cDNA Library Construction and Expressed Sequence Tags (ESTs) Analyses of an Alpine Plant Species Gentiana officinalis (Gentianaceae)

Gentiana officinalis, an alpine plant, one of the widely used Tibetan traditional medicines. In this study, total RNA was extracted from the whole plant of flowering individuals of this species, and a cDNA expression library was constructed using CreatorTM SMARTTM cDNA Library Construction Kit. The results showed that the titer of the cDNA expression library was 1.2×107 pfu/ml and the efficiency of recombination was 95.9%. The average length of insert fragments in the library was longer than 500 bp. A total of 181 valid ESTs were obtained from random sequencing of 343 clones. Further bioinformatic analyses suggested they represented 144 unique clonal sequences in which 55 sequences showed high homology to previously identified genes in Gentianaceae or other plants, 35 sequences matched to other uncharacterized expressed sequence tags (ESTs), and 54 sequences showed no well matches to available sequences in DNA databases. No protein matched to the latter two sorts of ESTs (89). Fifty-five ESTs with matched proteins were involved in a series of diverse functions: protein expression (35%), photosynthesis (22%), metabolism (18%), defense (11%), membrane transport (5%), cell division (5%), chromosome metabolism (2%) and signaling components (2%). At last, RT-PCR primers were designed according to the effective ESTs to amplified the cDNAs of G.officinalis, which further verified the accuracy of the ESTs. This cDNA library provided a critical basis for further analyses of functional genes and gene expression in this alpine species. In addition, these ESTs could be used to design functional nuclear primers for studying population genetics of this species and closely related species.