文献类型： 外文期刊

作者： Xu YueYu ¹ ; Zhou YuLei ² ; Song LinLin ⁶ ; Zhang Yan ³ ; Zhao MaoLin ¹ ;

作者机构： 1.Beijing Acad Agr & Forestry Sci, Beijing Agro Biotechnol Res Ctr, Beijing 100097, Peoples R China

2.Hunan Inst Engn, Dept Chem & Chem Engn, Xiangtan 411104, Peoples R China

3.Capital Normal Univ, Coll Life Sci, Beijing 100037, Peoples R China

4.Zhongkai Univ Agr & Technol, Coll Life Sci, Guangzhou 510225, Peoples R China

5.Gansu Agr Univ, Coll Grassland Sci, Lanzhou 730070, Peoples R China

6.Henan Inst Sci & Technol, Dept Biol, Xinxiang 453003, Peoples R China

关键词： Leymus multicaulis;megabase-size DNA;transformation-competent artificial chromosome (TAC);genomic library

期刊名称：SCIENCE IN CHINA SERIES C-LIFE SCIENCES （ 影响因子：1.61； 五年影响因子：1.148 ）

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收录情况： SCI

摘要： Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants.In order to clone genes highly tolerant to barley yellow dwarf virus (BYDV),Aphids,drought and salt from Leymus multicaulis,the two TAC genomic libraries I and II were constructed in vector pYLTAC17 and pYLTAC747H/sacS,which contain about 165000 and 236000 recombinant clones separately.The genome coverage of the two libraries was totally estimated to be about 3-5 haploid genome equivalents,as size selection of genomic DNA fragments was approximately from 9 to 300 kb.Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so,stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3x3 pattern using a GeneTAC~(TM) G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacS were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6x6 pattern after a replication of plates.Nineteen positive clones were detected by using the probe glutahione reductase gene of L.multicaulis.TAC libraries constructed here can be used to isolate genomic clones containing target genes,and to carry out genome walking for positional cloning.Once the target TAC clones were isolated,they could be immediately transferred into plant genomes with the Agrobacterium system.