Studies On Contemporary Biological Effects Of Trollius Chinensis Radiated By Nitrogen Ion

Trollius chinensis is one of the important Chinese medicinal materials with high medicinal value.In recent years,the demand of trollius chinensis has increased greatly,but wild trollius chinensis resource is limited and germplasm is single.In order to expand the germplasm resources,develop a new variety with high content of flavonoids（orientin and vitexin）and organic acids（veratric acid）,we surveyed and eveluated the germination rate,chlorophyll,malondialdehyde,protein and other physiological indexes and the contents of orientin,vitexin and veratric acid by high performance liquid chromatography-tandem four-stage mass spectrometry.Flavonoids are the main pharmacological components of trollius chinensis,and chalcone synthase（CHS）and chalcone isomerase（CHI）are the key enzymes in the flavonoid synthesis pathway.Therefore,we researched the genes related to flavonoid synthesis in depth.In this study,we used nitrogen ions with doses of 1,2,3,4,5,6,7,8×10¹⁵ ion/cm² to induce radiation mutagenesis on the seeds of trollius chinensis.Field statistics showed that the seed germination rate and germination index were the highest when nitrogen ion injection dose was 3×10¹⁵ ion/cm²;the germination potential was the highest when the dose was1×10¹⁵ ion/cm².The physiological indexes of trollius chinensis’s leaves was surveyed and evaluated by UV spectrophotometry.The results showed that malondialdehyde content was the lowest and protein content was the highest when nitrogen ion injection dose was 2×10¹⁵ion/cm².The chlorophyll content was the highest when the dose was 5×10¹⁵ ion/cm².The contents of total flavonoids and organic acids were determined by colorimetry and acid-base neutralization titration respectively.It was found that the contents of total flavonoids and organic acids were the highest when the dose was 1×10¹⁵ ion/cm².The content of orientin,vitexin and veratric acid was determined by High performance liquid chromatography-tandem quadrupole mass spectrometry.The highest content of orientin and vitexin was determined at 5×10¹⁵ ion/cm² and 2×10¹⁵ ion/cm² respectively.The content of vitexin was lower than that of control after nitrogen ion radiation treatment.Based on the above studies,we believe that 1×10¹⁵ N⁺/cm² is the appropriate dose for trollius chinensis after radiation mutagenesis.The results provided experimental basis for obtaining the mutant plants with high content of medicinal ingredients after radiation mutagenesis.We used real-time quantitative PCR to investigate the expression levels of CHS（Tc CHS1～Tc CHS8）and CHI（Tc CHI1～Tc CHI2）genes,which are key enzymes upstream of the flavonoid synthesis pathway.It was found that nitrogen ion radiation had a certain effect on gene expression.The accumulation of flavonoids was related to CHS and CHI gene expression.The production of flavonoids was negatively correlated with the expression of Tc CHS1,Tc CHS2,Tc CHS4,Tc CHS6 genes,and positively correlated with the expression of Tc CHS3,Tc CHS5,Tc CHS7,Tc CHS8,Tc CHI1,Tc CHI2 genes.The physicochemical properties,conserved domains,protein modification sites,subcellular localization,secondary structure,tertiary structure,clustering tree and protein interaction network of CHS and CHI proteins were analyzed by bioinformatics methods,providing reference for further study of the functions of CHS and CHI in the biosynthesis pathway of flavonoids in trollius chinensis.