Cloning And Gene Expression Analysis Of Grifola Frondosa Farnesyl Diphosphate Synthase Gene Sequence

Grifolafrondosa(Basidiomycete,Agaricomycetes,Polyporales,Meripilaceae,Grifola)as a kind of fungus crops,is commonly known as‘yunxun’,‘lizimo’,the mushroom is known by its Japanese name maitake(舞茸,"dancing mushroom").As a kind of important edible and medicinal fungi,Ithadreduced insulin resistance,enhanced the sensitivity of insulin to control blood sugar,enhanced the human immunityfunction,also inhibited the accumulation of fat cells,reduced blood pressure,inhibited the effect of AIDs.Triterpenoids were important secondary metabolites of G.frondosa,and also one of the important standardto measure the effect of G.frondosa.It hadbeen verified that farnesyl diphosphate synthase played a critical role in the synthetic pathway of triterpenoids via analysis of those pathways,and its activity and expression level coulddirectly impact the yields of subsequent products.Therefore,studiedon the genes and the regulatory mechanism of the expression of farnesyl diphosphate synthase in G.frondosacouldfurther interpret the molecular mechanism of triterpene synthesis in G.frondosa.Firstly,the amino acid sequences of farnesyl diphosphate synthase in species near in genetic relationship with G.frondosa were screened from NCBI’s Genbank,and the comparison of multiple sequences was conducted by DNAMAN,so as to find out the conserved regions of amino acids,on which basis the degenerate primers were designed.Secondly,polymerase chain reaction(PCR)amplification was performed with total clump cDNA of G.frondosa as the model,so as to obtain a chain of specific genetic segment of farnesyl diphosphate synthase in G.frondosa,with a length of 825 bp.Thirdly,based on the specific genetic segment,5’-RACE method was used to design specific premiers,so as to obtain the 5’-segment sequences of farnesyl diphosphate synthase in G.frondosa by amplification,after which 3’-RACE method was applied to amplify the 3’-segment sequences of farnesyl diphosphate synthase in G.frondosa.Fourthly,the 3’-and 5’-segment sequences were analyzed to find out the promoters and terminators,and to design the specific premiers around them.And then,complementary cDNA of G.frondosa was used as the model to obtain the complete sequence of farnesyl diphosphate synthase by amplification,with a total length of 1086 bp.Finally,the cDNA sequences of farnesyl diphosphate synthase in G.frondosa were cloned to pYF1845,a prokaryotic expression vector,and was then introduced to ergosterol-dependt yeast strain CC25.The successfully introduced positive transformants could grow successfully in SC(-ura)culture medium at 30℃.In the natural growing environment of G.frondosa,some unknown ingredients might promote the growth of G.frondosa and the accumulation of effective substances.Therefore,to explore the influence of exogenous microbial metabolites or growing environment on the yields of triterpenoids of G.frondosa during their synthetic pathways,the water decoction of barks and rhizosphere soil of chestnut,the host of G.frondosa,the jasmonic acid methyl ester solution,and the rhizobiumfermentation liquor andthe fermentation liquor ofCasuarina frankia were applied as inducers in this study to induce the growth of hypha of G.frondosa,after which the biomass of mycelium and the expression level of farnesyl diphosphate synthase were adopted as reference values to screen the final inducer and its concentration.The results revealed that fermentation liquor of Casuarina frankiahad the highest effect on the increase of mycelium biomass,followed by rhizobium fermentation liquor,jasmonic acid methylester solution,water decoction of barks of chestnut,and the water decoction of chestnut rhizosphere soil in sequence.The expression level of farnesyl diphosphate synthase was the highest when rhizobium fermentation liquor was the inducer,followed by Frankiaceae fermentation liquor of C.Frankia,barks of chestnut,and jasmonic acid methylester solution in sequence,whereas the water decoction of rhizosphere soil of chestnut had obvious inhibitive effect on the expression level of farnesyl diphosphate synthase of G.frondosa.