Cloning And Functional Research On Two Related Genes Of Salidroside Biosynthesis In Rhodiola Sachalinensis

Herbs of Rhodiola rosea L genus consist of over 90 species in the world and nearly 73 species in China. Rhodiola sachalinensis A.Bor, a perennial herb, belonging to Rhodiola rosea L genus, is one of the rare herbs used as a medicinal material in Changbai Mountain area in North China. Rhodiola sachalinensis A.Bor is suggested to function as a promising environmental adaptogen related to aerospace industry, marine assignments, desert work, highlands projects, sports and other industries, i.e. as a biologically-active compound to increase resistance in humans to different stress-related disorders and a variety of other diseases. In addition, salidroside has been reported to act as an anti-inflammatory, anticancer, anti-viral, anti-aging, anti-oxygen deficiency, anti-fatigue, anti-radiation and double-regulation multi-functional compound. But Rhodiola sachalinensis A.Bor has been endangered due to its harsh ecology, difficulty in pollination and other characteristics of its reproductive system and severe damages to its dependent natural environment caused over utilization of the land, therefore natural herbs of the genus have been decreased sharply in number.At present, China plays an important role in the research of Rhodiola rosea L genus herbs.Study fieldscover chemical composition, pharma -cologic action. resource conservation and artificial cultivating, tissue culture and intermediate propagation, antifreeze protein, cell culture and bioconversion, RAPD analysis, classification and identification etc. salidroside and tyrosol,as functional substance of Rhodiola rosea L genus herbs,heve made important progressare its metabolic pathway, molecule mechanism and cloning of enzymes involving in biosynthesize of functional substance or rate-limiting enzyme, etc. In this paper, two gene sequences related to salidroside and tyrosol have been identified by cDNA RACE methods to isolate genes from R. sachalinensis, besides, the characteristic and functional of the two gene sequences have been further investigated here.The mechanism of the last biosynthesis of salidroside in plants has been quite clear. Salidroside could be generated by uridine diphosphate glucose (UDPG) and tyrosol with the catalyzing of UDP-glucosyl- transferase. However, it is not clear about the mechanism of biosynthesis of tyrosol in plants. Biosynthesis step of alkaloids in secondary metabolite may be the key point to generate tyrosol.Tyrosine decarboxylase serves as one of the first enzyme which is associated with initial metabolism and a number of alkaloids biosynthesis, considered as the key enzynme and rate-limiting enzyme. In this research, tyrosine decarboxylase complete cDNA sequence was cloned from R. sachalinensis, named TyrDC1, with accession DQ471943 (Genbank). And the cDNA(1715 bp) had one open reading frame of 508 amino acids with 56,88kDa and pI 6.15. Bioinformatics analysis indicated that TyrDC1 is a new number of tyrosine decarboxylase in plants. TyrDC1 cDNA with known tyrosine decarboxylase in plants showed the range nearly between 46.91% (from Arabidopsis thaliana, NM₁19010) to 54.61 %(from Parsley, M95685). Conserved region located in middle and region of variability in ends, in special C-terminal. The amino acid sequence of TyrDC1 contains typical TyrDC structural domain and there is no transmembrane domain in amino acids sequence. Northern hybridization analysis showed TyrDC1 transcript levels are high in inducing callus, and higher than normal callus, whereas no expression phenomenon is in leaves. HPLC determined salidroside content in different tissues in which TyrDC1 gene expression levels are not equal. The results found that salidroside content is higher in induced callus than in normal callus, which is greatly higher than in leaves. So it can be concluded that there are direct correlation between different tissues and salidroside content in inducing conditions. In the subsequent research, pCATyrDC1, a highly effective vector was constructed, containing TyrDC1 gene sequencer, and then it was transformed to R. sachalinensis mediated by agrobacterium . It was identified that TyrDC1 gene integrated in the genome of transgentic R. sachalinensis by PCR, Real Time PCR and Real Time RT-PCR methods, respectively. Salidroside was detected quantitatively in transgenic cultures of R.sachalinensis by HPLC. The results indicated that the salidroside contents of leaves of transgenic plants and transgenic calli increased 1.88- and 2.43-fold compared to the untransformed controls, respectively.Glycosyltransferase (EC 2.4.x.y) extensively exists in biosystem, which determines conforming of glucosidic bond. Another important protein associated with secondary metabolite in plants is UDP(uridine diphosphate)—glucosyl transferase (UDPGT, UGTs) . It regulates majority glycosides synthetic in secondary metabolism path, and acts as key enzyme in salidroside synthesis. In this study, UDPGT complete cDNA sequence was cloned from R. sachalinensis, named UGT3 with accession number EQ508689(Genbank). The 1547bp UGT3 cDNA sequence contained an open reading frame encoding a predicted translation product of 453 amino acids with a predicted molecular mass of 51.18kDa, pI 5.37. Bioinformatics analysis demonstrated UGT3 is a new number of UGTs family, and contains a typical UGTs structural domain between 6-453 amino acid. Meanwhile, a 3'terminal UGTs structural domain contains conserved HCGWNS amino acids, beginning at the 342 amino aceds. UGT3 and other UGTs in plants share 24.23% to 54.24% homology. Analysis of other biology characters showed that UGT3 is a matrix protein and exists in single copy in R. sachalinensis by Southern hybridization. Northern hybridization analysis revealed that UGT3 has higher expression in callus, while trace expression in leaves. HPLC determined salidroside in different tissues found that salidroside content are higher in callus than in leaves. So the conclusion can be drawn that there is direct correlation in different tissues.In summary, isolation and identification of key genes in salidroside biosynthesis have provided a research foundation for illustration of bio -synthetic metabolism path and this study will greatly benefit scale pro -duction of salidroside in medical industry in the future..