Construction And Characterization Of A Full-length CDNA Library For North American Ginseng Root Tissues

North American ginseng ( panax quinquefolius L.) has been used for many years as atraditional medicine in many oriental countries. It is an herb with many active componentsand various clinical and pharmacological effects associated with its use have been reported.Root of this plant contain a variety of dammarane saponins,known as ginsenosides, which arethe characteristic major constituents of ginseng and believed to be responsible for most of theeffects . It has been reported that ginsenosides has many beneficial bioactive effects in humanhealth such as antitumor, antistress, antiaging and improved immune functions. Althoughmany reports have been published regarding the pharmacological effects of ginsenosides,little is known about the biochemical pathways operant in ginsenoside biosynthesis, or thegenes involved therein. In order to delineate these biosynthesis pathways and their regulationusing biochemical and molecular biological technique, preserve the resource informationdatabank of the panax ginseng. In this paper we describe the construction and validation of afull-length cDNA library of North American ginseng root tissues and some preliminaryresults.The total RNA was separated from North American ginseng root tissues using TRIZOLmethod. SMART technique and COSâ…¢/3'primer were used for first-strand cDNA synthesis.LD PCRs were then used to synthesis the double-strand cDNAs that were then digested bySfiI and fractionated by CHROMA SPIN-400 columns. The longer than 0.4kb cDNAs werecollected and ligated to λTripIEx2 vector. Then λphage packaging reaction and libraryamplification were performed. The qualities of both unamplified and amplified cDNAlibraries were strictly checked by conventional titer determination. Sixteen plaques wererandomly picked and tested using PCR with universal primers derived from the sequenceflanking the vector. The titer of primary cDNA library was 1.2×10⁶pfu/ml, the titer ofamplified library was2.6×10¹⁰ pfu/ml and the rate of recombinant was above 86% . The insertsize ranged from 0.3～2.0kb . Therefore, a cDNA library of North American ginseng root hasbeen successfully constructed.Then 96 clones were selected to sequence from the BM25.8 plasmids, 58 sequencesobtained from the plasmids were proved to be useful. After analyzing by using the BLASTprogrames in NCBI. 10ESTs were submitted to GenBank.The full-length cDNA library of North American ginseng root tissues has beenconstructed successfully by SMART technology, which is essential for screening and cloningof new genes. Furthermore, Accormplishment of this paper is an initial key work for buildingthe resource information databank of the North American ginseng.