钝齿棒杆菌天冬氨酸激酶基因的克隆和序列分析

Cloning and Sequence Analysis of Aspartokinase Genes From Corynebacterium crenatum

运用PCR方法 ,从野生型钝齿棒杆菌株 (Corynebacteriumcrenatum)AS1 5 4 2及具有AEC抗性的突变株CD945染色体上分别扩增出天冬氨酸激酶 (AK)基因 (ask) ,构建了重组质粒。核苷酸序列分析表明 ,C .crenatumAS1 5 4 2AK基因与C .crenatumCD945相比 ,第 1 1 99位的碱基由T变为C ,引起酶蛋白β亚基第 80位氨基酸从亮氨酸变成脯氨酸。该氨基酸的突变在蛋白结构上位于ACT结构域内 ,该区受赖氨酸调控。C .crenatumAS1 5 4 2的AK基因的编码区核苷酸序列与C .glutamicum、C .flavum及B .lactofermentum相比 ,同源性分别为 97 2 3 %、97 5 5 %和 97 6 2 % ,酶蛋白氨基酸序列的同源性分别为 99 76 %、99 5 2 %和 99 76 %。但在AK基因的启动子上游序列部分与其它棒杆菌相比有较大差异。

Aspartokinase genes ( ask ) from wild\|type Corynebacterium crenatum AS1.542 and an AEC\|resistant mutant Corynebacterium crenatum CD945 were cloned and sequenced.Analysis of ask sequence shows a exchange in a single base pair at position 1199 from T to C,leading to an amino acid change in the β subunit of Aspartokinase.Leu 80 in the wild\|type is converted to Pro 80 in the feedback\|resistant enzyme.The substitution is located in ACT domain,a region regulated by concentration of lysine.The ORF sequence of ask from C.crenatum AS 1.542 shows homologies of 97 23%,97.55% and 97.62% to those from \%C.glutamicum,C.flavum\% and B.lactofermentum .And the amino acid sequence deduced from ORF displays homologies of 99.76%, 99 52% and 99 76%,respectively.But there is much variation in the upstream sequence of C.crenatum AS 1 542 ask promoter compared to those from other Corynebacteria.