Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco

Abstract

The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5' control region of the napA gene were fused to the reporter gene beta-glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between -1101 and -309 resulted in increased GUS activity. In contrast, deletion of sequences between -309 and -211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between -152 and +44. Further 5' deletion of the fragment to -126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between -148 to -120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to -152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.

hpRNA-Mediated Targeting of the Arabidopsis FAD2 Gene Gives Highly Efficient and Stable Silencing

“…1) targeted at the FAD2 ⌬12-desaturase gene. All three constructs used the rapeseed (Brassica napus) napin promoter (Stalberg et al, 1993), which directs high-level transcription of the transgenes specifically in the embryo and endosperm of the developing Arabidopsis seed. A proportion of plants transformed with each construct produced seed with significantly reduced ⌬12 desaturation levels, and there were noticeable differences in effectiveness among the three constructs (Fig.…”

“…This fragment is a 5Ј-truncated version of the full FAD2 coding region and was used to prevent any overexpression that might otherwise result from the sense transcription and translation of the full coding region. Expression was directed to the developing seeds by driving the constructs with the truncated version (Fp1) of the seed-specific napin promoter from rapeseed (Brassica napus; Stalberg et al, 1993). Three types of gene-silencing constructs were evaluated: a CS construct consisting of the 1,103-bp coding region fragment and its complete 3Ј-UTR (Fig.…”

“…All constructs were assembled in the plasmid vector pBluescript SKϪ (Stratagene, La Jolla, CA) before cloning into the binary vector pBI121 (CLONTECH Laboratories) as HindIII-SacI fragments, replacing the ␤-glucuronidase-containing HindIII-SacI region of pBI121. The truncated Fp1 promoter, containing sequences between Ϫ309 and ϩ1 (Stalberg et al, 1993), was cloned into the HindIII-EcoRV site of pBluescript. For the CS construct, a 1,103-bp fragment was amplified from the FAD2 cDNA clone (Okuley et al, 1994) using PCR primers At1 and At2 and cloned into the EcoRV-EcoRI site behind the Fp1 promoter.…”

Two Acyltransferases Contribute Differently to Linolenic Acid Levels in Seed Oil

“…However, there are differences during seed development. At the heart stage, napin is expressed in the cotyledons but not in the embryonic axis, and at the torpedo stage, napin is expressed more strongly in the cotyledons than in the embryonic axis (Stålberg et al, 1993). It has been suggested that, for wild-type C. sativa, DGAT1 activity is strongest in the cotyledons, whereas PDAT is more important in the embryonic axis than in the cotyledons .…”

Enhanced Seed Oil Production in Canola by Conditional Expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in Developing Seeds

“…Previous studies on the napA promoter indicated that the sequences between 2152 and +44 are essential for the seed-specific expression pattern in transgenic tobacco plants, and several ciselements upstream from 2152 were found to act as positive or negative regulatory motifs (Stålberg et al, 1993;Ellerströ m et al, 1996). We made similar napA promoter GUS reporter constructs that were stably transformed into Arabidopsis plants.…”