Construction Of A Transformation-competent Artificial Chromosome(TAC)Library From The Nuclear Genome Of Glycine Soja

Vector DNA and large fragment of genomic DNA with high quality are two essential elements for the construction of plant genomic library, for they play a key role in the efficiency of subsequent ligation and transformation and the size of the inserted fragments.In this thesis, the nuclear DNA was extracted from the etiolated seedlings of Glycine soja, and the technique was groped for the construction of transformation-competent artificial chromosome (TAC) library from nuclear genome of Glycine soja with the pYLTAC747NH/sacB vector. The result showed that the pure vector DNA could be obtained after the plamid was extracted by the alkaline lysis method and purified by QIAGEN Plasmid Mini kit. The closed-loop vector DNA was digested by different units of HindⅢrespectively. The result examined by agarose gel electrophoresis indicated that one microgram vector DNA could just be digested fully by 2U HindⅢat 37℃for 30min. Subsequently, the linear vector DNA was dephosphorized by 0.5MBU and 1MBU HK thermolabile phosphatase respectively. The result from agarose gel electrophoresis, self-ligation reaction of the vector and eletroporation showed that the complete dephosphorylation of one microgram linear vector DNA occurred when it was processed by 1MBU HK thermolabile phosphatase at 30℃for lh. The concentration of the linear and dephosphorized vector DNA was up to 20-25ng/uL; the nuclear DNA was extracted from the etiolated seedlings of Glycine soja by filtration and centrifugation. Generally, the nuclei were embeded in low-melting-point agarose pulgs, digested with proteinase K and depurated with pulsed field gel electrophoresis to yield about two-Megabase-size DNA. The concentration of DNA solution could be up to 10 ng/μL after partial digestion by HindⅢ, elution by pulsed field gel electrophoresis, concentration and dialysis.The ligation product was obtained by processing the vector DNA and the fragment with T4 ligase by optimized ligation system and procedure. Whereafter, The ligation product was spotdialyzed on a VSWP filter and eletroporated into E.coli DH10B electrocompetent cells. The positive clones were screened by LB+Kan25mg/L+5%Sucrose which was coated with the bacteria solution after eletroporation; the transformation frequency was up to 1.92×10/mg DNA. About 30,000 positive clones were obtained by this method, the average size of the inserted fragments was 20-40kb.The positive clones could be stored at-80℃with 384-well plates.