Construction Of Leymus Multicaulis Transformation-competent Artificial Chromsome (TAC) Library

Construction of transformation-competent artificial chromosome libraries has been significantly improved and advanced technically since the PAC and YAC systems were established. Large genomic DNA fragments cloned in TAC vectors can be maintained stably in both Escherichia coli and Agrobacterium tumefadens,and transferred efficiently into the host genomes, faithfully transmitted to transgenic progeny. Once a genomic library of plant in the TAC vector is established,the target genes can be cloned by screening the library ,and the library should be applicable in positional cloning.These TAC clones are available for direct introduction into plants with the Agrobacterium transformation system and conducted functional experiment in revertant mutants,so as to accelerate target genes practical processes.Leymus multicaulis (2n=4X=28, XmXmNsNs) was disease resistance and stress tolerance sometimes. It is very necessary to clone and separate these resistance genes.Construction of transformation-competent artificial chromosome libraries of Leymus multicaulis consists of four parts: vector preparation, megabase DNA partial digestion and size selection, DNA transformation, and library assembly. For vector preparation, the vector DNA must be checked and highly purified, vector DNA digestion must be appropriate (neither over-digested nor under-digested), and dephosphorylation of the restricted vector DNA must be complete. Megabase or high molecular weight (HMW) DNA is essential for large DNA fragment cloning and complex genome analysis.To isolate nuclei, and then embed them in low-melting-point (LMP) agarose in the form of plugs, followed by cell lysis and DNA purification in the LMP agarose plugs . Intact nuclei prepared from etiolated leaves of Leymus multicaulis under ×40 phase-contrast objective lens include 29×10~7 /ml .Ligation of the size-selected DNA fragments to the TAC cloning vector and transformation of the Ligated DNA into E. coli DH10B was by Electroporation. The library consists of 500,000 colonies with 94% recombinants and 6% empty clones. The Library was stored as single clone and clone pools.