Cloning And Functional Identification Of GeCPR From Gastrodia Elata And Laccase Gene From Its Symbiotic Armillaria Mellea

Gastrodia elata?G.elata?is a kind of precious traditional Chinese medicine,which belong to fungus nutritive orchid plant.It can sedate and hypnotize,resist convulsion and vertigo,relieve pain,improve memory,treat senile dementia,and improve immunity.The main medicinal active ingredients of G.elata are gastrodin and p-hydroxybenzenyl alcohol.The total content of medicinal G.elata should not be less than 0.25%.The synthesis of active components in G.elata is related to cytochrome P450.Cytochrome P450 reductase?CPR?is an important component of cytochrome P450 enzyme family?CYPs?,which provides electrons for CYPs.Armillaeria mellea?A.mellea?is the most important large symbiotic fungus in the growth process of G.elata.The A.mellea invaded into G.elata will be digested by G.elata and transformed into nutrients for its own growth.The growth of A.mellea affects the yield and quality of G.elata.A.mellea mainly depends on the degradation of lignocellulose in the fungus material to small molecular organic matter such as glucose for providing its own nutrients of growth.The laccase activity of A.mellea is related to the utilization of bacterial material.At present,G.elata is a medicinal and edible plant.Market demand of G.elata is large,which being meted mainly by artificial cultivation of G.elata.However,the content of functional active ingredients and the efficiency of using bacterial materials in cultivating G.elata were lower.Therefore,this study using biological engineering technology,built A.mellea transgenic technology,and then through A.mellea transcriptome sequencing and based on the transcriptome data,screening out G.elata cytochrome P450 reductase gene?GeCPR?and A.mellea laccase genes?Lac?and finding out their CDS sequence;through the RACE-PCR amplification of the gene sequence,constructing Lac gene eukaryotic expression vector,and transforming it into A.mellea in order to improve the activity of laccase,which would improve the utilization efficiency of bacterial materiel and promote the growth of A.mellea.The eukaryotic overexpression vector of GeCPR gene was constructed and transferred into A.mellea to convert the precursor toluene into 4-hydroxybenzene-methanol,so as to improve the content of4-hydroxybenzene-methanol in A.mellea,and then the content of4-hydroxybenzene-methanol and gastrodin in G.elata was expected to increase by inoculation of G.elata,providing new cultivation techniques and methods for the production of G.elata.The main research results are as follows:The transgenic method of A.mellea was studied by using red fluorescent protein.The red fluorescent protein gene was amplified from plot1 vector,and the eukaryotic expression vector ph2gw-35s-mrfp1 was successfully constructed.Red fluorescence was observed in the transgenic A.mellea by agrobacterium-mediated transformation,indicating that agrobacterium-mediated method can be used for the transgenic method of A.mellea.Meanwhile,the influencing factors of agrobacterium-mediated method were optimized.Based on the transcriptome analysis data of A.mellea and G.elata,laccase genes of A.mellea for degradation of bacterial material were screened out and CDS sequences were found.The gene was cloned by RACE-PCR technology,which contain a total length of 1617bp and could encode 538 amino acid residues.The bioinformatics analysis of the protein predicted that the molecular weight of the protein was 59 kilodalton,and the theoretical PI value was 4.28.The protein was hydrophilic as a whole,with 42 amino acid sites showing different degrees of phosphorylation.Three-dimensional modeling of Lac protein showed that random curl was its main structure.In this experiment,the prokaryotic expression vector of laccase gene was successfully constructed and expressed in E.coli to obtain the recombinant protein.Recombinant protein on enzymology characteristics analysis found that:the optimum temperature for the enzyme to 40?,the optimal pH value of4.Metal ions,Fe²⁺,has inhibitory effect on the activity of the enzyme;Mg²⁺and Cu²⁺can promote laccase activity;Ca²⁺,K⁺,Na⁺and Zn²⁺ion have smaller influence on the activity of laccase.Cytochrome P450 reductase?GeCPR?related genes were screened based on the transcriptome analysis data of A.mellea and G.elata.Usin race-pcr technology cloned the gene with a total length of 2094bp,encoding 697 amino acid residues,among which leucine content was the highest.Based on bioinformatics analysis of the protein,it was predicted that the molecular weight of the protein was 77 kilodalton,the theoretical PI value was 5.28,and the protein was hydrophilic as a whole,with 67phosphorylation sites.In this experiment,the prokaryotic expression vector of GeCPR gene was successfully constructed and expressed in E.coli,and the recombinant protein was purified.Enzymology characteristics analysis showed that optimum temperature of 35?GeCPR enzyme;The optimal pH value is 8;Cu²⁺,Fe²⁺and Mg²⁺had inhibitory effect on enzyme activity;Zn²⁺had strong inhibitory effect on GeCPR activity;Ca²⁺,K⁺and Na⁺had no effect on enzyme activity.The eukaryotic expression vector of GeCPR gene pH2GW-35S-GeCPR was successfully constructed by Gateway technology.The strain can successfully catalyze the conversion of p-cresol to p-hydroxybenzenyl alcohol.