Identification And Expression Regulation Analysis Of Terpenoid Synthase Gene In Aquilaria Sinensis

Agarwood is a resin-containing wood produced by plants of the genus Aquilaria after being injured.It has a long history of being used as a Chinese medicinal material and incense.Researchers at home and abroad have focused on the chemical composition,pharmacological activity and artificially induced aroma technology of agarwood,but there are few studies on the molecular regulation of the process of agarwood.Aquilaria sinensis（Lour.）Spreng.is a plant of the genus Aquilaria that grows in my country,and it is also a source of domestic agarwood specified in the Chinese Pharmacopoeia.Sesquiterpenoids are the main components of Agarwood and one of the active components of Agarwood to exert pharmacological effects.However,the molecular mechanism of its biosynthesis and regulation is not fully understood.Terpene synthase（TPS）plays a key role in the biosynthesis of sesquiterpenes.This study identified TPS family genes at the genomic level,and analyze its system evolution,gene structure,protein domain,gene expression and regulation characteristics;4 ERF transcription factors were screened simultaneously and preliminary studies were carried out on their regulation of TPS gene expression.The research results lay the foundation for revealing the function and regulation mechanism of TPS in the biosynthesis of agarwood sesquiterpene,and also provide a theoretical basis for the establishment of efficient artificial fragrance technology.Through the analysis of the genome data of Aquilaria sinensis and the transcriptome data processed by the fragrance agent,a total of 26 TPS genes（AsTPS1-AsTPS26）with complete open reading frames were identified.The length of the protein encoded by the AsTPSs gene is between325 aa and 843 aa,and most of them are hydrophilic unstable proteins.The function prediction results showed that most TPS genes are sesquiterpene synthase coding genes,and a few are single-post coding genes.Evolutionary analysis shows that 26 AsTPS genes are distributed among the five subgroups of TPS-a,TPS-b,TPS-c,TPS-e/f and TPS-g,of which TPS-a is the largest subgroup,including 16 Genes;intron-exon gene structure and conservative motif analysis found that genes belonging to the same subgroup have similar intron-exon gene structure and conservative motifs.Almost all AsTPSs proteins contain DDxxD and NSE/DTE domains;the two subgroups of TPS-a and TPS-b have RRX₈W conserved domains.There are many cis-acting elements related to plant growth and development,plant hormone response,and biotic/abiotic stress in the promoter region of AsTPSs genes.Expression analysis based on transcriptome data showed that 8 TPS genes encoding sesquiterpene synthase（AsTPS1,AsTPS2,AsTPS3,AsTPS5,AsTPS11,AsTPS13,AsTPS15 and AsTPS23）were induced by fragrance agents;real-time PCR results it was shown that in addition to AsTPS23,the expression of these TPS genes was also significantly induced by methyl jasmonate,ethylene and injury treatments.Expression analysis based on transcriptome data showed that the expression of 22 ERF genes was induced by fragrance agents,similar to the expression of TPS genes related to Agarwood sesquiterpene biosynthesis.Evolutionary analysis shows that AsERF1,AsERF2,AsERF31 and AsERF4 belong to the ERF-B3 subgroup.Subcellular localization analysis showed that AsERF1-4 were located in the nucleus.Expression analysis showed that methyl jasmonate,ethylene and injury treatments can all induce the expression of AsERF1-4,and it is speculated that they may be related to the biosynthesis of sesquiterpenes.Yeast one-hybrid experiments further proved that AsERF1-4 can bind to AsTPS1 and AsTPS3 gene promoters.The results of transient expression of tobacco leaves showed that the transcription factors AsERF1,AsERF2,AsERF31,AsERF4 can increase the activity of the promoters of AsTPS1 and AsTPS3 genes in tobacco leaves.It provides the basis for future research on the function and regulation mechanism of TPS gene in the biosynthesis of agarwood sesquiterpene.It also provides a theoretical basis for the establishment of efficient artificial fragrance technology.