Construction Of A Full-length Cdna Library From Dove Tree Leaves Tissues

Dove tree (Davidia involucrate) is not only the relict species in tertiary ancient tropical flora but also the first-grade state protection of rare and endangered species. What's more, it is the most populer viewing woody flower. Untill now, there are lots of reports relevant to macroscopic view of Dove tree and few reports relevant to molecular leave. To study special kind gene and preserve resource information library of Dove tree, the full-length cDNA library of leaves of Dove tree involucrate were structured, and quality of library was assessed.The total RNA was separated from leaves of Dove tree using the method of Trizol and Rneasy Plant Mini Kit. The tested reselt showed that the method of Rneasy Plant Mini Kit was better.So the total RNA which was separated from leaves of Dove tree using the method of Rneasy Plant Mini Kit were the based stuff for cDNA Library.Full-length cDNAs were constructioned by SMART TM cDNA Library Kit, and then connected to theλTriplEx2 vector. TheλDNA were packaged with the Gigapack (?)ⅢGold Packaging reorganization. The cDNA library was constructed using E. coli XLl-Blue and BM25.8 infectioned by packaged phage particles, and the quality of cDNA library was tested. The total RNA from the leaves of Dove tree were transcribed into single-chain cDNA, and the double-strand cDNAs, which were digested by Proteinase K and SfiI Enzyme and fractionated by CHROMA SPIN-400 columns, were synthetized by LD PCRs. The longer than 0.5kb cDNAs were collected and ligated toλTripIEx2 vector, and then the Xphage packaging reaction were performed, the oral cDNA libraries were constructed. After the titer and reform rate was checked, the amplification library was constructed and saved.40 plaques were randomly picked up and tested using PCR with universal primers derived from the sequence flanking the vector. The titer of primary cDNA library was 6.3×106pfu/ml, and the rate of recombinant was above 97.4%, and the titer of amplified library was 5.16×107 pfu/ml. The insert size ranged from 0.4～2.0kb. All of length of insert ragments, library titer and recombination rate meet the requirements of cDNA library.The X phage cDNA library of leaves constructed from Dove tree successfully, not only preserve the genetic resources of Dove tree, but also was the foundations to develope EST library, clone new genes and start functional genomics research of Dove tree, furtherly.