Expression analysis of light-regulated genes isolated from a full-length-enriched cDNA library of Onosma paniculatum cell cultures.
Shikonin and its derivatives are formed in large amounts in dark-cultured Onosma paniculatum cells. In order to isolate and identify the genes regulating shikonin biosynthesis, we constructed and characterized a full-length-enriched cDNA library of dark-cultured cells by using the SMART (Switching Mechanism At 5'-end of RNA Transcript) cDNA synthesis and LD-PCR (long-distance PCR) strategies. The titer of the primary cDNA library was 1.04 x 10(6)pfu/mL with a recombination rate of 99.60%. Most of the cDNA inserts ranged from 1.0 to 2.5 kb, and 78.33% of the 76 randomly selected clones contained full-length coding regions. Expression analysis of randomly selected genes by small scale microarray revealed that 23 genes were down-regulated, including 17 genes with known functions, 2 genes with putative functions, and 4 novel genes, and that 3 genes were up-regulated (two-fold) in cells cultured under white light as compared with those cultured in the dark. Interestingly, two of the down-regulated genes, encoding aci-reductone dioxygenase (ARD)-like protein and ethylene responsive factor (ERF), are involved in ethylene biosynthesis and signal transduction, implying that ethylene might play an important role as a signal molecule in light-regulated shikonin formation. These data contribute to a better understanding of light-involvement in regulating the formation of plant secondary metabolites.
Qi
Jin-Liang
JL
Institute of Plant Molecular Biology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, PR China.
Zhang
Wen-Ju
WJ
Liu
Shao-Hua
SH
Wang
Hong
H
Sun
Deng-Yun
DY
Xu
Guo-Hua
GH
Shi
Ming-Wang
MW
Liu
Zhi
Z
Zhang
Ming-Sheng
MS
Zhang
Hui-Ming
HM
Yang
Yong-Hua
YH
D028082:Boraginaceae / Q000166:cytology* / Q000235:genetics* / Q000528:radiation effects; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005030:Ethylenes / Q000096:biosynthesis; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant / Q000528:radiation effects*; D015723:Gene Library; D017343:Genes, Plant; D008027:Light; D009285:Naphthoquinones / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D005030:Ethylenes; D009285:Naphthoquinones; C016101:shikonin; C036216:ethylene