Cloning And Functional Analysis Of MYC2 Gene

Dryopteris fragrans(L.)Schott is a deciduous fern in Dryopteris family of Dryopteris genus,which grows in the alpine region.It has been used for decades to treat skin diseases with the leaves of t Dryopteris fragrans.History,as a wild medicinal resource plant,A.sinensis has important application prospects.In recent years,studies have shown that there are many compounds such as phenols and terpenoids in the genus A.The preliminary work of this laboratory has proved that the steroidal metabolites of Dryopteris fragrans have antibacterial and anti-tumor effects.However,the current metabolic pathways for ferns have not been reported.Therefore,it is of great significance to study the metabolic pathways of t Dryopteris fragrans and its genus.The biosynthesis of terpenoids in plants is largely induced by insects or microorganisms.In production practice,exogenous application of jasmonic acid is often used to simulate insect and fungal diseases to induce indirect defense responses of plants to study the accumulation of steroid metabolites.MYC2 is an important transcription factor in plant response to the jasmonic acid metabolism pathway,and plays an important role in regulating plant secondary accumulation of metabolites such as terpenoids and inducing plant defense responses.Therefore,in this study,the jasmonic acid hormone JAs was used as an exogenous hormone to treat the leaves of Dryopteris fragrans,and the expression of MYC2 transcription factor was regulated,and the heterologous expression of MYC2 in Arabidopsis thaliana was explored.Metabolic pathways of Dryopteris fragrans.The main experimental results of this paper are as follows:1.A fragment annotated as the MYC2 gene was found in the transcriptome database of the whole line of Dryopteris fragrans.By screening,one of the four gene fragments was selected as a core fragment of the MYC2 gene.Based on the core fragment sequence,the full-length gene was 3130 bp,the open reading frame was 2496 bp,and it was named Df MYC2.The gene accession number on NCBI was MK193871.The expression of Df MYC2 gene was analyzed by roots,rhizomes,petioles and leaves.The results showed that the expression level was the highest in leaves,followed by petiole expression,and the lowest in roots and rhizomes.Then,Me JA was used to exogenously treat Dryopteris fragrans,and the results showed that the expression of MYC2 gene increased with time and decreased after Me JA treatment.3.Using bioinformatics technology to analyze and predict the primary structure,secondary structure and tertiary structure of MYC2 protein.The results showed that the MYC2 protein encodes 831 amino acids,contains three secondary structure types,and has a signal peptide,which is a secreted protein,and has no curly structure and signal peptide.4.Construction of plant expression vector The Arabidopsis thaliana wild type plant was genetically transformed to obtain Df MYC2 gene overexpressing plants,and the phenotype was observed.It was found that the overexpressed plants grew faster than the wild type plants in the same period,indicating Df MYC2 Gene expression can promote the growth of Arabidopsis thaliana plants;the number of epidermal hairs on the petiole of over-expressed plants is significantly higher than that of wild-type plants,and the expression levels of sesquiterpene synthase genes TPS11 and TPS21 are increased in overexpressing plants,demonstrating Df MYC2 gene The expression of the sesquiterpene synthase gene can also be promoted in Arabidopsis.The At MYC2 deletion plant was obtained by CRISPR/Cas9 technology,screened and observed for phenotype.The results showed that the deletion plant grew slower than the wild type Arabidopsis thaliana and the number of epidermal hair on the petiole was significantly reduced.It indicated that At MYC2 gene also affected the growth and epidermal hair development of Arabidopsis thaliana plants;while the expression levels of sesquiterpene synthase genes TPS11 and TPS21 were significantly decreased in the deletion plants.6.The Df MYC2 gene was transfected into the At MYC2 deletion homozygote to obtain Df MYC2 complementary At MYC2 deletion type Arabidopsis thaliana.The results showed that the At MYC2 gene-deficient plants grew slower than the wild-type Arabidopsis thaliana and the number of epidermal hairs on the petiole was significantly reduced,and the expression levels of the sesquiterpene synthase genes TPS11 and TPS21 in the deleted plants were significantly lower,indicating At MYC2.The gene not only affects Arabidopsis plant growth and epidermal hair development,but also affects the expression of the sesquiterpene synthase gene.7.Df MYC2 gene was transferred into tobacco for subcellular localization analysis.It was found that Df MYC2 gene was mainly expressed in the nucleus.