摘要
介绍了构建大腹圆蛛Fosmid基因组文库及牵引丝蛋白(MaSp)基因克隆筛选的全过程.采用改良CTAB法提取大片段基因组DNA,通过自主构建的电洗脱核酸回收装置分离回收30～40kbDNA片段,经补平磷酸化、与pCC2FOS载体连接、体外包装和转染EPI300TM-T1R,首次构建了无偏向性的大腹圆蛛Fosmid文库,其滴度为4.5×105cfu/mL,覆盖基因组倍数为10.以α-32P标记寡核苷酸探针对文库进行初步筛选,获得含MaSp基因的12个阳性克隆.该文库符合Fosmid文库的品质要求,为进一步筛选并研究大腹圆蛛MaSp基因序列奠定了基础.
The overall process for constructing fosmid genomic library in A raneus ventricosus and screening new Dragline silk gene （MaSp gene） was described. Genomic DNA prepared for library construction was extracted by improved method based on CTAB. was recovered by self-designed electroelution The DNA was randomly sheared and 30-40 kb fragment device. Fragments were end-repaired and ligated with pCC2FOS Vector. Recombinant molecules were packaged in vitro with the phage Packaging Extract, then transformed into E.coli EPI300TM-T1R. A fosmid library of Araneus ventricosus was constructed firstly with 4.5×10^5 clones and gene coverage ratio was about 10. Twelve positive clones were achieved by in situ colony hybridization with isotope [α-32P] labeling probe. This library is a typical fosmid library, and it will be used to screen new MaSp gene.
出处
《生命科学研究》
CAS
CSCD
2010年第1期21-26,共6页
Life Science Research
基金
国家高技术研究发展计划(863计划)项目(2006AA03Z451)
教育部博士点基金项目(08Y10512)
东华大学青年教师科研启动基金项目(105-10-0044018)
上海市大学生创新活动计划项目(X120704100)
