| Rhodiola crenulata is a valuable medicinal shrub, growing mainly in Tibet plateau4500meters above the sea level, belonging to the family Crassulaceae. One characteristic of Crassulaceae species is its red color in tissues including leaching flower, root and stem liquid. Due to its growing habits such as living in cold, hypoxia, strong sunlight and no pollution zone, and5folds higher content of salidroside and tyrosol than other plants of Rhodiola, Rhodiola crenulata is also known as "snow ginseng". The main secondary metabolites of Rhodiola crenulata not only have anti-radiation, anti-aging, anti-anoxia, anti-fatigue and anti-tumor function, but also could improve the function of cardiovascular system. Thus, Rhodiola crenulata has great development prospect in special areas where is suitable for its growth. However, the reduction of the number of natural wild plant resources and the low content of salidroside (0.5%～4.1%, DW) greatly limit the commercialized production of salidroside, leading to its unsatisfied demand for salidroside in the market. High cost and toxicity confine the applyment of chemical synthesis of salidroside to large scale production. Along with the cloning of genes encoding salidroside biosynthetic enzymes from Rhodiola crenulata, in recent years, gene engineering has become one of the most effective approaches to enhance the salidroside content. The present reseach provides new targets for regulating salidroside biosynthetic pathway of salidroside through metabolic engineering strategy.The full length cDNA sequence of UDP-glucosyltransferase was obtained by the RACE technology and genomic DNA sequence was obtained by hiTAIR-PCR technology. The results show that coding region of UDP-glucosyltransferase contains an81bp intron sequence. Bioinformatics analysis indicated that the amplified gene RcUDPGT has higher homology with other plant genes. The RcUDPGT has weak hydrophilic property and is located in cytoplasm.3-D structure prediction showed that the gene encodes a protein with UDPGT binding sites. Promoter analysis suggests that there may exist light-, anaerobic-, thermal-, temperature-, stress and defense-and anthocyanin synthesis-responsive elements in promoter region. Hairy roots system of Rhodiala crenulata was established with the optimized MS medium with1/2MS+0.2mg/L6-BA, and fluffy, rapid growth and negative geotropic hairy roots were obtained. The optimum suspension culture condition of hairy root is1/2MS liquid medium,20℃,110rpm cultured in darkness. Growth curve and Logistic growth kinetic equation is y=2.667/(1+839.79e-0.301t). The wet weight inflection point A/2is1.33g, days inflection point (lnB)/k is the22.36day, the curve degreeoffitting is R2=0.99270.The effect of variety of elicitors on expression level of three key enzymes (TyDC, PAL and UDPGT) of salidroside biosynthesis and the content of salidroside in hairy root of Rhodiola crenulat was detected. Results showed that the content of salidroside after SA and MeJA treatment was respectively7.63and4.1times of the control. Analysis combined with genes expression level data showed that the correlation coefficent of expression level between RcTyDC and RcUDPGT was0.9957, and the correlation coefficent between expression level of RcUDPGT and content of salidroside was0.8572, which indicated that in comparison with phenylpropanoid pathway, tyrosine decarboxylation pathway may play an more important role in salidroside synthesis metabolism. Content meausurement showed that salidroside content is highest in stem, followed by leaf, flower and root of Rhodiola crenulata.In summarize, the present study confirmed that tyrosine decarboxylation pathway is the key pathway for biosynthesis of salidroside. In addition, the hairy root system of R. crenulata was established and UDPGT and PAL genes were firstly cloned. Results obtained here will be beneficial to the future research of salidroside biosynthesis and metabolism in Rhodiola crenulata. |
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