Expression And Functional Analysis Of BHLH Differential Genes In Glycyrrhiza Inflata Based On Salt Stress Transcriptome Sequencing

Glycyrrhiza inflata is a perennial herb,distributed in Inner Mongolia,Gansu,Xinjiang,China.It is a traditional Chinese medicinal plant with salt tolerance,and it is one of the promising plants for improving saline-alkali wasteland.Many secondary metabolites are produced in the roots and rhizomes,with glycyrrhetinic acid and flavonoids as major components.Glycyrrhizic acid and flavonoids have the pharmaceutical properties such as anti-inflammatory,antibacterial,antiviral,antioxidant,and liver protection.This experiment was based on the transcriptome data of Glycyrrhiza inflata calluses under salt stress.The content of glycyrrhetinic acid and flavonoids was determined by high performance liquid chromatography.Biological functions of GibHLH differentially expressed genes studied by an experimental method of overexpression.The results were as follows:1.43 GibHLH genes were screened for the transcriptome data onto Glycyrrhiza inflata calluses under salt stress,including 10 differentially expressed GibHLH genes.Physicochemical properties of GibHLH differentially expressed proteins were analysised by the Expasy-Prot Param tool,and identified their structural domains by online analysis software of MEME,Motif.Differentially expressed GibHLH transcription factors were predicted to function in response to salt stress and participate in secondary metabolism by constructing evolutionary trees by MEGA7 and sequence alignment by DNAMAN.2.q RT-PCR was used to analyze the expression patterns of 10 GibHLH differentially expressed genes in Glycyrrhiza inflata calluses treated with 0.8% Na Cl and 15% PEG.The results showed that the differential expression of GibHLH7 and GibHLH10 was stable and responded to salt stress,and their functions can be further studied.3.The flavonoids and glycyrrhizic acid content in Glycyrrhiza inflata calluses treated with salt for 0 、 7 、 14 and 20 days were determined by high performance liquid chromatography.The results showed that the content of total flavonoids in Glycyrrhiza inflata calluses increased to the prolongation of salt treatment time,and the content of glycyrrhizic acid was very low in the four times periods.4.By overexpressing GibHLH7 and GibHLH10 genes,the functions of the two genes were analyzed at the gene level.PCR identified two genes successfully introduced into tobacco.By measuring the physiological and biochemical indicators of isolated tobacco leaves,the salt tolerance of tobacco overexpressing GibHLH10 was better than wild type,so GibHLH10 may regulate transport proteins in response to abiotic stress.q RT-PCR identification of GibHLH7 and GibHLH10 were successfully introduced into Glycyrrhiza inflata calluses.The callus of Glycyrrhiza inflata over-expressing GibHLH7 had higher CHS expression than wild type.GibHLH7 may promote flavonoid synthesis in response to abiotic stress by regulating the expression of CHS.In conclusion,this paper analyzed the differentially expressed GibHLH genes in the transcriptome of Glycyrrhiza inflata calluses under salt stress,and analyzed the functions of GibHLH7 and GibHLH10 by overexpression.Studies had shown that GibHLH7 positively regulated the expression of CHS,which may respond to abiotic stress through the accumulation of flavonoids.GibHLH10 may regulate transport proteins to improve the salt tolerance of plants.The above research not only lays the foundation for the study of bHLH genes in Glycyrrhiza inflata in response to salt stress,but also provides theoretical guidance of improving the accumulation of secondary metabolites.