4. Results
4.2. Grain proteome analysis of accessions from the Oregon Wolfe Barley mapping
4.2.3. Cloning and overexpression of candidate proteins
In the comparative proteome analysis of accessions from the OWB mapping population, two proteins, cytosolic 6-phospho-gluconate dehydrogenase and glucose/ribitol dehydrogenase homolog, were identified showing a higher abundance in salt tolerant cultivars. Their biological function is discussed later on, but it is very likely that they contribute to salt tolerance. The test the latter, a more detailed characterization was initiated by cloning and overexpression studies.
To obtain barley full-length clones, the nucleotide sequence of the identified proteins was blasted against the barley EST database CR-EST (http://pgrc.ipk-gatersleben.de/cr-est/index.php, release v1.5). This tool provides sequence and annotation data from crop EST projects at the IPK Gatersleben and contains about 40,280 barley consensus sequences.
Clones with the highest similarity to the proteins identified by the proteome approach were ordered and the sequences were verified by DNA sequencing. Comparison at the nucleotide level revealed a 99 % identity of the clone HS09N23 when compared to barley glucose/ribitol dehydrogenase homolog mRNA (gi:633889) and a 88 % nucleotide identity of the clone
Ubiquitin promotor (Ubi1p)
α-Gliadin promotor (AGp)
Binary vector (p6d35S)
Pa-G HS09N23 tNOS
879 bp
SmaI SmaI
Pa-G HI05J23 tNOS
1440 bp
SmaI SmaI
Pa-G HS09N23 tNOS
879 bp
SmaI SmaI
Pa-G HI05J23 tNOS
1440 bp
SmaI SmaI
Ubi-int HS09N23 tNOS 879 bp
EcoR I Sal I
Ubi-int HI05J23 tNOS 1440 bp
EcoR I Sal I
Ubi-int HS09N23 tNOS 879 bp
EcoR I Sal I
Ubi-int HI05J23 tNOS 1440 bp
EcoR I Sal I
Pa-G/Ubi-int HS09N23 tNOS
SfiI SfiI
LB RB
Pa-G/Ubi-int tNOS
SfiI SfiI
LB HI05J23 RB
879 bp
1440 bp Pa-G/Ubi-int HS09N23 tNOS
SfiI SfiI
LB RB
Pa-G/Ubi-int tNOS
SfiI SfiI
LB HI05J23 RB
879 bp
1440 bp
HI05J23 when compared to Oryza sativa cytosolic 6-phosphogluconate dehydrogenase mRNA (gi:30313360). Based on these high similarities both clones were selected for overexpression in the salt sensitive barley cv. Golden Promise.
Each clone was fused to the promoter of the maize ubiquitin for constitutive expression (Ubi1p) or to the promoter of the wheat α-gliadin for grain-specific expression (AGp) (Figure 33). This approach should allow the functional characterisation of candidate proteins not only overexpressed in the whole plant, but also confined to grain-specific cell types.
Figure 33: Schematic diagrams of expression vectors used for barley transformation. Clones coding for glucose/ribitol dehydrogenase homolog (HS09N23) and cytosolic 6-phosphogluconate dehydrogenase (HI05J23) were fused to the maize ubiqutin promoter or to the wheat α-gliadin promoter, respectively.
Expression cassettes were subcloned into a binary vector for Agrobacterium tumefaciens-based transformation. The vectors were kindly provided by Drs Götz Hensel (Lig154(pNOS+PaG) containing AGp) and Axel Himmelbach (pUBI-ABM containing Ubi1p), IPK and DNA Cloning Service, Hamburg (p6d35S).
The resulting expression cassettes were finally cloned into a binary vector (p6d35S). The Plant Reproductive Biology group at IPK performed the A. tumefaciens-based gene transfer to immature barley embryos resulting in stable transgenic plants. About 100 immature embryos were used for transformation and subsequent selection on hygromycin containing media.
