遗传 ›› 1999, Vol. 21 ›› Issue (1): 19-22.

• 论文 • 上一篇    下一篇

ENU诱导带LacZ靶基因的λgt11

吴涛②曹佳;钱频;杨明杰 WU Tao;CAO Jia;QIAN Pin;YANG Ming-Jie   

  1. Department of Preventive Medicine, Third Medical Military University, Chongqing 400038
  • 收稿日期:1900-01-01 出版日期:1999-02-10 发布日期:1999-02-10

Mutation Study of λgt11 DNA with LacZ Induced by ENU

  • Received:1900-01-01 Online:1999-02-10 Published:1999-02-10

摘要: 采用ENU(乙基亚硝基脲)作用于裸露的 λgt11 DNA,经体外重包装,转染宿主菌 E. coli Y1090,在含底物X-gal,诱导剂IPTG的选择性培养基上铺皿,发现被处理的 λgt11 DNA除了使噬菌体存活率下降外,还出现了靶基因“LacZ”较高频率的突变。其中以二甲基亚砜(DMSO)为溶剂,当存活率分别为3.5×10-3、1.6×10-3和5.5×10-4 时,相应的突变率依次为1.1×10-3、3.2×10-3和5.2×10-3,DMSO溶剂对照突变率则<5.0×10-5 。对ENU诱导的5个阳性突变体进行了扩增,以PCR产物为模板,采用正向引导,对阳性突变体靶基因LacZ进行了部分测序,在被测序的260bp范围内,发现了9个位点的碱基突变。碱基突变的类型有颠换(67%)、转换(11%)和移码突变(22%)。颠换主要以A→T、G→C为主。似乎胞嘧啶(C)更易发生突变(占43%)。
Abstract To construct molecular mutation detective system of λ DNA with LacZ, naked λ gt11 DNA was treated with mutagen ENU (Ethylnitro sourea). The ENU-damaged DNA was added to Lambda packaging extracts and the resulting phage were grown in host E.coli Y1090 on a selective plate containing substract X-gal and inducer IPTG. Under these conditions, the results showed that the higher the viability ratio was, the lower the frequency of clear-plaque mutants occured. In our study, when survival ratios of the host cell survival ratio were 3.5×10-3、1.6×10-3 and 5.5×10-4 respectively, the mutation ratio were 1.1×10-3、3.2×10-3 and 5.2×10-3 accordingly, and the mutation ratio by DMSO (negative control ) was below 5.0×10-5. 260 bases from ENU―induced LacZ gene were subjected to DNA sequence analysis. There were several mutation sites: transversion (6, 67%), transition(1, 11%), frame shift(2, 22%)(both were insert mutation). Transversions mainly consisted of A→T, G→C. Among the four bases, cytosine seemed to be more sensitive to ENU (43%).

关键词: LacZ基因,  突变,  诱变剂,  DNA测序 Key words KLacZ,  体外重包装, 小麦染色体, 显微噪作, 激光切割

Key words: Wheat chromosome, Microoperation, Microdissection by laser beam