Regulation Of The Secondary Metabolism Of Glycyrrhiza Uralensis By Methyl Jasmonate

Glycyrrhizae Radix Et Rhizoma as one of most popular Chinese medicine become more and more widespread with the development of modern pharmacological researching, resulting in a dramatic increasing in global demand. Currently, the wild Glycyrrhizae resource has been depleted, and the artificial cultivation quality was poor because of lacking cultivation techniques and having many influence factors. Therefore, adopting manual intervention method is an effective way to improve the quality of Glycyrrhiza uralensis in appropriate cultivation period.In this experiment, we were tested triterpenoid and flavonoids active ingredient, and related gene expression of G. uralensis which was induced by methyl jasmonate(MeJA). Discuss the regulation and mechanism of secondary metabolism of G. uralensis by MeJA. Provide a basis for improving the quality of cultivated licorice through chemical regulation. And Selected GubAS to be cloned and constructed expression vector according to the result. Laying the foundation of obtaining transgenic plants or transgenic hairy root to study genes function, to plant good varieties and produce active ingredient of G. uralensis through biotechnology.The main research methods and results are as follows:1. The regulation of active ingredient accumulation in G. uralensis by MeJA: We were used 20, 50, 100 μmol/L MeJA processing 6 months growth G. uralensis aerial part ecery three day, and altogether process three times. Measure the active ingredient glycyrrhizic acid and liquiritin through HPLC. G. uralensis roots and rhizomes treated by MeJA had a significant effect on its active ingredient accumulation, especially at a concentration of 20, 50 μmol/L MeJA which promote the accumulation of glycyrrhizin significantly. 20, 50 μmol/L MeJA had no significant effect on the content of liquiritin, and 100 μmol/L MeJA treat inhibited the accumulation of liquiritin. It is suggested that MeJA in suitable concentration will promote glycyrrhizin accumulation, but have no effect or inhibitory effect to liquiritin accumulation of G. uralensis roots and rhizomes.2. The regulation of key biosynthetic gene expression in G. uralensis by MeJA: We were used 20, 50, 100 μmol/L MeJA spraying 6 months G. uralensis aerial part and harvested in 2, 6, 12, 24, 36, 72 h treatment, used real-time quantitative PCR analysising triterpenes biosynthetic pathway key enzymes genes β-amyrin synthase(GubAS), β-amyrin-11-oxidase(Gub AO), and flavonoid biosynthetic pathway key enzyme gene 6-deoxychalcone synthase(GuDOCS) relative expression level of G.uralensis root. The result proved that 20 μmol/L of MeJA treated 0 ~ 6 h had a role in promoting GubAS, GuDOCS gene expression levels, and processed 2 ~ 6 h can increased the amount of expression in GubAO of licorice. 50 μmol/ L MeJA treated 0~2 h improved the 3 gene expression levels, but 6 h treatment came up GuDOCS expression suppression. 100 μmol/ L MeJA had no significant effect on GubAS and GubAO; GuDOCS expression improved when 100 μmol/L of MeJA was treated 0~2 h, then decreased gradually. It is suggested that MeJA in low concentration and short treatment will promote active ingredients accumulating and its’ biosynthetic genes expressing in G. uralensis roots and rhizomes, and will have no effect or inhibitory effect with MeJA concentration increased and processing time extended.3. GubAS cloned and constructed expression vector: We used full-length cDNA sequences of GubAS which got in previous transcriptome sequencing study to obtain recombinant clone pMD-GubAS. Purifying and double digesting pMD-Gub AS to get the target gene GubAS, and connected GubAS to pBI121 expression vector fragment which have the same restrictions endonuclease enzyme restriction sites in order to construct expression vector pBI-GubAS. Then, we detected accuracy of pBI-GubAS by PCR amolification and sequence technique. The plasmid construction providing the basis for obtaining transgenic plants or transgenic hairy root to study genes function, to plant good varieties and produce active ingredient of G. uralensis through biotechnology.