油菜油体钙蛋白基因BnClo1的克隆和表达

Cloning and Expression of BnClo1 Gene from Brassica napus

应用同源序列克隆法设计同源简并引物,结合RT-PCR和RACE-PCR技术,从甘蓝型油菜中分离克隆了编码28.1kD油体钙蛋白(caleosin)的基因BnClo1。其全长1058bp的BnClo1 mRNA(GenBank中序列号为AY966447)包含完整的开放阅读框和3′末端Poly(A)尾巴结构,染色体DNA结构上含6个外显子和5个内含子。Northern杂交结果表明,油菜中BnClo1在种子形成中期开始丰富表达,在种子形成后期,即种子开始脱水成熟时期,高量稳定地表达。半定量PCR结果显示,BnClo1在油菜种子吸水膨胀后前2d的茎中明显表达。证明在油菜种子发育期间,BnClo1对mRNA的转录表达是由胚胎发育来调控的,具有显著的时空特性,并与油体的形成和积累密切有关。推测的caleosin蛋白为245个氨基酸残基(GenBank中序列号为AAY40837)组成的两性蛋白质,主要含3个结构域即由N末端1～16位氨基酸残基组成的α-螺旋和17～61位氨基酸残基组成的强亲水性的随机卷曲构成的N-末端亲水性结构域;由80～120位氨基酸残基组成的中间疏水性结构域和C-末端亲水性结构域。N-末端亲水性结构域包含一个潜在的结合Ca2+的EF-手结构。中间疏水性结构域包含一个潜在的脯氨酸-结(proline-knot)模体,在92～114位氨基酸残基组成的α-螺旋跨膜区域,推测在caleosin蛋白与单层磷脂层和油体锚定结合上及增加种子油体的稳定性上起着重要的作用。

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 （Sopl）, 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 BnClol 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 BnClol was also obtained by PCR cloning （accession No. DQ140380 in GenBank）. Rapeseed genomic sequence of BnClol 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, BnClol was expressed in a spatially co-cordinated and temporally regulated manner. BnClol 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 clays 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 Ca^2＋-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.