Between 15 and 26 T0-lines were generated per construct and verified by PCR for the hygromycine resistance gene. Transgene copy numbers were assessed by hybridisation of HindIII digested DNA with a (32P)-dCTP-labelled probe for hygromycine resistance gene (Figures 34 and 35). In order to analyse the transgene expression of constructs driven by the ubiquitin promoter, total RNA from leaf tissue was hybridised with the respective
gene-specific probe (Figure 34). In leaves of Golden Promise wild type plants, no transcript for glucose/ribitol dehydrogenase homolog was detected and the expression level of 6-phosphogluconate dehydrogenase transcripts was also very low in this tissue. When compared to wild type plants, most transgenic plants showed an accumulation of transcripts for glucose/ribitol dehydrogenase homolog and 6-phosphogluconate dehydrogenase, respectively, although no apparent phenotype was observed.
A
B
Figure 34: Southern and Northern blot analysis of ubiquitin promotor-diven expression cassettes for glucose/ribitol dehydrogenase homolog (AGL1p6d35S-Ubi1p::HS09N23) (A) and 6-phosphogluconate dehydrogenase (AGL1p6d35S-Ubi1p::HI05J23) (B). DNA and RNA were extracted from leaf material.
Digested DNA was hybridized with a probe for hygromycine resistance gene, RNA blots were hybridized with the respective gene-specific probe. Transcripts for glucose/ribitol dehydrogenase homolog and 6-phosphogluconate dehydrogenase were more abundant in transgenic barley as compared to wild type (WT) plants.
AGL1p6d35S-Ubi1p::HS09N23
1 WT 2 3 4 5 7 9 10 12 13 14 15 WT 17 18 19 20 23 25 26 Southern Blot Analysis
Northern Blot Analysis
1 WT 2 3 4 5 7 9 10 12 13 14 15 17 18 19 20 23 25 26
AGL1p6d35S-Ubi1p::HI05J23
1 2 3 WT 4 5 6 7 9 10 11 12 13 14 15 WT 17 1 2 WT 3 4 5 6 7 9 10 11 12 13 14 15 17
Southern Blot Analysis
Northern Blot Analysis
The transcript levels of barley lines transformed with constructs driven by the α-gliadin promoter were evaluated by RNA analysis of mature grains (Figure 35).
A
B
Figure 35: Southern and Northern blot analysis of the α-gliadin-diven expression cassettes for glucose/ribitol dehydrogenase homolog (AGL1p6d35S-AGp::HS09N23) (A) and 6-phosphogluconate dehydrogenase (AGL1p6d35S-AGp::HI05J23) (B). DNA was extracted from leaf material and RNA was extracted from mature grains. Digested DNA was hybridized with a probe for hygromycine resistance gene, RNA blots were hybridized with the respective gene-specific probe. Transcripts for glucose/ribitol dehydrogenase homolog were more abundant in transgenic barley as compared to wild type (WT) plants.
mRNA abundance of 6-phosphogluconate dehydrogenase in transgenic lines was not altered to the same extent.
Here, several plants failed to develop grains, which is probably due to the transformation process, rather than to transgene expression. For that reason Northern Blot analysis was
AGL1p6d35S-AGp::HS09N23
Southern Blot Analysis
2 3 4 WT 5 7 10 11 12 13 15 17 18 19 20 21 23 24 25 26
Northern Blot Analysis
WT 4 7 10 11 12 19 20 23 24 25
AGL1p6d35S-AGp::HI05J23
1 2 3 4 WT 5 6 7 8 9 10 11 12 13 14 15 17 18 19 22 Southern Blot Analysis
Northern Blot Analysis
WT 3 4 6 7 8 9 14 18 19 22
performed for a smaller number of transgenic plants as compared to the ubiquitin promoter-driven transgenic plants. A high abundance of glucose/ribitol dehydrogenase homolog trancripts was found in non-transgenic grains of the cultivar Golden Promise, indicating a grain-specificity of this gene. When the gene was fused to a α-gliadin promoter for overexpression of glucose/ribitol dehydrogenase homolog, transgenic lines were identified with a higher transcript level as compared to wild type plants. In wild type plants, the expression of 6-phosphogluconate dehydrogenase was lower in mature grains as compared to leaf tissue. The transcript accumulation was only moderately enhanced by overexpression of this gene under control of the grain-specific α-gliadin promoter.
Currently, the next generation of transgenic lines is growing and grains of this generation will be characterized in future stress experiments.
4.3. Proteome analysis of accessions from the Steptoe Morex mapping