Cao, Zh. Zh.

Chen, P.

Xu, Y. Y.

Zhao, M. L.

Zhou, Y. L.

English

University of Kentucky

University of Kentucky UKnowledge International Grassland Congress Proceedings XXI International Grassland Congress / VIII International Rangeland Congress Construction of a Transformation‐Competent Artificial Chromosome (TAC) Library of Leymus multicaulis Y. L. Zhou Zhongkai University of Agriculture and Technology, China Y. Y. Xu Hunan Institute of Engineering, China M. L. Zhao Beijing Academy of Agriculture and Forestry Sciences, China Zh. Zh. Cao Gansu Agriculture University, China P. Chen Zhongkai University of Agriculture and Technology, China Follow this and additional works at: https://uknowledge.uky.edu/igc  Part of the Plant Sciences Commons, and the Soil Science Commons This document is available at https://uknowledge.uky.edu/igc/21/12-2/46 The XXI International Grassland Congress / VIII International Rangeland Congress took place in Hohhot, China from June 29 through July 5, 2008. Proceedings edited by Organizing Committee of 2008 IGC/IRC Conference Published by Guangdong People's Publishing House This Event is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in International Grassland Congress Proceedings by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. 　 Multifunctional Grasslands in a Changing World 　 Volume Ⅱ 　 瞯 ]427　 瞯Grasslands/Rangelands Production Systems——— Developing Improved PlantsConstruction of a transformation‐competent artificial chromosome ( TAC) library of LeymusmulticaulisY .L . Zhou1 ,4 ,Y .Y . Xu2 , M .L . Zhao3 , Zh .Zh . Cao4 ，P . Chen11 College o f A griculture and Gardenning , Zhongkai University o f A griculture and Technology , Guangz hou Guangdong510225 , PR China ,E‐mail :z houyl＠ yahoo .cn2 H u�nan Institute o f Engineering , X iangtan Hu�nan 411104 , PR China3 Bei j ing A griculture Biotechnology Research Center , Bei j ing A cademy o f A griculture and Forestry Sciences , Bei j ing100089 , PR China4 Gansu A griculture University , L anz hou Gansu 730070 , PR China .Key words :Leymus multicaulis ,transformation‐competent artificial chromosome ( tac) , genomic library , high molecular weightdna , insert sizeIntroduction Leymus multicaulis ( ２n ＝ ４x ＝ ２８ ＝ １４II , XmNs ) is an important forage of Leymus Hochst of T riticaea ofGramineae plant (Wang Shijin and LI Jianhua ,１９９３ ) , which possesses resistance characteristics to dry , salt , barley yellowdwarf virus , budworm , barren conditions and pollution . For utilizing the resistance characteristics of Leymus multicaulis , weconstructed a transformation‐competent artificial chromosome ( TAC ) genomic library of Leymus multicaulis in thepYLTAC７４７H vector . Such genomic libraries should be useful for gene cloning in Leymus multicaulis and other crops .Materials and methods High molecular weight ( HMW) DNA was prepared from tender leaves of Leymus multicaulis etiolatedseedlings using the nuclei‐based method as described ( Y .L . Zhou et al . ,２００７ ) . TAC vector DNA preparation ,and purity ,partial digestion , size selection of Leymus multicaulis HMW DNA , and the ligation and transformation of vector DNA withHMW DNA used the method as described ( Y .G . Liu et al . , ２００２) .Results We successfully isolated very pure HMW nuclear DNA which was over ２Mb in size ( Figure １) . A TAC genomic librarywas constructed from nuclear DNA of Leymus multicaulis . The library consisted of ２ .４ × １０５ clones which were collected asbulked pools each containing ５００ clones and stored in ５ × ９６‐well plates . The library has insert sizes of genomic DNA of approximately５Kb ～ ２００Kb and average insert size of ５０Kb ( Figure ２) , representing approximately ２ ～ ４ haploid genome equivalents .Figure 1 Size determination o f Leymus multicaulisHMW DNA by p lused‐f ield gel electrophoresisM :Yeast Chromosome PFG Marker ( NEB) , 1 .5ug ; 1 :1 p lug ; 2 :1 /2 p lug .　 　Figure 2 Recombinant TAC clones digestedw ith restriction enz yme H indI I IL ine1‐8 :clones w ith TAC747H vector ; M : λDNA‐H indI I I molecular marker .Conclusions Most isolated Leymus multicaulis DNA was greater than ２ Mb , and was suitable for constructing a TAC library .Vector pYLTAC７４７H , which is １８ ,９００ bp , was used to construct the TAC library . Constructing a large‐insert genomic DNAlibrary is essential for gene map‐based cloning . Such libraries will be used to clone many genes .With its high stability and goodgenomic coverage , the TAC library provides an efficient platform for gene cloning and functional complementation of targetgenes in Leymus multicaulis .ReferencesSh .J . Wang J .H . Li , １９９３ . A preliminary introduction and evaluation of forage germplasm resources in tribe triticeae . A ctaPrataculture Sinica ２(１) , ６０‐６９ .Y .L . Zhou , Y .Y Xu , M .L .Zhao , Zh .Zh . Cao , Zh .P . Wang , ２００７ .An Efficient Protocol for Preparation of High MolecularWeight DNA of Nuclei from Leymus Multicaulis .Pratacultural Science ２４(１０) , ５２‐５６ .Y .G . Liu , HM Liu , L .T . Chen , W .H . Qiu , Q .Y . Zhang , H . Wu , C .Y . Yang , J . Su , Z .H . Wang , D .S . Tian , M .T .Mei ,２００２ . Development of new transformation‐competent artificial chromosome vectors and rice genomic libraries forefficient gene cloning . Gene ２８２ (１‐２) , ２４７‐２５５ .

Construction of a Transformation‐Competent Artificial Chromosome (TAC) Library of \u3ci\u3eLeymus multicaulis\u3c/i\u3e

https://uknowledge.uky.edu/cgi/viewcontent.cgi?article=3351&amp;context=igc

Construction of a Transformation‐Competent Artificial Chromosome (TAC) Library of \u3ci\u3eLeymus multicaulis\u3c/i\u3e

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