In this study, we selected two known pathogen-inducible cis-acting elements, F and E17, to construct synthetic pathogen-inducible promoters for analysis in transformed canola (Brassica napus L.). The synthetic promoter approach was used, which involved the insertion of dimers and combining two cis-acting elements (E17 and F) upstream of the minimal CaMV 35S promoter. Canola plants were transformed by three constructs, pGEE, pGFF, pGFFEE containing synthetic promoters (SP), SP-EE, SP-FF and SP-FFEE, respectively. Analyses of histochemical and fluorometric GUS expression indicated that synthetic promoters responded to fungal elicitors and phytohormone treatments. The SP-FF promoter showed high responses against methyl jasmonate and Sclerotinia sclerotiorum, while SP-EE demonstrated inducibility only in response to salicylic acid and Rhizoctonia solani. The SP-EE promoter similar to SP-FFEE, did not respond to S. sclerotiorum and methyl jasmonate. However, SP-FFEE was highly induced by R. solani elicitors and showed that the level of GUS expression was greater than that by either of E17 or F elements alone. These three synthetic promoters did not activate the expression of the reporter gene in response to cold, heat, UV and wounding.
Differential gene expression at the transcriptional level was examined as an initial step in the investigation of the P(i) starvation response of Brassica nigra suspension cells. Total RNA was extracted from 7-day old cells grown in media containing either no P(i), 1.25 mM or 10 mM Pi. In vitro translation was carried out using their respective poly(A)+ RNA isolates and the resultant polypeptides were separated on a high-resolution SDS-PAGE gel. Scanning densitometry identified four polypeptides (ca. 31.7, 32.3, 52.5 and 64.8 kDa) present only in the P(i)-starved samples. Screening by differential hybridization was performed on a cDNA library constructed from mRNA isolated from P(i)-starved cells. Probes prepared from mRNA from P(i)-deficient and P(i)-sufficient cells identified a number of clones representing mRNA species that were preferentially transcribed under P(i) deficiency. These phosphate starvation-responsive (psr) clones were placed into eleven groups as determined by cross-hybridization. Northern blots showed that the corresponding genes are inducible in both mild and severe P(i) starvation conditions. Preliminary sequencing identified one of the clones as being homologous to beta-glucosidases from several plant species. The possible role of beta-glucosidase during Pi starvation and the identities of the other psr genes are discussed.
We have previously isolated a phosphate starvation-response (psr) cDNA clone, psr3.1, from Brassica nigra which encodes a beta-glucosidase. Southern blots of Arabidopsis thaliana genomic DNA probed with the psr3.1 cDNA indicated that this gene exists as a single locus. A genomic library of A. thaliana was screened at high stringency to isolate the corresponding genomic clone. The resultant clone was coined psr3.2 because of its sequence divergence from isolated psr3.1 cDNA clones. Northern blotting with probes derived from the coding region of the genomic clone showed that this gene is expressed at high levels in P(i)-starved roots and the enhancement occurred within two days of growth in medium lacking P(i). The expression of this gene is repressed by heat shock and anaerobic conditions, and it is not significantly induced by high salinity, or by nitrogen or sulfur deprivation. Sequence analysis of the genomic clone revealed the existence of 13 exons interrupted by 12 AT-rich introns and it possessed a high homology with the B. nigra psr3.1 as well as various other beta-glucosidase genes from other species. Sequence similarity and divergence percentages between the deduced amino acid sequences of the psr3 clones and other beta-glycosidases suggests that they should be included along with two other Brassicaceae genes in a distinct subfamily of the BGA glycosidase gene family. The presence of an endoplasmic reticulum retention signal at the carboxy terminus indicates the likely cellular location of PSR3.2. The possible metabolic and regulatory roles of this enzyme during the P(i)-starvation response are discussed.
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