Construction and characterization of the transformation-competent artificial chromosome (TAC) libraries of Leymus multicaulis  

作　　者：XU YueYu 1,2,3,ZHOU YuLei 4,5 ,SONG LinLin 6 ,ZHANG Yan 3 &ZHAO MaoLin 1 1 Beijing Agro-Biotechnology Research Center,Beijing Academy of Agriculture and Forestry Science,Beijing 100097,China 2 Department of Chemistry and Chemical Engineering,Hunan Institute of Engineering,Xiangtan 411104,China 3 College of Life Science,Capital Normal University,Beijing 100037,China 4 College of Life Science,Zhongkai University of Agriculture and Technology,Guangzhou 510225,China 5 College of Grassland Science,Gansu Agricultural University,Lanzhou 730070,China 6 Department of Biology,Henan Institute of Science and Technology,Xinxiang 453003,China 

出　　处：《Science China(Life Sciences)》2008年第7期604-613,共10页中国科学（生命科学英文版）

基　　金：the National Natural Science Foundation of China(Grant Nos.30370905 and 30571135);Natural Science Foundation of Beijing City(Grant No.5032009)

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/sacB,which contain about 165000 and 236000 recombinant clones sepa-rately.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 3×3 pattern using a GeneTAC?G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 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.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/sacB,which contain about 165000 and 236000 recombinant clones sepa-rately.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 3×3 pattern using a GeneTAC?G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 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.

关 键 词：LEYMUS multicaulis megabase-size DNA transformation-competent artificial chromosome(TAC) genomic library 

分 类 号：Q943[生物学—植物学]