【作者】 柯涛；

【导师】 马向东；

【作者基本信息】 河南农业大学 ， 微生物学， 2003， 硕士

【摘要】 引导蛋白质功能进化常用的方法是模拟和加速蛋白质基因自然重组的进程，即在蛋白质的基因中引进随机突变。因此，蛋白质基因体外随机突变的方法影响着引导蛋白质功能进化的效果。本研究采用5-溴脱氧尿苷三磷酸(5-BdU)部分取代脱氧胸苷三磷酸(dTTP)，在PCR的过程中对克隆的野油菜黄单胞菌的α-淀粉酶基因进行了体外诱变，诱变结果表明：5-BdU浓度越高，诱变越强；浓度越低，诱变越弱。当5-BdU浓度为dTTP的0.1％时，可以得到最多的正诱变结果。本研究用LBSP鉴别培养基直接筛选技术，仅一个循环就获得了大量无酶活的重组质粒和酶活提高了20倍的突变基因。进行第二个循环后筛选得到酶活提高40倍的突变基因。分别挑选出无酶功能的、酶活是野生型基因1/4的、和酶活是野生型的40倍的三个突变基因和野生型基因进行测序和同源性分析，结果显示：此淀粉酶基因的DNA序列全长1808bp，由DNA推测的α-淀粉酶前体蛋白由475个氨基酸组成，前体α-淀粉酶的N-端为信号肽序列，长度为35个氨基酸，具有典型的信号肽序列的特征。与嗜水气单胞菌(Aeomonas hydrophila)的α-淀粉酶氨基酸序列有70％同源性。三个突变基因的突变位点共13个，转换、颠换、添加和缺失四种突变类型都有。其中，无酶功能基因突变位点最多，导致全长编码区内5个氨基酸突变，低酶活和高酶活基因各两个突变位点。其中低酶活基因编码区内一突变位点导致氨基酸序列的提前终止。高酶活编码区位点突变导致C-端序列变化和终止子的后移 本诱变方法克服了用碱基类似物在体内诱变由于核酸复制酶等的校正作用而造成诱变无效的难题，为基因的诱变找到了一条新途经。更多还原

【Abstract】 Directed evolution is becoming increasingly important in biological research. It is used to probe the relationship between the structure and activity of proteins; to define the roles of particular proteins and protein assembles in the cell; to determine the logic and order of molecular events during differentiation and morphogenesis; to improve the activity of enzyme, antibiotics and so on.The routine method for inducing protein function evolution is imitating and speeding protein gene mutation. In this research α -Amylase gene was mutated in vitro by PCR with dNTP in which deoxythymidine triphosphate (dTTP)was partially replaced by 5-bromo-2’-deoxyuridine-5’-triphosphate(5-BdU). The higher the concentration is, the stronger its inducing ability is in a certain range. The experimental results showed that the suitable ratio for the 5-BdU and dTTP was about 1:1000. In this thesis the LBSP identification medium screening techniques was used. Only after one cycle, large quantities no-function recombination plamid and enzyme activity increasing 20 times recombination plasmid were obtained. In the second cycle the mutation type of enzyme activity increased 40 times was found.We sequenced three type mutation genes. One is no-function. The second amylase activity is 1/4 of the native amylase. The other amylase activity is 40 times of native amylase. The non-amylase activity gene has 9 base changes, resulting in 5 amino acid changes from the native enzyme. The low-enzyme activity gene has 2 base changes, resulting in short amino acid sequence with native enzyme. The high-enzyme activity has 2 base changes, resulting in long amino acid sequence with native amylase.This inducing method resolved the problem of non-effective induction as in base analogue induction. And the method we used provide a new measure for this kind of work.更多还原