【作者】 曾庆平；

【导师】 郭勇；

【作者基本信息】 华南理工大学 ， 发酵工程， 1997， 博士

【摘要】 为了发展我国的抗衰老和抗艾滋病药物生物技术，研究了大蒜SOD的高效诱导与协同调控以及野生稻TI基因的体外重组与异源表达。结果表明，物理应激与化学应激均可提高培养细胞中的SOD活力。其中，冷胁迫与热休克诱导时细胞的SOD活力分别提高67％和30％，变温冷胁迫和间隙热休克诱导细胞的SOD活力则分别是对照的1.72倍和1.71倍。经水杨酸诱导的细胞，其SOD活力比对照提高84％，水杨酸与冷胁迫以及热休克互作诱导的SOD活力为对照的2.08倍和3.65倍。这项成果为SOD的规模化发酵生产奠定了基础，并为培养和诱导条件的进一步优化做好了准备。研究发现，物理或化学应激首先激发胞外H₂O₂的瞬时释放，然后再诱导胞外POD活力的持续升高。在氧化应激初期，胞内外POD活力呈现“外升内降”的动态变化。自由基引发剂与自由基清除剂互为拮抗，二者从正负两个方面影响调节着胞外POD活力。甲基紫精诱导的胞外POD活力高于对照，而硫脲诱导的胞外POD活力则低于对照。水杨酸能抑制CAT活力而使H₂O₂水平升高，半胱氨酸则能诱导CAT活力并使H₂O₂水平降低。GSSG和NADP可能具有提高H₂O₂水平的作用，而GSH和NADPH可能具有降低H₂O₂水平的作用。胞外H₂O₂水平可能受POD控制，而胞内H₂O₂水平可能由CAT调节。因此，H₂O₂可能是植物细胞氧化应激信号传导分子。 野生稻TI基因的序列分析表明，它不仅与栽培稻TI高度同源（氨基酸水平的同源性高达74％），而且与所比较的其它植物TI和某些种子贮藏蛋白（特别是一些具有重要生理功能的植物蛋白质）部分同源，并且与动物及人TI的活性位点周围的氨基酸残基的种类相似。同时，根据种子贮藏蛋白超基因家族提出了种子贮藏蛋白系统分类法。另一方面，通过对克隆的野生稻TI基因的体外重组和遗传转化，已将其成功地转移到E.coli菌株中，并经IPTG诱导和SDS-PAGE检测观察到外源TI基因表达的融合蛋白。经细胞裂解粗提取，发现外源基因表达产物对胰蛋白酶具有抑制活性。这项成果将有助于今后深入进行蛋白质结构与功能的研究，并为TI基因的蛋白质工程改良与基因工程生产和育种打下基础。更多还原

【Abstract】 For developing national pharmaceutical biotechnology of anti-ageing and anti-AIDS disease, induced overproduction and cooperative regulation of garlic SOD as well as in vitro recombination and heterologous expression of wild rice TI gene were investigated. Results have shown that both physical and chemical stress were capable of effectively increasing their SOD activity in cultured cells, in which, the cellular SOD activity induced by chilling and heat shock were raised for 67% and 30%, respectively, and the SOD activity in cells induced by the disturbed chilling and interrupted heat shock were as 1.72 and 1.71 folds as the control. In cells induced by salicylic acid, the SOD activity was increased by 84%, and by the interaction of salicylic acid with chilling or with heat shock, elevated by 2.08 or 3.65 times, when compared with the control. These results would provide a basis for the large-scaled fermentative production of SOD, and might be a prerequisite for the further optimization of cell culture and induction. It was also found that physical or chemical stress initially invoked a transient release of H2O2 into the medium, and then stimulated a continuous elevation of extracellular POD activity. During the striking early phase of oxidative stress, both activities of extra- and intracellular POD were dynamically changed as a mode of "outside-elevation and inside-decline". The free radical generators, in a positive pattern, and free radical scavengers, in a negative pattern, functioned as antagonists, and thereby affected and regulated levels of H2O2. The induced extracellular POD activity by methyl viologen was higher, but by thiourea was lower, than the control.. While salicylic acid raised the level of H₂O₂ by inhibition of CAT activity, cysteine declined it by the induction of this enzyme. GSSG and NADP might act as positive effectors for controlling the H2O2 level, and GSH and NADPH, however, the negative effectors. The extra- and intracellular levels of H₂O₂ might be individually controlled by POD and CAT. As a conclution, H₂O₂ an oxidative stress signal transduction molecule in plant cells might be convinced.The sequence analysis of the wild rice TI gene have indicated that not only a extensive homology to cultivated rice was found （the homology was 74% at the amino acid level）, but partially homologies to other compared plant TI and some other seed storage proteins （especially certain physically impotant plant proteins） were disclosed. The similarity of wild rice TI with animal and human TI in the kind of amino acid residues flunked the active site of TI was discovered. A standard classification system of seed storage proteins was also suggested according to the更多还原