黑龙江野鲤细菌人工染色体基因组文库构建  

Construction of a bacterial artificial chromosome (BAC) genomic library of Heilongjiang River carps (C. carpio haematopterus)

作　　者：耿波[1] 关云涛[2] 孙效文[1] 

机构地区：[1]中国水产科学研究院黑龙江水产研究所农业部北方鱼类生物工程育种重点实验室,黑龙江哈尔滨150070 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国水产科学》2009年第2期165-172,共8页Journal of Fishery Sciences of China

基　　金：国家计划(973)项目“重要养殖鱼类品种改良的遗传和发育基础研究”(2004CB117405);黑龙江水产研究所基本科研业务费项目“鲤鱼BAC基因组文库克隆”(2007HSYZX-SJ-12)

摘　　要：为了开展鲤基因组研究,深入研究遗传连锁图谱遗传标记的定位及数量性状的定位和克隆,并最终为分子育种提供服务,本实验构建了鲤的BAC基因组文库。通过采集黑龙江野鲤(Cyprinus caxpio haematopterus)全血细胞制备琼脂糖凝胶包埋块的方法获得了高分子量(HMW)DNA。通过BamHⅠ部分酶切和PFGE电泳分离选择获得100～300kb的DNA片段,用电洗脱和透析的方法对产物进行浓缩和纯化。纯化的HMWDNA连接到大小为7.2kb的克隆载体pEZBAC上。为了评估构建的文库的质量使用一系列引物对文库进行了验证。本研究首次构建黑龙江野鲤的BAC文库,该库含有46656个克隆,插入片段大小在50～300kb之间,平均大小在100kb左右,覆盖鲤全基因组2.45倍,调取任一基因的概率为90%左右。获得的BAC克隆,保存在378块96孔板和27块384孔板中。建立了高效稳定的2步3维PCR筛选黑龙江野鲤BAC文库的方法。本实验为今后进一步进行鲤的功能基因定位及染色体步行、BAC-FISH等研究工作打下重要基础。As the development of the genome,it is important to identify the genomic mechanisms for specific traits of fish. Traits of importance might be related to growth,disease resistance,good conversion efficiency,color or taste. To identify genomic regions reponsible for specific traits,genomic large insert libraries have previously proven to be of crucial importance. Here we constructed a highly redundant genomic bacterial artificial chromosome （BAC） library using high molecular weight DNA from a Heilong River wild carp （Cyprinus carpio haematopterus） mainly distributing in Heilong River. Its popular name is carp or oil carp. C. carpio haematopterus is groundfish and likes to live in sligthly turbid water. It has a broad distribution and is one of the chief catch fishes and cultural fishes. C. carpio haematopterus has fine characters such as strong antireversion force （cold-resistance and disease resistance）,genetic diversity and so on and often used to cross breeding and cells and embeding in low-melting-point （LMP） agarose plugs. Firstly we determined the optimal partial digestion conditions for BAC cloning by varying the concentration of the restriction enzyme,the time of digestion with the same amount of the restriction enzyme. The partial digestion of HMW DNA is to generate equality size DNA fragments followed twice size selection. Finally we performed size-selection for BamHⅠrestricted DNA Fragments from 100-300 kb with PFGE and eluted DNA with electroelution from the agarose gel slice and then purified them with dialysis and condensed with PEG8000. The purified HMW DNA was ligated to the vector pEZ BAC with 7.2 kb. Screening of the BAC library with mix primers was carried out to confirm the library quality. We constructed the BAC library of Heilong River carps for the first time. This BAC library consists of 46,656 BAC clones in total, and the insert DNA size is about 50～300 kb,the average of size is about 100 kb. The genome coverage of the BAC library is 2.45 and the possibility to find a s

关 键 词：黑龙江野鲤 细菌人工染色体(BAC) 基因组文库 超级池 

分 类 号：S965.116[农业科学—水产养殖]