Study On The Effect Of Methyl Jasmonate On The Ganoderic Acid Biosynthesis And Differently Expression Of Genes In Response To Methyl Jasmonate In Ganoderma Lucidum

Ganoderma lucidum is a traditional medicinal fungus. Modern researches showed that the chemical composition of G. lucidum complex have a wide range of pharmacological activities. Triterpenes in G. lucidum, also called ganoderic acid, which is one kind of the secondary metabolites, are considered as the main active ingredient. They have important medicinal value, such as:anti-tumor inhibits histamine release, anti-HIV and absorption effects and so on. Ganoderic acids (GAs)have the most important pharmacological activity, so their contents have been taken as an important measurement of the quality of G. lucidum. In the condition of very low amounts of GA, it is an effectively solution to improve the production of GA by changing environmental factors.As an endogenous regulator, methyl jasmonate (MeJA)plays an important roles for regulating the stress response, plant growth and development. This is the first time study to assess the novel use of methyl jasmonate (MeJA)to elicit GA biosynthesis in Ganoderma lucidum and the resulting experiments demonstrated that MeJA was indeed a potent inducer. To maximize GA synthesis, a statistical methodology called uniform design (UD)was used to optimize inducement conditions, which were determined to be254μM MeJA solubilized in Tween-20that was added to the culture on day6. The resulting GA yield was4.52mg/100mg dry weight.To characterize the effect of MeJA on GA biosynthesis, quantitative real-time PCR was used to measure transcription levels of several genes in the synthesis pathway including hydroxy-3-methylglutaryl-Coenzyme A synthase (hmgs), hydroxy-3-methylglutaryl-Coenzyme A reductase (hmgr), mevalonate-5-pyrophosphate decarboxylase (mvd), farnesyl pyrophosphate synthase (fps), squalene synthase (sqs) and oxidosqualene cyclase (osc). These data indicated that hmgr、fps、sqs were important genes in the GA biosynthesis.In this work, cDNA-AFLP was used to identify differential expressed genes in response to MeJA.64primer sets, each of which amplified to60transcript-derived fragments (TDFs), were used. Out of the total3910transcript derived fragments (TDFs) obtained using cDNA-AFLP with64primer pairs,919(23.5%)displayed altered expression patterns after induction, of which703showed up-regulated and216down-regulated.390TDFs produced reliable sequences after sequencing of485TDFs selected, of which91(18.8%) had known functions through BLAST searching the GenBank database. Compared with the publicly available databases,91TDFs presented some significant similarity with known sequences, whose functions were involved in metabolism, gene regulation, transcription factor, signal transduction, stress defense, etc.20homologous genes were further selected for validation of cDNA-AFLP expression patterns using qRT-PCR analyses. Results confirmed the altered expression patterns of20genes revealed by the cDNA-AFLP technique.According to the TDFs from the results of cDNA-AFLP, three signal transduction relative genes were successfully cloned using the RACE PCR and SEFA PCR techniques. These genes were MAPK (1789bp)、Rho (1182bp)and mob (1079bp), encoding small GTPase, mitogen-activated protein kinase and protein kinase activator, respectively. The promoter sequences were obtained from genomic DNA by SEFA PCR. Using PlantCARE software, TATA box and CAAT boxes were found. The potential responsive elements associated with light, MeJA, and stress factors were also found in the promoter region.In this study, the Gl-rho over-expression vector was transformed into G. lucidum by Agrobacterium tumefaciens-mediated transformation system. The gene was silenced by the dual promoter co-silencing system. The results show that, the expression level of Gl-rho gene in OE-rho9(B33)approximately2.5times than that in wild-type strain (wt). The expression level of Gl-rho gene in RNAi-rho-5(GTPi5) is approximately0.25times than that in wild-type strain (wt). The results show that, the triterpenes content was not changed in OE-rho transformants compared with wt. The content of triterpenes in RNAi-rho-5(GTPi5)approximately reached at131%of wt.