中国南荻基因组大片段人工细菌染色体文库的构建  

Construction of a large-size fragments bacterial artificial chromosome library of China Miscanthus lutarioriparius

作　　者：姚瑶 崔腾腾 张丽敏 陈翠霞[1,2] 

机构地区：[1]山东农业大学农学院,山东泰安271018 [2]作物学国家重点实验室,山东泰安271018

出　　处：《中国科技论文》2017年第12期1427-1433,共7页China Sciencepaper

基　　金：高等学校博士学科点专项科研基金资助项目(20123702110015);山东省科技发展计划资助项目(2013GNC11102)

摘　　要：以二倍体中国南荻为实验材料,以pIndigoBAC536-S为载体,以HindIII为限制性内切酶,经过载体的制备、高质量大片段DNA的获得、部分酶切最佳条件的确定、2次大片段选择和连接转化等过程,成功构建了中国南荻人工细菌染色体(bacterial artificial chromosome,BAC)文库。该文库共包含130 560个克隆,空载率为2%,同时构建了340个超级池,便于以后针对目的基因片段的BAC筛选。对随机挑取的100个单克隆检测,结果显示,插入片段分布集中,平均大小为106kb,覆盖了南荻基因组的5.6倍。随机挑取的5个克隆经过100代左右的繁殖,DNA的酶切图谱保持一致,表明BAC克隆的稳定性良好。该文库能够为该品种的基因组研究提供1个有效的平台,将对芒属植物优良基因的克隆、基因组物理图谱的构建以及比较基因组学等方面的研究发挥重要作用。A bacterial artificial chromosome(BAC)Library of China Miscanthus lutarioriparius was successfully constructed through the procedure of vector preparation,obtention of high quality large DNA fragments,determination of optimal conditions for partial digestion,twice successive selections of large fragment,connection and transformation using Chinese Miscanthus lutarioriparius as experimental materials,pIndigoBAC536-S as the carrier,HindIII as the restriction enzyme.The BAC library consists of 130 560 clones;the rate of empty clone is about 2%.340 superpools were also constructed for the future BAC screening of target gene fragments.One hundred clones were randomly selected for testing.The results sho that the average size of insert fragments is 106 kb.The BAC library covers 5.6 times of M.lutarioroparius genome.Five randomly selected clones could be steadily inherited after about 100 generations' cultivation,and their digestion profiles are consistent,indicating the stability of the BAC clone.This library will provide an effective platform for genome research of Miscanthus,and play an important role in gene cloning,physical map construction and comparative genomics research of Miscanthus.

关 键 词：作物遗传育种 南荻 基因组 人工细菌染色体文库 

分 类 号：Q943.2[生物学—植物学] S578[农业科学—作物学]