关键词: 油体钙蛋白, RACE, 基因克隆, 同源简并引物, 序列分析, Northern杂交

Abstract:

Triacylglycerols (TAGs) is stored in seeds as a nutrient for germination and postgerminative growth of seedlings. TAGs storage is confined to discrete spherical organelles called oil bodies. Plant seed oil bodies comprise a matrix of TAGs surrounded by a monolayer of phospholipids embedded with abundant oleosins and some minor proteins. Three minor proteins, temporarily termed caleosin(Sop1), steroleosin(Sop2) and Sop3, have been identified in oil bodies of diverse species. With the rapid development of molecular biology, the more wide application of rape seed oil bodies on genetic engineering, the more attention to Sops 1–3. To reveal the biological function and provide scientific basis of application on plant genetic engineering for rape caleosin proteins, a gene BnClo1 encoding caleosin protein was isolated by homology-based candidate gene method combined with RT-PCR and RACE-PCR from B. nupus. The full-length cDNA clone (accession No. AY966447 in GenBank) comprised 1058 nucleotides consisting of a 36-nucleotide 5’-untranslated region, an open reading frame of 738 nucleotides, and a 284-nucleotide 3’-untranslated region. The open reading frame encoded a putative caleosin protein. The corresponding genomic sequence (1 676 nucleotides) of BnClo1 was also obtained by PCR cloning (accession no. DQ140380 in GenBank). Rapeseed genomic sequence of BnClo1 comprised six exons with five introns conservatively inserted in their coding regions. The splicing model of introns accords with the GT/AG rule in eukaryotes. In B. napus, BnClo1 was expressed in a spatially co-cordinated and temporally regulated manner. BnClo1 expression appeared to be highly regulated through embryogenesis. Northern blot demonstrated that BnClo1 mRNA was not detected in the earliest embryos, i.e. 20 DAF (day after flower) and presented in maturing rapeseeds at approximately 25 DAF. BnClo1 expression increased dramatically in the latter stages of embryogenesis, and this mRNA maintained a substantial level thereafter until the rapeseeds started to desiccate in a mode similar to oleosin mRNA. There was a single size class of BnClo1 transcript whose expression was regulated through embryogenesis. Semi-quantitative PCR showed that caleosin mRNA was only detected in the stem at two days after germination of rapeseeds. It is presumably revealed that BnClo1 is transcribed along with oleosin and steroleosin genes during seed maturation when oil bodies are actively assembled in diverse species. The deduced polypeptide of the rapeseed clone comprises 245 amino acid residues with molecular weight of 28.1 kD. Caleosin protein documented GenBank with accession no. AAY40837 might be an amphipathic protein associated with rapeseed oil bodies. Hydropathy plot and secondary structure analysis suggested that caleosin is comprised of three distinct structural domains: an N-terminal hydrophilic domain with a single Ca²⁺-binding EF-hand motif, a central hydrophobic anchoring domain of some 40 residues, and a C-terminal hydrophilic domain with conserved protein kinase phosphorylation sites. In addition, the central hydrophobic domain of caleosin also contained a proline-rich region with the potential to form a proline knot motif of the type that appears to be important in the lipid-body targeting. A potential amphipathic alpha helix, e.g. residues 92–114 of caleosin might play a role in their binding both of single layer membrane and lipid bodies. Caleosin protein closely integrating with lipid bodies through embryogenesis plays an important role in the biogenesis process of oil bodies. With calcium-binding, the physiological function of caleosin protein may be responsible for decomposition of oil bodies and mobilization of triglycerides during seed germination.

Key words: Caleosin, RACE, Gene cloning, Homology-Degenerate primer, Sequence analysis, Northern blotting