Heterologous Synthesis Of Ginsenoside Rh2 In Tobacco

Panax herb mainly includes Panax ginseng,Panax notoginseng and Panax quiquefolium.It is a traditional Chinese herb commonly used in China and has the highest output value in the world.The main active components of Panax herb are triterpene saponins.While the outstanding pharmacological activities in some rare monomer saponins offer good potential for medicinal development,their low content in herb restricts the systematic and in-depth pharmaceutical research and development and the application of these saponins.In recent years,the rapid development of synthetic biological technology provides a new way and a new idea for the heterologous synthesis of rare ginsenosides.Among the currently known rare ginsenosides of Panax,ginsenoside Rh2 has attracted much attention because of its excellent antitumor activity,but its content in Panax herb is very low.In order to obtain more ginsenoside Rh2 for research and development,synthetic biological technology can be used for heterologous expression in the species that are easy to cultivate(plant)and have large biomass.In this study,the heterologous synthesis of ginsenoside Rh2 in tobacco was successfully realized by artificially constructing a three-step enzyme catalytic reaction in tobacco.In this study,we first cloned damanediol synthase gene(PnDDS),protopanaxadiol synthase gene(CYP12H)and glucosyltransferase gene(UGTPn3)from Panax notoginseng cells by PCR.The three genes were connected with the expression vector pCAMBIA2300s respectively,and constructed the plant expression vector pCAMBIA 2300s-PnDDS/CYP12H/UGTPn3.At the same time,the prokaryotic expression vector of UGTPn3 was constructed and the three-dimensional external enzyme catalytic system was established.The constructed overexpression vector was transformed into Agrobacterium tumefaciens,and the tobacco leaves were soaked by leaf disc method.The positive tobacco plants were screened at the DNA and RNA levels of transgenic tobacco.The contents of damanediol(DD),protopanaxadiol(PPD)and ginsenoside Rh2 in the roots,stems and leaves of transgenic tobacco were detected by HPLC.The vitro enzymatic studies showed that UGTPn3 could catalyze the glycosylation of PPD C-3 to produce ginsenoside Rh2.We screened a variety of transgenic tobacco plants expressing PnDDS alone,PnDDS and CYP12H at the same time,and pndds,CYP12H and UGTPn3 at the DNA and RNA levels.It was gradually proved that transgenic tobacco has the ability to synthesize DD,PPD and ginsenoside Rh2,and the content of DD,PPD and ginsenoside Rh2 in the roots,stems and leaves of transgenic tobacco plants was positively correlated with the expression of PnDDS,CYP12H and UGTPn3.The distribution of ginsenoside Rh2 in transgenic tobacco was specific to the organ,and the content was the highest in roots(5.30 μg/g),followed by the middle leaf(3.30 μg/g),the lowest content in stem(2.00 μg/g).On the basis of obtaining transgenic tobacco plants synthesizing ginsenoside Rh2,we further studied the ability of tobacco cells to synthesize ginsenoside Rh2 in vitro.The results showed that there was no significant difference in cell growth between transgenic tobacco cells and wild-type tobacco cells in vitro.To a certain extent,the three foreign genes transferred in this study had little effect on the normal physiological activity of tobacco cells.In addition,plant hormones can increase the expression of key genes in the main metabolic flow of saponin synthesis in tobacco cells and promote the synthesis of ginsenoside Rh2.Among them,2,4-D has the best effect,and the content of ginsenoside Rh2 in cells is the highest,which is 38.67 μg/g.