甘薯近5种Ipomoea trifida（Kunth）G. Don基因组Fosmid文库构建及PCR筛选体系建立

摘要

为挖掘有价值的基因资源,对甘薯近缘种I. trifida的基因组文库进行了研究。通过流式细胞法测定I. trifi-da(2x,编号DLP4597)基因组大小为531.699 Mb。以叶片为材料采用包埋法提取基因组DNA,通过物理剪切进行基因组DNA片段化并进行末端平滑化和磷酸化处理,脉冲场电泳回收33~48 kb的DNA片段,然后连接到Fosmid载体pCC1FOS(Epicentre)上,包装转化大肠杆菌EPI300,构建了I. trifida的基因组Fosmid文库,该文库包含101952个单克隆,保存于1062个96孔培养板中,平均插入片段35 kb,覆盖基因组约6.7倍,理论上任意片段筛出率达到99.88%。以20个96孔板为一组构建三维PCR筛选体系,共分为53个组(第53组包含22个96孔板),每组包含1个超级池,20个板池,12个列池,8个行池,理论上最多通过93个PCR反应即可筛选到1个阳性单克隆。随机选择来自I. trifida的10个基因进行文库克隆筛选,平均阳性克隆数为8.2个,最少阳性克隆数为3个,最多为16个。%Ipomoea trifida (Kunth) G. Don(2n=2x=30) and the cultivated sweetpotato are both belonged to group B in Batata section. This species has been used as model research and improve the sweetpotato with the char-acteristics of the lower ploidy level,chromosome number and elite resistance. In order to enhance the genomics study and explore excellent genes resource of I. trifida, it is very valued to construct a genomic library. This work identi-fied I. trifida the genomic size as 531. 699 Mb by carrying out flow cytometer analysis. The I. trifida genomic DNA was isolated by low melting agarose embedding method. Then Shear the DNA by physical method and end-repair the sheared DNA to blunt,5′-phosphorylated ends. Recycle the fragment between 33 to 48 kb. Ligate the blunt-ended DNA to the Cloning-Ready CopyControl pCC1 FOS vector ( Epicentre ) . Package the ligated DNA and transfection EPI300-T1R cells. The genomic Fosmid library containing 101 952 clones in 106 2 96-well plates was constructed. The average size of the inserted DNA in recombinant plasmids was 35 kb. The library coverage was at least a 6. 7-fold genome equivalent and the probability of harboring any gene in the genome of the strain was 99 . 88%. With 20 plates as a group,we build the PCR screen system. Every group contained 1 super-pool,20 plate-pools,12 column-pools and 8 row-pools. A positive clone would be found by at most 93 PCR reactions. 10 genes were used in this screen system to identify the efficiency of the system. The average positive clone number was 8. 2,in which the low-est was 3 and the highest was 16 .