通过同源重组对根霉属米根霉进行整合转化

Integrative transformation of the zygomycete Rhizomucor pusillus by homologous recombination.

作者信息

Wada M, Beppu T, Horinouchi S

机构信息

Department of Biotechnology, University of Tokyo, Japan.

出版信息

Appl Microbiol Biotechnol. 1996 Jun;45(5):652-7. doi: 10.1007/s002530050743.

DOI:10.1007/s002530050743

PMID:8766701

Abstract

For development of a homologous transformation system for the zygomycete fungus, Rhizomucor pusillus, the isopropylmalate isomerase (leuA) gene was cloned from R. pusillus IFO 4578 by the DNA-probing method with the leuA sequence of Mucor circinelloides as probe. The nucleotide sequence revealed that leuA of R. pusillus encoded a 755-amino-acid protein of 82.5 kDa with no intron. The leuA gene on pUC19 (plasmid pRPLeu10) was introduced by polyethyleneglycol-assisted transformation into protoplasts of a leuA- mutant of R. pusillus that was obtained by UV mutagenesis. Transformation under optimal conditions yielded 20 Leu+ transformants (micrograms pRPLeu10 DNA)-1 (1 x 10(6) viable protoplasts)-1. Blot analysis of DNA from the transformants showed that the pRPLeu10 sequence was integrated into the genome by homologous recombination at the leuA locus.

摘要

为了开发一种适用于接合菌纲真菌米根霉的同源转化系统，以卷枝毛霉的亮氨酸基因（leuA）序列为探针，通过DNA探针法从米根霉IFO 4578中克隆了异丙基苹果酸异构酶（leuA）基因。核苷酸序列分析表明，米根霉的leuA编码一个由755个氨基酸组成、分子量为82.5 kDa的蛋白质，且无内含子。通过聚乙二醇辅助转化，将pUC19上的leuA基因（质粒pRPLeu10）导入经紫外线诱变获得的米根霉leuA-突变体的原生质体中。在最佳条件下进行转化，每微克pRPLeu10 DNA可产生20个亮氨酸营养缺陷型回复突变转化子（每1×10⁶个活原生质体）。对转化子的DNA进行印迹分析表明，pRPLeu10序列通过在leuA位点的同源重组整合到基因组中。