The higher plant stearoyl-acyl carrier protein desaturase (SACPD, EC 1.14.99.6) is a soluble, nuclear encoded enzyme found in plastids responsible to catalyze the conversion of stearoyl-ACP (18:O ACP) to oleoyl-ACP (18: 1 ACP). The SACPD mRNA transcripts were previously found to be abundant during early seed embryogenesis in the rapeseed (Brassica napus) for storage lipid synthesis. A partial SACPD cDNA fragment (250 bp) was cloned using total RNA isolated from B. napus cv. Cyclone dark germinated seedling cotyledons by employing the reverse-transcription polymerase chain reaction (RT-PCR) technique. After that, a digoxigenin-labeled probe (SDPCR250) was made by PCR based on the cloned fragment. Then, a cDNA library was constructed using mRNA isolated from the above tissues and screened...[ Read more ]

The higher plant stearoyl-acyl carrier protein desaturase (SACPD, EC 1.14.99.6) is a soluble, nuclear encoded enzyme found in plastids responsible to catalyze the conversion of stearoyl-ACP (18:O ACP) to oleoyl-ACP (18: 1 ACP). The SACPD mRNA transcripts were previously found to be abundant during early seed embryogenesis in the rapeseed (Brassica napus) for storage lipid synthesis. A partial SACPD cDNA fragment (250 bp) was cloned using total RNA isolated from B. napus cv. Cyclone dark germinated seedling cotyledons by employing the reverse-transcription polymerase chain reaction (RT-PCR) technique. After that, a digoxigenin-labeled probe (SDPCR250) was made by PCR based on the cloned fragment. Then, a cDNA library was constructed using mRNA isolated from the above tissues and screened by SDPCR250. Four categories (type I to type IV) of positive cDNA clones encoding four full-length isoforms of SACPD precursor polypeptide were found. Four typical cDNA clones were then chosen and sequenced completely showing more than 90% identity to each other. Furthermore, after screening a B. napus cv. Bridger genomic library using SDPCR250, three positive clones (λBRSDG06, λBRSDG33 and λBRSDG36) were identified. They were predicted to encode for type II, III and IV cDNA-like sequences based on their sequence differences around the putative translational start region. The 5' flanking regions from the above 3 plaques were cloned and characterized, designated as SDpl, SDp2 and SDp3 respectively. The sequence of SDpl was found to be distinct from SDp2. However, SDp2 sequence was predicted to be highly homologous to SDp3 based on preliminary restriction mapping. Nevertheless, some cis-acting DNA motifs (such as abscisic acid responsive element, late pollen-specific regulatory sequences) were found in both SDpl and SDp2. By linking SDpl and SDp2 with β-glucuronidase reporter gene, transient expressions using biolistic were done to study their promoter activities in seedling cotyledons and microspore-derived embryos.