适用于链霉菌大片段基因组DNA 克隆和异源表达的细菌人工染色体(BAC)载体的构建及应用

Construction of a new bacterial artificial chromosome (BAC) vector for cloning of large DNA fragments and heterologous expression in Streptomyces

摘要:

摘要:【目的】很多链霉菌来源的天然产物的生物合成基因簇往往很大，用传统的cosmid 载体很难完整的克隆和异源表达。本研究通过载体改造，成功构建出一个新的细菌人工染色体(BAC)载体，用于链霉菌来源的天然产物生物合成基因簇的克隆及异源表达实验。【方法】从复合型载体pCUGIBAC1出发，通过λRED介导的PCR-targeting 方法，用链霉素抗性基因替换掉原有的氯霉素抗性基因标记，同时插入链霉菌中常用的安普拉霉素抗性标记、转移起始位点oriT、φC31 整合酶基因int、整合位点attP 等元件。【结果】成功构建出可装载链霉菌大片段DNA 的BAC 载体pMSBBACs。使用pMSBBACs 构建出链霉菌U27的基因组BAC文库，平均插入片段大小为100 kb。选取其中一个大小为140 kb的BAC 质粒进行功能验证，实验证明通过接合转移和原生质体转化的方法都能够将这个大型BAC 质粒导入链霉菌模式菌株，并通过位点特异性重组整合到染色体中进行异源表达。【结论】BAC 载体pMSBBACs 可成功用于放线菌大片段基因组DNA 的克隆和异源表达实验。

Abstract:

Abstract:［Objective］Many natural product biosynthetic gene clusters are too large to be entirely cloned into one cosmid for heterologous expression． Because bacterial artificial chromosome (BAC) vectors are well known for their capacity of cloning large DNA fragments，we constructed a new BAC vector for cloning and heterologous expression of natural product biosynthesis gene clusters in Streptomyces．［Methods］The chloramphenicol resistance gene on the original BAC vector pCUGIBAC1 was substituted with a streptomycin resistance gene via λ RED-mediated PCR-targeting technique．The streptomycin resistance gene was then excised by digestion with NheI and the left gap was filled with the origin of transfer (oriT)，the φC31 integrase gene，the integrating attP site，and an apramycin resistance gene．［Results］We achieved the final BAC vector pMSBBACs． To test the newly established vector，pMSBBACs was used to build up a genomic BAC library of Streptomyces U27． The average size of inserts in the library is about 100kb． A 140 kb BAC plasmid as a representative was successfully introduced into heterologous hosts，S．lividans and S．albus，by either conjugation or protoplast transformation． It demonstrated that the BAC plasmids constructed by pMSBBACs could be integrated into chromosomes via site-specific recombination for heterologous expression．［Conclusion］The newly constructed pMSBBACs was verified to be a good BAC vector for cloning of large DNA fragments and heterologous expression in Streptomyces．