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Gene targeting in cyanobacteria utilizing I-TevI endonuclease

利用 I-TevI 核酸内切酶对蓝藻进行基因打靶

基本信息

批准号：
11660330
负责人：
MATSUOKA Masayoshi
金额：
$ 1.79万
依托单位：
Sojo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Chromosomal gene replacement in cyanobacteria often relies upon the availability of drug-resistant markers, and thus multiple replacements have been restricted. We report here a versatile gene replacement system without this restriction in a unicellular cyanobacterium Synechococcus sp. PCC 7942. The strategy is based upon the dominance of streptomycin-sensitive rps12 gene encoding a ribosomal S12 protein over a streptomycin-resistant rps12-R43 allele with Lys43Arg substitution. To demonstrate the utility of this method, a cassette consisting of the wild-type rps 12 gene and a kan gene conferring kanamycin resistance was integrated in the rps12-R43 mutant at the psbAI locus encoding photosystem II D1 protein, resulting in streptomycin-sensitive merodiploids. Despite a spontaneous gene conversion in these merodiploids to form streptomycin-resistant progeny at frequencies ranging from 1 x 10^<-5> to 5 x 10^<-5>, we could induce homologous recombination by transforming the merodiploids with template plasmids carrying psbAI 5' and 3' non-coding sequences flanking the D1-coding sequence which was replaced by either gfp ORF for a green fluorescent protein or a precise deletion. Depending on the replication ability of the template plasmids, at most 3 to 16 % of streptomycin-resistant progeny from the merodiploids after transformation turned out homogenote recombinants with concomitant loss of the kan gene even for polyploid cyanobacteria. The rps 72-mediated gene replacement thus makes it possible to construct mutants free from drug-resistant markers, and opens a way to create cyanobacterial strains bearing an unlimited number of gene replacements.

在蓝藻中，染色体基因替换往往依赖于耐药标记的可用性，因此多次替换受到限制。我们在这里报告了一个多功能的基因替代系统，没有这种限制，在单细胞蓝藻聚藻球菌sp. PCC 7942。该策略是基于编码核糖体S12蛋白的链霉素敏感rps12基因优于具有Lys43Arg替代的链霉素耐药rps12- r43等位基因。为了证明该方法的有效性，在编码光系统II D1蛋白的psbAI位点上，将野生型rps12基因和赋予卡那霉素抗性的kan基因组成的卡带整合到rps12-R43突变体中，产生了链霉素敏感的merodiploides。尽管在这些二倍体中自发的基因转化形成链霉素抗性后代的频率从1 × 10^<-5>到5 × 10^<-5>，我们可以通过在d1编码序列两侧携带psbAI 5‘和3’非编码序列的模板质粒来诱导同源重组，d1编码序列被绿色荧光蛋白的gfp ORF或精确缺失所取代。根据模板质粒的复制能力，即使是多倍体蓝藻，转化后最多3%至16%的merodiploids链霉素抗性后代也会出现同源重组，同时kan基因也会丢失。因此，rps 72介导的基因替换使得构建不含耐药标记的突变体成为可能，并开辟了一种方法来创建承载无限数量基因替换的蓝藻菌株。