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

油籽废蛋白的利用及品质改良

基本信息

批准号：
12839006
负责人：
ADACHI Motayasu
金额：
$ 2.3万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12839006/
关键词：
cruciferin glycinin crystal structure physicochemical function transgenic plant

项目摘要

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.

本研究的第一个目的是用x射线晶体学方法测定油菜籽中十字花科植物素的晶体结构。由于天然十字花科植物素形成至少由三种亚基组成的异源六聚体，因此实际上难以制备其晶体用于X射线晶体学。因此，我们构建了E。大肠杆菌表达系统中制备重组前十字花科荧光素。利用从其发育种子中纯化的mRNA进行RT-PCR反应后获得cDNA。对不同表达条件的研究结果表明，重组前红花素在大肠杆菌中的表达量占总表达量的15%。大肠杆菌蛋白作为可溶性蛋白。利用该表达系统，制备了野生型前红花素和一些突变体，以制备晶体并表征其理化性质。突变形成盐内桥的氨基酸残基和C287残基。此外，修饰了前萤光素的可变区。另一个目的是生产在其种子中表达大豆球蛋白的工程油菜种子植物。由于大豆球蛋白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预期其改善效果。采用农杆菌法转化油菜种子的子叶，通过western blotting检测大豆球蛋白在种子中的表达。最终获得开花的F1代。