Construction Of A CDNA Library On The Root Of Medicago Sativa L. And Analysis Of Partial Expressed Sequence Tags

Medicago sativa L.has been planted as one of forage grass in large range for more than two thousands years.It belongs to superior perennial Leguminosae,which has good harvest,high nutritional value,strong adaptability and nice palatability,and therefore Medicago sativa L.is being called "King of forage grass" Its creeeping-rooted character derive from wild yellow flower alfalfa of Siberia,this character has excellent drought and low temperature resistance,which can develop creeeping-rooted branches base on horizontalroot.So it's very impotant to exploit this excellent character.Although many reports have been published regarding the ecological character of Medicago sativa L.,little is known about the biochemical pathways operant in Medicago sativa L.biosynthesis,or the genes involved therein.In order to delineate these biosynthesis pathways and their regulation using biochemical and molecular biological technique, preserve the resource information databank of Medicago sativa L.,in this paper we describe the construction and validation of a full-length cDNA library of Medicago sativa L. root tissues and some preliminary results.The total RNA was separated from Medicago sativa L. root tissues using Modified CTAB method,TRNzol method and other two methods.Then mRNA was isolated and purified.SMART technique and COSIII/3'primer were used for first-strand cDNA synthesis.LD PCRs were then used to synthesis the double-strand cDNAs that were then digested by Sfi I and fractionated by CHROMA SPIN-400 columns.The longer than 0.4kb cDNAs were collected and ligated to pDNR-LIB vector.Then switched to competent cell DH5a and library amplification were performed.The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination.Sixteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector.The titer of primary cDNA library was 1.12×106cfu/ml,the titer of amplified library was 1×1010 cfu/ml and the rate of recombinant was above 86%.Therefore,a cDNA library of Medicago sativa L. root has been successfully constructed.Then 102 clones were selected to sequence from the DH5αplasmids,54 sequences obtained from the plasmids were proved to be useful.Then analyzing by using the BLAST programes in NCBI.The full-length cDNA library of Medicago sativa L. root tissues has been constructed successfully by SMART technology,which is essential for screening and cloning of new genes.Furthermore, Accormplishment of this paper is an initial key work for building the resource information databank of the Medicago sativa L.