molecular cloning and vector construction - Vectors for stable expression of antifungal
genes were prepared from the Ubi/GUS+Ubi/BAR vector pAHC25 by replacing the GUS
gene. For stable tissue-specific expression of Hth, a barley promoter/Hth fusion will
replace the Ubi/GUS fusion in pAHC25. Transient expression studies of promoter
activity are conducted with the pAHC17 vector, which contains the Ubi promoter but has
no selectable marker. We have modified this vector so that the Gfp gene occurs
downstream from the Ubi promoter, allowing constitutive expression of Gfp
(pAHCSGFP vector). To test our promoters, the Ubi promoter is removed and replaced
with lemma/palea or pericarp promoter candidates. The modified vectors are used to
transform barley through the biolistic (gene gun) approach. The particle delivery and
tissue culture is performed essentially as described by Wan and Lemaux (1994).
Screening of putative transformants was conducted using PCR on leaf DNA extracted by
the CTAB procedure.
Transient expression analysis of promoters and antifungal testing - The putative
promoter regions typically include up to 1500 bp of upstream sequence (with respect to
the mRNA 5' end). In addition to the upstream region, regulatory sequences are
sometimes found in the mRNA 5' UTR and in the first intron. Initially, the upstream
region are subcloned from BAC clones or from Gene Walker PCR products and ligated in
front of the Gfp gene in our pAHCSGFP vector (above). Particle bombardments of
spikelets from emerged spikes, pericarps of intact seeds (after removing lemma and
palea), and leaves are conducted and monitored for up to 72 h with short-wave blue light.
Sequencing of cDNAs and transcribed regions of BAC clones is used to determine the
location of introns. We have now encountered two genes, Lem2 and EpiLTP, in which
introns near the 5’ end may influence expression. In these cases, additional tests are
conducted by joining 5' UTR and/or first intron sequences with the upstream sequence, in
which the first intron is ligated between the promoter region originally tested and the Gfp
coding sequence.
In order to detect Hth gene expression in transformants, northern blots and RT-PCR were
conducted. Total RNA was isolated from leaves by the GT procedure. First strand
cDNA was made by priming a reverse transcriptase reaction with oligo d(T). For RT-
PCRs, reactions were then conducted with an upstream Hth primer and a downstream
NOS primer. As an internal control, actin primers were used simultaneously. Since
much more mRNA was produced in Hth2 transformants, northern blots were used to
analyze expression.