Studies On The Effect Of MeJA On Flavonoid Accumulation In Euphorbia Kansui And The Function Verification And Expression Regulation Of EkFLS(Flavonol Synthase) Gene

Euphorbia kansui is a perennial herb of Euphorbia?Euphorbiaceae?,which is widely used for curing pleural effusion,ascites of cirrhosis,cough and asthma,edema,tumor and other diseases as a Chinese unique medicinal plant.It is generally believed that its medicinal ingredients mainly include terpenes?diterpenes,triterpenes?,steroids and flavonoids.There is no doubt that flavonoid synthesis pathway is one of the important metabolic pathways in plants,and is of great significance for plant growth and resistance to external abiotic stress.In addition,clinical research has proved that flavonoids in various plants have antibacterial and antitumor activity and can prevent diabetic nephropathy and regulate blood lipids.Therefore,flavonoids are widely used in industrial production and medical-health fields with important scientific research and commercial value.The synthesis of the secondary metabolites in plants is affected by environmental factors and signal hormones.The Jasmonate compounds?JAs?is a typical signal regulator.As a new type of plant endogenous hormone,JAs has been found to regulate flavonoid anabolism in plants.Many literatures have reported that flavonol is the main type of flavonoids in Euphorbiaceae,which is derivatived of flavone and catalyzed by flavonol synthase?FLS?.FLS is one of the key enzymes for synthesizing flavonoids in plants.However,the effect of Methyl jasmonate?Me JA?on flavonoid accumulation in Euphorbia kansui and the function verification and expression regulation of Ek FLS?flavonol synthase?gene have not been systematically studied.In this study,E.kansui was selected as the experimental materials to analyze the effect of Me JA on the accumulation of flavonoids and the related gene expression trends.After performing the informatics analysis and prokaryotic expression,Ek FLS gene was transferred into Arabidopsis thaliana for overexpression.Through the overexpression of Ek FLS gene and corresponding stress treatment analysis,the function of Ek FLS was clarified,which laid a theoretical foundation for the in-depth study on the molecular regulatory mechanism of FLS gene and the molecular mechanism of plant stress resistance,improving the content of plant flavonoids and enhancing plant stress resistance.The main results are as follows:1. After being treated with 100?mol/L Me JA,the content of the total flavonoids in E.kansui roots,stems and leaves of the treated group and the control group was measured by ultraviolet spectrophotometer.The results showed that there were significant differences in the total flavonoid content among different tissues,which showed that the leaf of had the highest total flavonoid content?2.286 mg/g on average?,followed by the stem?0.428 mg/g on average?and the root?0.184 mg/g on average?.After Me JA induction,the total flavonoid content in the leaf increased by2.4%-23.8%,and reached the maximum value at 36 h after treatment.The total flavonoid content in the root increased by 24.4%-73.3%,and reached the maximum value at 12 h after treatment.The content of total flavonoid in the stem was not significantly changed.In addition,flavonols of E.kansui were extracted and it was found that E.kansui contains quercetin and kaempferol testing by TLC and HPLC,with the content of 0.128 mg/g and 2.165 mg/g respectively.2. Five key enzyme genes in the flavonoid synthesis pathway were screened from the E.kansui transcriptome database?NCBI Sequence Read Archive database:SRP126436?:chalcone synthase?CHS?,anthocyanin synthase?ANS?,dihydroflavonol reductase?DFR?,chalcone isomerase?CHI?and flavonol synthase?FLS?.RT-q PCR technique was used to analyze the gene expression levels in different times after Me JA treatment,and the results showed that Ek CHS?unigene045546?,Ek CHS?unigene 052162?,Ek DFR?unigene 080108?,Ek FLS?unigene078670?,Ek ANS?unigene 075390?were up-regulated significantly,and the expression reached the highest level at 12 h after treatment;the expression of Ek DFR?unigene 080109?increased to the highest level after 36 h of treatment;Ek ANS?unigene 075388?was quickly reduced and then gradually returned to normal;Ek CHI?unigene 040233?showed no obvious change.3. The encoding sequence?CDS?of FLS was found from E.kansui transcriptome database?NCBI Sequence Read Archive database:SRP126436?,and the full-length c DNA of Ek FLS was cloned by RT-PCR and gene specific primers.Then two vectors for prokaryotic expression?p DE1:T7-His-FLS?and transgenic overexpression?p CAMBIA1300:35S-FLS?were built respectively.4. In order to further understand the mechanism of Ek FLS and its encoded protein,bioinformatics analysis on Ek FLS were conducted:The coding sequence?CDS?of1023 bp encoded a 340 amino acids polypeptide with a molecular weight of38.67 k Da and an isoelectric point?p I?of 6.19.The Ek FLS protein belongs to the flavonol synthetase superfamily.It is an acidic non-transmembrane protein located in the cytoplasm,whose main structural elements are alpha-helix and irregular curl.Phylogenetic analysis revealed that Ek FLS has a close relationship with FLS protein from Citrus and has a higher degree of evolution than these proteins in Citrus.SDS-PAGE analysis showed the optimal conditions for the prokaryotic expression of Ek FLS:the medium accounted for 10%of taper bottle volume,when bacteria growth to OD₆₀₀=0.9,added IPTG with 0.6 mmol/L final concentration,then cultivated in37?for 6 hours.5. The encoding region of Ek FLS was cloned into the vector under the control of the 35S promoter and transformed into Arabidopsis thaliana to obtain the transgenic plant.In order to further prove the regulation effect of Me JA on plant flavonoids,100?M Me JA was used to treat the wild-type and transgenic Arabidopsis thaliana,results showed that Me JA effectively up-regulates the expression of flavonoid pathway-related genes and the accumulation of flavonoids.In addition,Na Cl and PEG were used to simulate salt and drought environment.Compared with the wild type,the relative expression of genes related to the flavonoid synthesis and ROS scavenging pathway in transgenic Arabidopsis thaliana were significantly upregulated,the content of flavonoids and the enzyme activities of POD,SOD was also significantly increased.These results indicated that Arabidopsis thaliana with over-expressing Ek FLS increases the content of the total flavonoid and flavonol by up-regulating the relative expression of genes of key enzymes in flavonoid synthesis,and also enhances the activity of antioxidant enzymes by up-regulating the expression of genes of key enzymes in ROS scavenging pathway,then enhances the antioxidant capacity of plants,thereby improves the plant tolerance to Me JA,salt and drought.