Construction Of CDNA Library And Analysis On Expressed Sequence Tags Of Calamus Simplicifolius

In order to obtain the RNA from calamus simplicifolius, author attempt modified CTAB method,Guanidnium method and Trizol method, The results indicated that modified CTAB method for RNA is perfect. After three kinds of RNA were equally mixed,the full-length cDNA library were constructed by using SMART method. The quality of cDNA library were detected: the titer of primary cDNA library and amplified cDNA library were 1.40×105cfu/mL and 5.82×106cfu/μL.Sixteen monoclonal were randomly picked, the inserted fragment were amplified with M13 primers, the recombination rate of the library was 87.5%,the inserted fragment were distributed from 1.0kb to 4.0kb.Totally 2860 clones were randomly selected from cDNA library of calamus Simplicifolius,and partially sequenced by ABI3130XL capillary sequence machine, 2556 readablesequences that are more than 100bp were produced by analysis with Phred software.And theaverage length of the 2556 ESTs was 491.49bp,the average quality is 36;the GC content is 46.88%.The 2860 ESTs of calamus Simplicifolius has been analyzed and assembleed into 2043 UniGene including 408 contigs and 1635 singlet by the Phrep software and cross match software. Redundancy rate of ESTs of calamus Simplicifolius is 39.99%.EST analysis of significant homology with the non-redundant protein database suggested that about 10.78% of the 408 contigs were high expression abundance genes (expression frequency≥5),25.24%were moderate abundance genes(expression frequency 3-4),and 63.97%were low abundance genes(expression frequency1-2).The results indicated that most genes in calamus Simplicifolius were low or moderate expression abundance.The effective ESTs were blasted against the non-redundant nucleotide and protein databaseof NCBI.33.62%of the total effective ESTs had significant homology with the nucleotide database and 77.27%with the protein database.by the basis of NCBI database searches.