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

doi:10.7668/hbnxb.2014.02.008

华北农学报 ›› 2014, Vol. 29 ›› Issue (2): 45-50. doi: 10.7668/hbnxb.2014.02.008

所属专题： 薯类作物；生物技术；

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

李昂¹, 吴志明², 周志林¹, 赵冬兰¹, 张安¹, 马代夫¹, 李亚栋², 唐君¹, 曹清河¹

1 中国农业科学院甘薯研究所江苏徐州甘薯研究中心农业部甘薯生物学与遗传育种重点实验室江苏徐州 221131;
2 河北省农林科学院经济作物研究所河北石家庄 050051

收稿日期:2013-12-24 出版日期:2014-04-28
通讯作者: 曹清河(1975-),男,江苏徐州人,副研究员,主要从事甘薯种质创新和遗传改良研究。唐君(1960-),女,河南信阳人,研究员,主要从事甘薯种质资源研究。
作者简介:李昂(1990-),男,河南开封人,在读硕士,主要从事基因挖掘与功能验证研究。
基金资助:
国家自然科学基金项目(31371681;30800698);国家甘薯产业技术体系(CARS 11-B-02);农业部948项目(2011-G1-20);国家“863”项目(2012AA101204-1-8)

The Construction of Genomic Fosmid Library and Library PCR Screening System on Ipomoea trifida(Kunth) G. Don

LI Ang¹, WU Zhi-ming², ZHOU Zhi-lin¹, ZHAO Dong-lan¹, ZHANG An¹, MA Dai-fu¹, LI Ya-dong², TANG Jun¹, CAO Qing-he¹

1 Institute of Sweetpotato Research, Chinese Academy of Agricultural Sciences, Jiangsu Xuzhou Sweetpotato Research Center, Xuzhou 221131, China;
2 Institute of Commercial Crops Research, Hebei Academy of Agricultural Sciences, Shijiazhuang 050051, China

Received:2013-12-24 Published:2014-04-28

摘要/Abstract

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.trifida(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101 952个单克隆,保存于1 062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。

关键词: 甘薯近缘野生种, 基因组, Fosmid文库, 文库筛选体系

Abstract: 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 pCC1FOS vector (Epicentre) . Package the ligated DNA and transfection EPI300-T1 R 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.

Key words: Wild sweetpotato relatives, Genomics, Fosmid library, PCR screening system

中图分类号:

李昂, 吴志明, 周志林, 赵冬兰, 张安, 马代夫, 李亚栋, 唐君, 曹清河. 甘薯近缘种Ipomoea trifida (Kunth) G.Don基因组Fosmid文库构建及PCR筛选体系建立[J]. 华北农学报, 2014, 29(2): 45-50. doi: 10.7668/hbnxb.2014.02.008.

LI Ang, WU Zhi-ming, ZHOU Zhi-lin, ZHAO Dong-lan, ZHANG An, MA Dai-fu, LI Ya-dong, TANG Jun, CAO Qing-he. The Construction of Genomic Fosmid Library and Library PCR Screening System on Ipomoea trifida(Kunth) G. Don[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2014, 29(2): 45-50. doi: 10.7668/hbnxb.2014.02.008.

http://www.hbnxb.net/CN/Y2014/V29/I2/45

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

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

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