Creation Of Genetically Engineered Armillaria Mellea Producing Gastrodin

Gastrodia elata(G.elata)is a traditional Chinese medicinal herb that can be used both as a medicine and as a food.It has various pharmacological effects such as anti-epilepsy,anti-fatigue,and improving learning and memory.The main components of G.elata are gastrodin and 4-hydroxybenzyl alcohol,which cannot be lower than 0.25% according to the pharmacopoeia.Gastrodin is extracted from G.elata,but the content of Gastrodin in G.elata is low and the growth cycle is long,making extraction more inconvenient;If obtained through chemical synthesis,the by-products are more toxic and prone to environmental pollution,so the advantages of biological extraction method are more obvious.Using engineering bacteria to produce gastrodin can shorten the production cycle of gastrodin and reduce environmental pollution.Armillaria mellea(A.mellea)is a fungus of the genus Armillaria,which can effectively degrade components of plant cell walls(such as lignin,cellulose,etc.),and its degradation products provide nutrients for plants as humus in the soil.A.mellea not only can provide nutrition for G.elata,Polyporus umbellatus,etc.,but also has medicinal value.The construction of genetically engineered A.mellea producing gastrodin not only has its own value,but also has the pharmacological activity of gastrodin,or may provide certain conditions for alleviating the economic pressure of gastrodin production.In this paper,we analyzed the possible synthesis pathway of gastrodin in G.elata through literature review and transcriptome analysis and screened the transcriptome of G.elata,to obtain the differentially expressed genes cytochrome CYP450(CYP450)and glucosyltransferase gene(UGT).These gene were cloned and functional identified.The two genes were transferred into A.mellea to construct a pathway for producing gastrodin in A.mellea.The main results obtained are as follows:1.Based on the transcriptome data from the previous laboratory and the literature reviewed,the differential gene cytochrome CYP450(CYP450)was obtained from the transcriptome data of G.elata.The full-length gene sequence was amplified and obtained,with 2094 bp,encoding 697 amino acid residues,and a molecular weight of76.78 k Da.Bioinformatics analysis showed that the main components of its protein secondary structure were α Helical and irregularly curled,tertiary structure is oligonucleotide monomer,not signal peptide is a transmembrane protein,but a hydrophilic protein with 68 phosphorylation sites.The prokaryotic expression vector p EGX-4T-CYP450 was constructed to induce protein expression.Analysis showed that the protein was an inclusion body protein.A subcellular localization overexpression vector p RI101-35s-GFP-CYP450 with a GFP tag was constructed and transiently transfected into tobacco.It was found to be localized in the endoplasmic reticulum and nucleus.2.Based on the transcriptome data from the previous laboratory and literature reviewed,the differential gene glucosyltransferase gene(UGT)was obtained from the transcriptome of G.elata.The full-length sequence of the gene was amplified,with1419 bp,encoding 472 amino acid residues,and molecular weight of 52.03 k Da.Bioinformatics analysis showed that the main components of its protein secondary structure were α Helical and irregularly curled,the tertiary structure is an oligonucleotide monomer,not a signal peptide or transmembrane region,and is a hydrophilic protein with 40 phosphorylation sites.Construct a prokaryotic expression vector p ET-32a-UGT,induce protein expression,and obtain UGT enzyme protein.A subcellular localization overexpression vector p RI101-35s-GFP-UGT with a GFP tag was constructed and transiently transfected into tobacco.It was found that it was localized on the cell membrane.3.Construction of transgenic A.mellea: Construction of eukaryotic overexpression vectors p H2GW7-35s-CYP450 and p H2GW7-35s-UGT,transfer of CYP450 and UGT genes into A.mellea through Agrobacterium tumefaciens transfection method,screening of marker genes to obtain A.mellea strains,extraction of their DNA for PCR identification,confirming that A.mellea has been transformed into the target gene,obtaining single transformed CYP450 and single transformed UGT genetically engineered A.mellea,and starting from the UGT transformed A.mellea,PRI101-35s-GFP-CYP450 was transfected into A.mellea using Agrobacterium tumefaciens to obtain engineering A.mellea with dual CYP450 and UGT transformation.At the same time,in order to prove that the engineered A.mellea has the function of converting the substrate into 4-hydroxybenzyl alcohol and gastrodin,the substrate was added to the liquid culture medium for a period of time,and the efficiency of the engineered bacteria in converting the substrate into a product was measured using HPLC methods.Using the gene of G.elata to construct a pathway for the synthesis of gastrodin in A.mellea has certain significance for the production of gastrodin,and provides a certain foundation for the efficient production of gastrodin from G.elata.