OBJECTIVETo clone and analysis a new 3-hydroxy-3methylglutary CoA reductase cDNA from Salvia miltiorrhiza (SmHMGR3).

METHODTranscription database of S. miltiorrhiza was used and a new regulatory gene from terpene secondary metabolic pathway has been cloned. ORF Finder was used to find the open reading frame of SmHMGR3 cDNA and ClustalW has been performed to analysis the multiple amino acid sequence alignment. Phylogenetic tree has been constructed using MEGA5.0. RT-PCR has been applied to detect the transcription level of SmHMGR3 in roots, stems and leaves from flowering S. miltiorrhiza plant. The mRNA level of SmHMGR3 gene from hairy roots was detected after elicitor Ag+ supplied.

RESULTThe SmHMGR3 cDNA sequence was obtained. The total length of SmHMGR3 cDNA was 1,692 bp encoding 563 amino acids. The homology rate was 75.04% and 80.64% comparing with SmHMGR1 and SmHMGR2 respectively. QRT-PCR results showed that the highest mRNA level existed in leaves of S. miltiorrhiza. After induced by Ag for 24h, the transcription level reached the highest value.

CONCLUSIONA new SmHMGR3 gene has been obtained for the first time, and which can provide the new target for the further studies about tepenes metabolism.

Amino Acid Sequence ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Hydroxymethylglutaryl CoA Reductases ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry ; classification ; enzymology ; metabolism ; Sequence Homology, Amino Acid

OBJECTIVETo establish a culture system for transgenic Salvia miltiorrhiza hairy roots.

METHODInvestigated the success rate of different explants, different infection time and different co-culture time to induce hairy roots of S. miltiorrhiza. Co-cultured explants were sterilizated with 400 g x L(-1) Cef water for 5 min, inoculated on MS solid medium supplied with 400 mg x mL(-1) cef and 2.5 g x L(-1) Hyg, and then transfered to the 67-V liquid medium with 2.5 g x L(-1) Hyg after complete sterilization. GFP fluorescence detection was performed to detect positive hairy root lines. PCR method to detect rolC gene which is the specific gene of hairy root. Biomass was determinated in different growth periods of root lines. HPLC was conducted to measure the content of dihydrotanshinone I of transgenic hairy roots.

RESULTLeaf base of S. miltiorrhiza was used as a perfect explant to Induce hairy roots, the success rate can reach 93.3%. Inducing efficiency was up to 63.3% after Agrobacterium infection for 10 min. Co-culture for 2-3 d can reach the best induced effect. It is a high credibiliy to use PCR method combined with detection of GFP fluorescence to identified positive transformants. There is a close contact between biomass increases and secondary metabolite accumulation of transgenic hairy roots.

CONCLUSIONSuccessfully in vitro culture system has been established in transgenic S. miltiorrhiza, and this research can lay foundations for the further genetic engineering applications.

Cells, Cultured ; Culture Media ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; genetics ; growth & development ; metabolism ; Salvia miltiorrhiza ; genetics ; growth & development ; metabolism ; Tissue Culture Techniques ; methods