Effects Of Low Temperature Stress On Growth Of Gastrodia Elata By Transcriptome And Metabolome Analysis And Its Cloning And Functional Identification Of SOD And GS Genes

Gastrodia elata is a heterotrophic perennial herb symbiosis with fungi,and it is also a precious traditional Chinese medicinal material.Gastrodin and 4-hydroxybenzyl alcohol are the main medicinal components of G.elata,which have the effects of preventing and treating Alzheimer’s disease,Alzheimer’s disease,depression,stroke and improving memory.Due to its rich nutrition,G.elata is a popular health food at present.In recent years,with the continuous destruction of the ecological environment and manual mining,wild G.elata resources have decreased or are on the verge of extinction.At present,the supply of G.elata is in short supply,and artificial cultivation of G.elata has become the only effective way to meet market demand.Climatic conditions affect the growth and development of G.elata.Temperature is one of the key factors affecting the growth of G.elata.It affects the yield,quality and distribution of cultivated G.elata.Long-term low temperature stress will cause dark spots on G.elata,which will rot and die in severe cases.However,the physiological molecular mechanism of the effect of low temperature on the growth and development of G.elata has not been reported yet.This study takes G.elata grown at different temperatures as the research object,and uses transcriptome and metabolome analysis to study the gene expression levels of G.elata and metabolite composition changes in the body at different temperatures.Looking for the differential expression of key genes from different metabolites,inferring the regulation and influence of the growth and development of G.elata at different temperatures.Gene cloning and functional identification of the differentially expressed genes（DEGs）superoxide dismutase（SOD）and glutamine synthetase（GS）.The research results provide a theoretical basis for improving the yield and quality of G.elata and cultivating new cold-resistant G.elata varieties.The main results are as follows:1.This study used 13℃ female hemp（NS）,23℃ female hemp（TB）and 23℃ arrow hemp（SS）as experimental materials.A joint analysis of transcriptome and metabolome was performed to clarify the regulation of temperature on the growth and development of G.elata.Transcriptome analysis obtained 126787 Unigenes,and the number of Unigenes annotated to 6 major databases was 59501,which accounting for46.93%of the total Unigenes.Based on the threshold of significant difference expression|log2（multiple change）|≥1 and（P<0.05）,there are 17201,17162 and 10322differences in NS-vs-SS,NS-vs-TB,SS-vs-TB,respectively DEGs.Enrichment analysis of KEGG pathway showed that DEGs are mainly involved in metabolic pathways,biosynthesis of secondary metabolites,and RNA transport.A total of 437metabolites were detected in the metabolome,of which NS-vs-SS,NS-vs-TB and SS-vs-TB obtained 200,204 and 87 metabolites with different content,respectively.Among them,52,51,and 34 metabolites were up-regulated,and 148,153,and 53metabolites were down-regulated.These differences are distributed in sugar alcohols,amino acids,organic acids,lipids,nucleotides,phenolic acids and vitamins.Metabolomics data analysis shows that under the low temperature of G.elata,most of the levels of sugar alcohol metabolites,amino acids and their derivatives were down-regulated.They provide few nutrients for the growth of arrow hemp and can only maintain self-reliant life activities,which is not conducive to G.elata growth.The content of most lipids,organic acids,nucleotides and their derivatives,and phenolic acid metabolites were up-regulated.They were to prevent low temperature stress lipid antioxidant,phenolic acid secondary metabolism to eliminate ROS and increase,and organic acid and nucleotide metabolism to slow down and accumulate.The combined analysis of transcriptome and metabolome found that the content of metabolites was positively correlated with synthetase genes,and negatively correlated with decomposing enzyme genes.Through conventional analysis of MDA,H₂O₂,GSH and starch content of 4℃female hemp（FS）,NS,TB and SS,The results showed that with the decrease of temperature,the content of MDA,H₂O₂and GSH in G.elata increased,while the content of starch decreased.2.Based on the analysis of transcriptome data,superoxide dismutase（SOD）with significantly DEG was screened and the CDS sequence was found.The full-length sequence of SOD gene was 720 bp amplified by RACE-PCR technology,encoded 239amino acid residues.The prokaryotic expression vector of SOD gene of G.elata was constructed.Prokaryotic expression analysis showed that SOD is a soluble protein with a molecular weight of 47.4 k Da.The enzyme activity analysis showed that SOD is a metalloenzyme,the optimum temperature is 60℃,the thermal stability is better,and the optimum p H is 6.0.Different metal ions have different effects on SOD enzyme activity,and high concentrations of metal ions have a greater impact on SOD enzyme activity than low concentrations.The eukaryotic overexpression vector of SOD gene of G.elata was constructed.Transform into Arabidopsis sod mutant by Agrobacterium method to verify gene function.Transfect Armillaria with the Agrobacterium method to obtain engineered Armillaria transfected with SOD gene that can resist low temperature stress.Improve the antioxidant activity of genetically modified armillaria,and provide an experimental basis for further cultivation of cold-resistant G.elata.3.Based on transcriptome data analysis,screen out the significantly differentially expressed glutamine synthetase（GS）genes,find out the CDS sequence.The full-length sequence of GS gene was 1062 bp amplified by RACE-PCR technology,encoded 342amino acid residues.Construct the prokaryotic expression vector of G.elata GS gene.Prokaryotic expression analysis showed that GS is a soluble protein with a molecular weight of 59.94k Da.The enzyme activity analysis showed that the optimum temperature of GS is 50℃,the thermal stability is better,and the optimum p H is 4.0.Different metal ions have different effects on the enzymatic activity of GS.High-concentration metal ions have a greater impact on the enzymatic activity of GS than low-concentration.The eukaryotic overexpression vector of G.elata GS was constructed.Through transfection of Armillaria with Agrobacterium to obtain genetically engineered GS-transfected Armillaria.It can improve the resistance of engineered bacteria to low temperature stress.It is speculated that Armillaria containing the transgenic GS gene can promote nitrogen assimilation,reduce the oxidative damage caused by low temperature in the growth of G.elata,and increase the yield of G.elata.This study provides a theoretical basis for the cultivation of a new cold-resistant variety of G.elata.