小葱植物内生链霉菌线型质粒pYY8L的端粒与复制区的克隆和鉴定

Cloning and identification of telomere and internal origin of the endophytic Streptomyces linear plasmid pYY8L from Allium fistulosum

摘要:

摘要：从小葱植物中分离到一株编号为36R-2-1B的链霉菌菌株，该菌株含有一个约为280 kb的线型质粒pYY8L。【目的】克隆、测序和分析pYY8L新的端粒和复制区。【方法】采用改良的“在凝胶中进行DNA碱处理与酶切”的方法来克隆大的线型质粒pYY8L的端粒，通过构建基因组柯斯文库和次级克隆的方法来缩小和鉴定pYY8L的复制区。【结果】在小葱植物内生链霉菌36R-2-1B中检测到约为280 kb的线型质粒pYY8L，克隆了pYY8L的端粒。其末端的152 bp包含6个小的回文序列，可以形成复杂的二级结构

Abstract:

Abstract: Strain 36R-2-1B was isolated from Allium fistulosum and identified as Streptomyces spp., harboring a ~280 kb linear plasmid designated pYY8L. [Objective] Cloning, sequencing and analysis of telomere and internal replication origin of pYY8L. [Methods] pYY8L telomere was cloned by a modified procedure ─“alkaline treatment and enzyme digestion in gel”. The internal replication origin of pYY8L was cloned by construction of a cosmid library and then sub-cloning. [Results] A ~280 kb DNA band (pYY8L) of strain 36R-2-1B was detected by pulsed-field gel electrophoresis. The 152-bp telomere, containing six small palindromes and potentially forming complicated secondary structure, was cloned and sequenced. The replication origin of pYY8L was initially cloned on a cosmid and then sub-cloned as a 4891-bp autonomous replication sequence. This sequence was predicted to contain six genes, two of them resembling replication genes of Streptomyces linear plasmid SCP1, but their adjacent iterons were different. [Conclusion] New telomere and internal replication origin of Streptomyces linear plasmid pYY8L were cloned and identified. This is the first example of report on telomere and replication genes of linear plasmid from endophytic Streptomyces.