Cloning And Transformation Into Tobacco Of Key Enzyme Gene HMGR In Pseudostellaria Heterophylla

Pseudostellaria heterophylla, Taizishen (TZS), is a root of Pseudostellaria plants in caryophyllaceae family, which has high-medical values such as anti-fatigue, anti-stress, promoting the immune system in traditional Chinese herbal medicine. Saponins are largely distributed in nature with significant pharmacological activities, which is a kind of important secondary metabolites in plants, its main function is involved in plant’s stress resistance and protecting against the dangers of pathogens and herbivores. Saponins in Taizishen are the main medicinal composition, which has function of anti-fatigue, oxidative resistance, low temperature resistance, weighting the immuneorgan and activating the phagocytosis of reticuloendothelial system.3-hydroxy-3-methylglutaric acyl coenzyme A reductase (HMGR) is one of limited enzyme in MVA pathway of plants. Cloned HMGR from Taizishen and researched its function is of significance in helping understand the synthesis and metabolism of saponins in Taizishen, so the content of saponins will be increased and a better economic benefit will be created. Cultivation was concentrated in recent research, with a lack study of the active ingredient in molecular biology.In this study, HMGR full length cDNA from Taizishen is isolated by RT-PCR and RACE. The expression vectors of HMGR were built and transferred into tobacco K326 by agrobacterium mediated method. then extracted and analyzed the total triterpenes content and HMGR enzyme in genetically modified and non-genetically modified tobacco. The main results were as follows:1、HMGR is the key enzyme gene of saponins synthetic in Taizishen and HMGR full length cDNA from Taizishen is isolated and identified by RT-PCR and RACE. The full length of HMGR is 2137 bp, with a 1674 bp open reading frame that encodes a protein of 557 amino-acid was obtained. The coding ORF has 59.327 kDa molecular weight and 7.58 pI, which was hydrophobic protein. The HMGR amino acids from Taizishen shared 76.7% homology with Panax ginseng C. A. Mey, shared 77.1% homology with Eleutherococcus senticosus and Raphanus sativus L., shared 77.7% homology with Arabidopsis thaliana, which candidate sequence has been speculated.2、The recombinant vector pRi-HMGR was gain by connecting the ORF of HMGR and pRi 101-AN, and then transferred into tobacco K.326 with agrobacterium-mediated method. There are 30 positive tobaccos under 93.75% transformation rate through PCR.3、Extracted and detected the saponins content in genetically modified and non-genetically modified tobacco by the method of microwave and Vanillin-perchloric acid reaction system. Compared with the non-genetically modified plants, the saponins content of genetically modified tobacco were higher significantly. The maximum content of genetically modified plants was 16.64%,2 times at most to non-genetically modified plants. They showed HMGR has been expressed in the tobacco, and has improved the saponins content of plants.4、In this study, the assay system of HMGR enzyme activity has been optimized, as well as HMGR enzyme activity of genetically modified and non-genetically modified tobacco has been detected. The result showed its optimum pH was 6.0, optimum temperature was 26℃, and it has enzyme activity until 45 min ultraviolet radiation. The HMGR enzyme activity of genetically modified tobacco was higher significantly than non-genetically modified ones. The most of HMGR enzyme activity of genetically modified plants was 0.036 U, three times to non-genetically modified tobacco.