Cloning And Functional Analysis Of Genes Encoding Key Enzymes Of Flavonoid Synthesis From Atriplex Canescens

Soil salinization can cause various harms such as ion imbalance,osmotic stress and oxidative damage to plants and is one of the most serious abiotic stress factors affecting crop growth and limiting land resource utilization.To adapt to the harsh saline desert environments,plants inhabiting in these regions for a long time have evolved a series of unique mechanisms for salt resistance.Investigating the salt resistance mechanisms and isolating the key genes related to salt tolerance in these species will lay theoretical foundations for the improvement of crops and forages.Atriplex canescens,a typical halophyte with strong salt tolerance and drought resistance,grows in extremely extremely barren saline desert areas for a long time.Flavonoids are a kind of important antioxidants with strong ROS-scavenging ability and are widely involved in the process of plants stress resistance.Our previous transcriptome sequencing analysis showed that the expressions of a large mount of genes encoding key enzymes involved in flavonoid synthesis in leaves and roots of A.canescens were significantly up-regulated under salt condition,so we preliminarily speculated that flavonoids may be closely related to the strong salt tolerance of this species.Meanwhile,previous studies on the salt tolerance mechanism of A.canescens mainly focused on ion transport,osmotic regulation,photosynthesis,water transport and antioxidant enzymes,while few studies focused on the roles of antioxidants in its salt tolerance.In the present study,therefore,we tried to preliminarily determine whether flavonoids are related to salt tolerance of A.canescens by analyzing the content of flavonoids in leaves and roots of A.canescens under NaCl treatments.Then,three genes encoding key enzymes involved in flavonoid biosynthesis of A.canescens were cloned,and their expression patterns under salt treatments were analyzed.Finally,two genes were transferred into Arabidopsis thaliana to explore their roles on salt and drought tolerance of transgenic lines.The main results are as follows:1.DPBA staining showed that the fluorescence intensity of flavonoids in young leaves and roots of A.canescens seedlings treated with 100 and 300 m M NaCl was obviously higher than that in the control.At the same time,the content of flavonoids in mature leaves and roots of 4-week-old seedlings under salt treatments was significantly higher than that of control.These results indicated that flavonoids might be involved the process of A.canescens in response to saline environments.2.The full length cDNA of chalcone synthase gene AcCHS,chalcone isomerase gene AcCHI and flavanone-3-hydroxylase AcF3H were cloned.The ORFs of AcCHS,AcCHI and AcF3H were 1182 bp,741 bp and 1119 bp,encoding 393 amino acids,246amino acids and 361 amino acids,respectively.3.Expression pattern analysis showed that AcCHS was mainly expressed in roots,while AcCHI and AcF3H were mainly expressed in mature leaves,of A.canescens.The three genes were significantly induced by NaCl treatments in the main expression tissues,indicating that they were involved in the response of A.canescens to salt stress.4.After overexpressing AcCHI in A.thaliana,the expression abundances of some genes related to flavonoids synthesis pathway were significantly increased,and the flavonoids content in transgenic plants also showed a significant increase.Under 50m M NaCl and-0.15 MPa treatments,compared with wild type,transgenic plants grew better,had higher biomass,as well as present lower H₂O₂ content and relative plasma membrane permeability in leaves.The results indicated that the overexpression of AcCHI promoted the synthesis of flavonoids and enhanced the ability of ROS scavenging in transgenic A.thaliana,which finally improved the resistance of transgenic lines to salt and drought stress.5.In transgenic A.thaliana overexpressing AcF3H,the expression abundance of certain genes in flavonoids synthesis pathway and the content of flavonoids were significantly higher than those in wild type.Under salt and osmotic stress,the growth and biomass of transgenic lines overexpressing AcF3H were better than those of wild type,and the content of H₂O₂ and relative plasma membrane permeability in leaves were significantly lower than that in wild type.Therefore,the overexpression of AcF3H promoted the synthesis of flavonoids and improved the ability of ROS scavenging in transgenic A.thaliana,thus effectively enhancing its tolerance to salt and drought.In conclusion,the flavonoids and the key genes encoding key enzymes of flavonoid synthesis AcCHS,AcCHI and AcF3H,are involved in A.canescens responding to salt stress,and the overexpression of AcCHI and AcF3H can significantly improve the salt and drought tolerance of transgenic A.thaliana.This study provided a theoretical foundation for systematically revealing the salt tolerance mechanism of A.canescens,and the genetic improvement of forages and crops stress resistance.