【作者】 张继星；

【导师】 李彦舫；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 红景天甙是一种新型高效适应原性药物，提取于我国珍稀药用植物高山红景天(Rhodiola sachalinensis A．Bor)。由于野生红景天植物资源日渐濒危，且红景天中红景天甙含量极低，远不能满足市场的需求，因此，利用生物工程和基因工程手段提高红景天甙含量引起了研究者极大的兴趣。根据相关文献报道和本实验室对红景天甙和甙元酪醇生物合成途径研究的相关进展，本文采用cDNA末端快速克隆技术(RACE)，以高山红景天为试材，分离得到与酪醇和红景天甙生物合成相关的两个基因，对其基因特性、基因功能进行了研究。酪醇在植物体内的合成目前尚不清楚。据我们实验室研究结果，酪醇的合成极有可能来自于生物碱合成途径中的酪氨酸代谢途径。本文首次从高山红景天中分离了在植物次生代谢中起关键作用的酪氨酸脱羧酶全长cDNA序列，命名为TyrDC1。对TyrDC1进行基因特性分析的基础上，构建了植物高效表达载体pCATyrDC1并利用农杆菌介导法转化回高山红景天，经过分子鉴定证明TyrDC1已经整合到高山红景天基因组DNA分子中并在转录水平表达。转TyrDC1基因高山红景天愈伤组织和植株的红景天甙含量HPLC测定结果表明，TyrDC1的过表达使红景天甙含量增加。酪醇糖基化后生成红景天甙是红景天甙生物合成途径中的最后一个关键步骤。这一关键反应是由UDP-葡萄糖基转移酶催化合成的。借鉴本试验室已有的工作基础，本文从高山红景天中克隆了UDP-葡萄糖基转移酶全长cDNA序列，命名为UGT3。对其基因特性、表达模式以及UGT3基因差异表达组织中红景天甙含量进行了分析，结果表明，UGT3在愈伤组织的红景天甙含量高于叶片的红景天甙含量。综上，通过对红景天甙生物合成途径上游、下游相关关键基因的分离、鉴定、研究为探索红景天甙生物合成代谢途径分子机制提供新的证据，为最终阐明红景天甙的生物合成代谢通路奠定了基础。更多还原

【Abstract】 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..更多还原