Screening and identification of novel genes involved in biosynthesis of ginsenoside in Panax ginseng plant

The root of Panax ginseng plant undergoes a specific developmental process to become a biosynthesis and accumulation organ for ginsenosides. To identify and analyze genes involved in the biosynthesis of gisenoside, suppression subtractive hybridization (SSH) between mRNAs of 4- and 1-year-old root tissues was performed, and a subtracted cDNA library specific to 4-year-old roots was constructed. Forty cDNA clones selected randomly from the subtracted cDNA library were sequenced. Sequence information of all clones was evaluated by Nucleotide Blast analysis in GenBank/DDBJ/EMBL. The results showed that six subtracted cDNA clones represented the novel genes (ESTs), because no sequence homology with any known sequences was found in the database. Expression in 4-year-old P. ginseng root tissues was verified by reverse Northern dot hybridization for the six clones. These six novel genes were named GBR1, GBR2, GBR3, GBR4, GBR5, and GBR6, and-their Accession numbers of GenBank are AF485334, AF485335, AF485336, AF485337, AF485332, and AF485333, respectively. Finally, Northern blot analysis and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) confirmed that these six novel genes were differentially expressed in the defined development stage of P. ginseng plant roots. It is possible that their overexpression may play an important role in the ginsenoside biosynthesis. In addition, most of transcripts of all genes could also be detected in other P. ginseng plant tissues such as stem, leaf and seed. Our results provided a basis for obtaining the full-length cDNA sequences of such six novel genes, and for identifying their function involved in the biosynthesis of ginsenoside.