Transcriptome Metabolome Association Analysis And Flavonol Synthase Gene (AvFLS) Function Of Apocynum Venetum L. Under Salt Stress

Apocynum venetum L.is a perennial herb of the oleander family Apocynum,and the whole plant can be used as medicine.Flavonoids are the main active components of A.venetum,and play an important role in the resistance of A.venetum,to environmental stress.At present,the change pattern of flavonoids in A.venetum,under salt stress,as well as key differential genes and key differential metabolites are still unclear.In this study,we planned to analyze the changes of flavonoids under salt stress through physiological experiments,and combined transcriptomic and metabolomic analysis to screen the key regulatory gene flavonol synthase gene(Av FLS),carry out Av FLS expression pattern analysis,transform Av FLS into Arabidopsis thaliana,and the salttolerance function of transgenic A.thaliana was initially studied.The main results obtained are as follows:1.Under salt stress,the chlorophyll content of A.venetum increased at first and then decreased.Antioxidant enzymes actively respond to remove excess free radicals in the body;The content of flavonoids increased significantly with salt concentration and treatment time,and maintained osmotic pressure balance with soluble sugar and proline.The results showed that flavonoids played an important role under salt stress.2.Through the correlation analysis of A.venetum transcriptome and metabolites under salt stress,key differential metabolites such as flavonol,eriodictyol and rutin were screened.Screening key differential genes such as Flavonol synthase(Av FLS),chalcone isomerase(Av CHI),dihydroflavonol 4-reductase gene(Av DFR)and UDP-glycosyltransferase gene(Av UGT);Correlation analysis showed that 59 MYB transcription factors had significant positive regulation on 42 flavonoid synthesis genes.Among them,MYB transcription factor(Cluster-7324.22368)plays a positive role in regulating 11 flavonoid synthetases.The above-mentioned differential genes,metabolites and MYB transcription factors play an important role in the metabolic regulation of flavonoids in apocynum venetum under salt stress,so it is necessary to further study them.3.In this study,four different algorithms(Best Keeper,Norm Finder,ge Norm and ref finder)were used to evaluate the expression stability of 10 candidate reference genes under salt stress.The results showed that the most stable gene expressed in A.venetum under salt stress was Actin.Taking actin as the internal reference gene,the relative expression level of the key differential gene Av FLS was detected.The gene expression of Av FLS gene in leaves was significantly higher than that in roots and stems,and increased with salt stress.This study provides a reference for analyzing the expression pattern of A.venetum gene and selecting reference genes under salt stress,and shows that Av FLS plays an important role in the response of A.venetum to salt stress.4.The results showed that overexpression of Av FLS gene in A.thaliana can increase the content of flavonoids and photosynthetic rate,reduce the content of MDA,and significantly improve the salt tolerance of transgenic A.thaliana.In conclusion,Av FLS gene in A.venetum is induced by salt stress and plays an important role in resisting abiotic stress.The above research results lay a certain foundation for the development and utilization of A.venetum as a feed additive in the field of animal husbandry and the molecular breeding and improvement of forages in saline alkali land.