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目的建立一种高通量筛选具有产生安莎类抗生素潜能的放线菌的分子筛选方法。原理3-氨基-5-羟基-苯甲酸(AHBA)是安莎类抗生素生物合成的前体。在通过氨基莽草酸途径生物合成AHBA的过程中,AHBA合酶是催化5-氨基-3-脱氢-5-脱氧莽草酸形成AHBA的一个特异性关键酶。AHBA合酶基因的存在可以作为放线菌菌株具有合成安莎类抗生素能力的一个必要而非充分的条件。AHBA合酶基因高度保守,通过PCR方法可以建立一种针对AHBA合酶基因存在与否的、具有安莎类抗生素产生潜能的放线菌菌株高通量筛选方法。方法根据已知AHBA合酶序列的相似性,设计了针对AHBA合酶基因中高度保守的一段755bp片段大小的PCR筛选寡核苷酸引物,用于从土壤中分离的未知放线菌高通量筛选。结论从1900株土壤分离的未知放线菌中筛选获得33株AHBA合酶基因阳性菌株,即具有安莎类抗生素产生潜能的放线菌菌株。
OBJECTIVE To establish a high-throughput molecular screening method for screening actinomycetes with the potential of producing Ansar antibiotics. Principle 3-Amino-5-hydroxy-benzoic acid (AHBA) is a precursor of ANSA antibiotic biosynthesis. AHBA synthase is a specific key enzyme that catalyzes the formation of AHBA from 5-amino-3-dehydro-5-deoxyshikimate to AHBA through the amino-shikimate pathway. The presence of the AHBA synthase gene may serve as a necessary but not sufficient condition for actinomycete strains to have the capacity to synthesize ANSA antibiotics. AHBA synthase gene is highly conserved. A high-throughput screening method of actinomycete strains with the potential of Ansar antibiotics can be established by PCR method for the presence or absence of AHBA synthase gene. Methods Based on the similarity of known AHBA synthase sequences, a PCR-amplified oligonucleotide primer targeting to a highly conserved segment of AHBA synthase gene of 755 bp was designed for high-throughput unknown actinomycetes isolated from soil filter. Conclusion A total of 33 strains of AHBA synthase-positive strains were screened from unknown actinomycetes isolated from 1900 soils, that is, actinomycete strains with the potential of Ansar antibiotics.