Utilization of waste protein from oil seed by improvement for its quality

本研究的第一个目的是用x射线晶体学测定菜籽中十字花素的晶体结构。由于天然十字花素是由至少三种亚基组成的异质六聚体，因此用x射线晶体学方法制备其晶体实际上是困难的。因此，我们构建了重组原十字花素的大肠杆菌表达体系。利用其发育种子中纯化的mRNA进行RT-PCR反应获得cDNA。通过各种表达条件的研究，重组原十字花素作为可溶性蛋白占大肠杆菌总蛋白的15%。利用该表达系统制备了野生型原十字花素和部分突变体，并对其进行了晶体制备和理化性质表征。形成盐桥的氨基酸残基和C287残基发生突变。此外，还对原十字花素的可变区域进行了修饰。另一个目标是生产在其种子中表达大豆甘氨酸的工程油菜种子植物。由于甘氨酸具有较好的功能和营养特性，因此将其添加到油菜籽中可以提高油菜籽的品质。采用农杆菌法对油菜种子子叶进行转化，并用免疫印迹法观察甘氨酸在种子中的表达。最终得到开花的F1代。

The first objective of this study was determination of the crystal structure of cruciferin from rapeseed by x-ray crystallography. Since native cruciferin forms heterogenic hexamer composed of at least three kinds of subunit, it is actually difficult to make its crystal for x-ray crystallography. Thus, we constructed the E. coli expression system to prepare recombinant procruciferin. The cDNA was obtained after RT-PCR reaction using mRNA purified from its developing seeds. As a result of investigation for various expression conditions, the recombinant procruciferin occupied 15% of total E. coli protein as a soluble protein. Using this expression system, wild type procruciferin and some mutants were prepared to make crystals and characterize their physicochemical properties. The amino acid residues forming intra salt bridge and C287 residue were mutated. In addition, variable regions of the procruciferin were modified. Another objective was the production of engineered rape seed plant that expresses soybean glycinin in its seed. Because the glycinin has better functional and nutritional properties, we can expect the improvement by adding the better properties to the rape seed. The cotyledon of the rape seed was transformed by agrobacterium method, and the expression of glycinin in the seed was observed by western blotting. Eventually, flowering F1 generation was obtained.