[Functional genomics studies of Salvia miltiorrhiza II--gene expression profiling of different stage of hairy root].

OBJECTIVE

Studying the gene expression profiling of different stage hairy root of Salvia miltiorrhiza, in order to find functional genes.

METHOD

The contents of second metabolites were determined by HPLC and gene expression profiling was detected by cDNA microarray. cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine's linear RNA amplification method and subsequent enzymatic reaction. The microarrays were scanned with a ScanArray Express scanner using ScanArray 2.0 software and quantified by signal intensities of individual spots from the 16-bit TIFF images using GenePix Pro 4.0. The linear normalization method was used for data analyze. Northern blot was used to test the gene expression results obtained by microarray. Different expressed genes were sequenced and analyzed by gap4 software, and then they were analyzed with BLASTX, BLASTN, GO and KEGG.

RESULT

Growth rate and second metabolites analysis indicated that the stage from 30 d to 45 d was the growth stage, while the stage from 45 d to 60 d was the second metabolites accumulation stage. Accordingly 30 d hairy root was chosen as a reference, which was hybridized with 45 d and 60 d hairy root separately. Total 203 different expressed genes were obtained. Northern blot showed that the result was identical with the microarray result. After sequenced, there were 172 genes clustered into 114 clusters (Unigenes). Among them, 62 unigenes had known functions, 34 unigenes were hypothetical protein, 9 unigenes were homologues with no similarity and 9 unigenes were unidentified protein with low similarity. Total 67 genes were classified into cellular component ontology, molecular function ontology and biological process ontology based on GO analysis. Total 26 genes, which represented 29 metabolic-related enzymes, were located in metabolic maps based on KEGG pathway classification.

CONCLUSION

Several important functional genes related to second metabolite synthesis were cloned such as P450 and copalyl diphosphate synthase genes. cDNA microarray was a useful tool for functional genomics of traditional Chinese medicine.