【作者】 丁勇；

【导师】 甘莉；

【作者基本信息】 华中农业大学 ， 植物学， 2006， 硕士

【摘要】 除了丰富蛋白质oleosin，油菜种子油体中还存在有三种微量蛋白质，caleosin，steroleosin和Sop3。编码两亲性oleosin蛋白的基因已经在甘蓝型油菜中被分离克隆。本论文应用同源序列克隆法设计同源简并引物，结合RT-PCR和RACE-PCR等技术首次从甘蓝型油菜中成功分离克隆了编码caleosin蛋白的基因，分析了caleosin蛋白和caleosin基因的结构特征。从甘蓝型油菜各种发育阶段的种子中提取mRNA，用甘蓝型油菜caleosin基因特异性探针进行Northern杂交研究了caleosin基因mRNA的表达特征和表达转录体带型。Caleosin基因被成功插入到表达载体pTYB12中，并在大肠杆菌ER2566中完成了原核表达。本论文主要的研究结果如下。 提出了一种从油菜种子中有效地提取高质量总RNA的方法——热CTAB-LiCL联用法。提取的RNA样品具有较高的纯度和完整性。变性胶电泳后为28S和18SrRNA二条完整的带型，A260／A280比值在1.8-2.0之间，得率高约为1mg_（RNA）／g_（样品）。提取的RNA完整无降解，并克服了多酚、多糖及次生物质等的干扰，完全适合RT-PCR、全长基因的克隆及Northern Blotting等分子生物学实验的要求。 甘蓝型油菜caleosin基因mRNA序列登陆GenBank，序列号为AY966447。mRNA全长序列为1058bp，包含了完整的开放阅读框和3’末端Poly（A）尾巴结构。mRNA开放阅读框区域为第37位至774位之间的738bp的序列片断，能够编码245个氨基酸残基的caleosin蛋白。Caleosin基因mRNA序列已经达到了3’最末端。但是通过序列分析没有发现caleosin基因mRNA序列5’端具有帽子结构。这有两种可能：一种是5’RACE-PCR并没有扩增到caleosin基因mRNA序列5’最末端；另一种是甘蓝型油菜caleosin基因转录mRNA后序列5’端本身就没有加上帽子结构。 甘蓝型油菜caleosin基因组DNA序列长为1676bp，登陆GenBank，序列号为DQ140380。Caleosin基因在基因组序列结构上具有6个外显子和5个内含子。内含子的剪接符合真核生物内含子剪接的GT／AG规则：即内含子的5’端总是GT和3’端总是AG。 甘蓝型油菜caleosin基因编码的油体结合蛋白caleosin在GenBank上的序列号为AAY40837。Caleosin蛋白由245个氨基酸残基组成，包含了20种常见氨基酸，分子量为28.096kD，等电点5.81。蛋白质性质分析表明甘蓝型油菜caleosin蛋白为两性蛋白质，带电荷氨基酸和疏水性氨基酸的含量比较高且基本相同。亲水性图和二级结构分析表明caleosin蛋白主要可以划分为三个结构域：由N末端1-16位氨基酸残基组成的α—螺旋（Alpha helix）和17-61位氨基酸残基组成的强亲水性的随机卷曲（Random coil）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有一个潜在的脯氨酸-结（Proline-Knot）更多还原

【Abstract】 Besides abundant oleosin, three minor proteins, caleosin, steroleosin, and Sop3 are present in rapeseed oil bodies. A gene encoding an amphipathic protein named oleosin associated with oil bodies has been cloned from Brassica nupus. In this study, a gene encoding caleosin protein was first isolated by homology-based candidate gene method combined with RT-PCR and RACE-PCR from B. nupus. Caleosin gene was subsequently over-expressed in Escherichia coli. Structural characterization of caleosin protein and caleosin gene was described in the paper. Total RNA was extracted from maturing rapeseeds at various days after flowering （daf）. After blotting, the membrane was hybridized with a ³²P-labeled specific probe containing the full-length sequence of caleosin mRNA from B. nupus. Northern hybridization was subsequently used to determine expression pattern of caleosin mRNA accumulation in rapeseed. The paper’s main study results are as follows:A highly efficient method of isolating total RNA from rapeseed was developed in this study. Polyphenols, polysaccharides and protein, which are rich in rapeseed cells, could be effectively removed. With the method, two clear bands of 28S rRNA and 18S rRNA isolated from rapeseeds could be found by formaldehyde denaturalization agarose gel electrophoresis. It was proved that RNA was intact, without degradation. The ratio of A260/A280 is 1.8-2.0. The results showed that the total RNA acquired from rapeseed by this method had higher purity and quality and could be directly used in molecular biological research, like RT-PCR, full-length cDNA cloning and Northern blotting analysis, etc.An incomplete cDNA clone presumably encoding rape caleosin protein was obtained by homology-based candidate gene method, and the upstream sequence and downstream sequence of the clone was completed by RACE-PCR. The full-length cDNA clone （accession no. AY966447 in GenBank） was linked by ligation of the three overlapping fragments. The cDNA fragment comprises 1,058 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 encodes a putative caleosin protein. This encoded protein and its gene have not been reported in rape.The corresponding genomic sequence （1,676 nucleotides） of this putative caleosin gene was also obtained by PCR cloning （accession no. DQ140380 in GenBank）. Rape genomic sequence of caleosin gene comprises six exons with five introns conservatively inserted in their coding regions. The splicing model of introns accords with the GT/AG更多还原