基因体外诱变的一种新方法  

A New Method for in vitro Mutagenesis ——Deoxynucleotide Triphosphate Substitute Method

作　　者：马向东[1,2] 柯涛[3] 黄春华[1] 周俊初[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室 [2]河南农业大学生物技术与食品科学学院郑州450002 [3]河南农业大学生物技术与食品科学学院

出　　处：《微生物学报》2004年第3期378-381,共4页Acta Microbiologica Sinica

基　　金：国家自然科学基金 ( 3 9970 0 2 2 ) ;国家"973计划"( 0 0 1CB10 89)~~

摘　　要：在PCR的过程中 ,采用 5 溴脱氧尿苷三磷酸 (BrdUTP)部分取代脱氧胸苷三磷酸 (dTTP)的方法 ,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。结果表明 ,BrdUTP浓度越高 ,诱变越强 ;浓度越低 ,诱变越弱。当BrdUTP浓度为dTTP的 0 1 %时 ,可以得到最多的正诱变结果。用LBSP鉴别培养基初筛 ,然后用Yoo改良法测定酶活 ,仅一轮诱变就获得了其表达产物α 淀粉酶的酶活分别降低了 5倍和提高了 2 0倍的两个突变基因。再以后者为PCR模板进行第二轮诱变 ,从而筛选到了α 淀粉酶的酶活提高 4 0倍的突变体。此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 。Cloned α-amylase gene of Xanthomonas campestris pv. campestris 8004 was mutated in vitro using a PCR technique in which deoxythymidine triphosphate was partially replaced by 5-bromo-2′-deoxyuridine-5′-triphosphate. The results showed that the suitable ratio concentration for the 5-bromo-2′-deoxyuridine-5′-triphosphate and deoxythymidine triphosphate was 1∶1000, LBSP selection medium was used to choose the transformants, the 20 times higher, 5 times lower and none α-amylase enzyme activity of gene products were got by only one round PCR. The mutant with 40 times higher activity was obtained by using primary PCR products as a template for a second PCR reaction. This inducing method resolved the problem of non-effective induction as in base analogue induction. And the method provides a new measure for molecular breeding.

关 键 词：PCR 体外诱变 5-溴脱氧尿苷三磷酸(BrdUTP) Α-淀粉酶基因 

分 类 号：Q78[生物学—分子生物学]