Transcriptomes Sequencing Of Aquilaria Sinensis Treated With A Agarwood-inducing Reagent And Cloning And Expression Analysis Of Its Sesquiterpene Synthase Gene

Agarwood is dry wood containing resin from the Thymelaeaceae plants,which has extremely high medicinal value and economic value.Aquilaria sinensis(Lour.)Spreng.belongs to the Aquilaria Lam.(Thymelaeaceae)and is a unique medicinal plant in China.It is the most important plant resource for producing agarwood in China.At present,the research on Aquilaria sinensis mainly focuses on the analysis of the chemical composition and structure of agarwood,but the molecular mechanism of agarwood formation is still unclear.Sesquiterpene is one of the main characteristic components of Aquilaria sinensis with various medicinal values and biological activities.The sesquiterpene synthase is one of the important enzymes in the sesquiterpene biosynthesis process.It has important theoretical and practical applications to study the structure and function of the sesquiterpene synthase,for the future use of biotechnological methods to improve the yield of the agarwood value.In this study,a transcriptome sequencing was carried out by using the stem of Aquilaria sinensis treated by our patent agarwood-inducing reagent.A sesquiterpene synthase gene was cloned by differential expression analysis between the before and after treatment.The bioinformatics analysis and expression analysis of the gene was performed.The main results are as follow:(1)A transcriptome sequencing was carried out by using the stem of Aquilaria sinensis treated by our patent product.A total of 1,190,225,503 clean reads were obtained.There may be 41 differentially expressed unigenes that may be involved in the biosynthesis of sesquiterpene,of which the gene c32690_gl_i3 was significantly upregulated.(2)Primers were designed according to the the up-regulated expression gene sequence obtained from the sequencing analysis of the transcriptome.A Sesquiterpene synthase gene in Aquilaria sinensis was cloned by RT-PCR.The gene is 1632 bp and encodes 543 amino acids.Blast analysis showed that the protein encoded by this reading frame has an amino acid identity of 91%with the SesTPS1 of Aquilaria malaccens and it was named as AsVS.(3)Bioinformatics analysis showed that AsVS has the conserved domains of sesquiterpene synthase,conserved as DDXXD,NST/DTE and R(R)X8W,with a relative molecular weight of about 62 kD and a theoretical pI of 5.57.Phylogenetic tree analysis showed that AsVS was closest to the sesquiterpene synthase SesTPS1 in Aquilaria malaccensis,reaching 91%.The results of cluster analysis of terpenoid synthase indicated that the protein encoded by As VS belongs to the Tpsa subfamily,which belongs to the sesquiterpene synthase family.(4)AsVS expression analysis showed that our patent agarwood-inducing reagent can promote the expression of AsVS gene by using quantitative real-time PCR.(5)The prokaryotic expression vector pET28a-AsVS was successfully constructed in this study and transformed into E.coli BL21(DE3).The protein was induced by IPTG and the protein was purified by nickel affinity chromatography to catalyze the synthesis of sesquiterpene substrate FPP.The reaction products were detected by GC-MS.No sesquiterpenoids were found.