Research Of Transferring Ginsenoside Biosynthetic Key Enzyme βAS Gene Into Rice

Belonging to Araliaceae ginseng species, Ginseng is a traditional precious herb, and has the functions of enhancing immunity, anti-tumor, anti-atherosclerosis to protect the myocardium. Ginsenoside, the main components of ginseng, is dammarane tetracyclic triterpenoids or hong resin alkyl type pentacyclic triterpene, which has not yet been synthesized artificially. Rice and ginseng have similar metabolic pathways with a common precursor substances-squalene. The squalene of rice synthesizes sterols under the catalysis of central a-tuen alcohol synthase(CS) in rice, while the squalens of ginseng synthesize triterpenoids saponins like saponin under the catalysis of ginsenoside biosyntheticβ-amyrin synthase (βAS). Therefore, by transferring the key gene of ginsenoside synthesis into rice, we can produce quality rice with saponin. The main results of this study are as follows:1. By PCR method, from the Panax ginseng C.A.Mey, up to 15% of which is total saponin , we cloned the open reading frame (2286bp) of ginsenoside biosynthetic key enzymeβAS gene, encoding 762 amino acids.2. By a safety strategy of double T-DNA genetic cotransformation,βAS gene and selectable marker genes were respectively constructed in two different T-DNA insections of the same vector, with a T-DNA carrying the selectable marker gene hpt, the other T-DNA carring theβAS gene promoter (GluB-1 promoter). Both sides of the geneβAS had two positive sequences of tobacco Rb7 MARs, which improved the expression ofβAS. We made a plant expression vector "pCDMAR-βAS-hpt" that can expressβAS gene effectively in rice endosperm.3. Using Agrobacterium-mediated method, the recombinant vector pCDMAR-βAS-hpt was transferred into high-quality rice "Taijing 9". By the resistant screening to hygromycin, we obtained 20 resistant plants, of which there were 6 positive plants after PCR detection.