Preliminary Study On The Formation Mechanism Of Aquilaria Sinensis And Functional Study Of The Sesquiterpene Synthase Gene

The peeled and dry root,stem and twigs derived from Syringa pinnatifolia Hemsl.,which belongs to Oleaceae family,genus Syringa,were named as Shanchenxiang(SCX).SCX can take effect with suppressing Heyi,clearing heat and alleviating pain in Mongolian traditional medicinal theory in China and the pharmacology studies also reveal that SCX has various effects including myocardial protection,acesodyne,anti-tumor,anti-bacteria,anti-inflammation,anti-oxidation.In Mongolian traditional treatment,SCX was thought as an alternative to agarwood.Similar to agarwood,there is also a concept called “Jiexiang” in SCX.The woody stem of S.pinnatifolia whose heartwood are thick with high proportion are adopted as the source of SCX clinically.Considering the slow growth and instability of source of wild S.pinnatifolia,it has been listed in rare protective list.At the beginning of this century,the cultivation of S.pinnatifolia has been developed,however,the phenomenon of Jiexiang was hardly found in the cultivated woods.The cultivated S.pinnatifolia are not suitable as the source material of SCX used in clinic and the source was limited,so that the study on the mechanism of Jiexiang would be very important.Focusing on the sesquiterpenes,this study analyzed the SCX whose progress of Jiexiang has been complete(JX)and the other sample which has not been complete(WJX)using microscopic observation,volatile metabolome,transcriptome based on the precondition that the dominant pharmacodynamic substance in SCX is sesquiterpenes.In addition,some sesquiterpene synthase genes were screened out to be used for clone and expression analysis.Overall,this study discussed the mechanism of Jiexiang through all of the above means.The results in this paper are listed below:1.The microscopic observation of JX and WJXTaking woody stem which is the main medicinal part of SCX as the studying object,the microscopic observation of JX and WJX was carried out with histochemical staining.It was found that the main characteristics were in xylem ray.Compared to WJX,there were lots of purple fillers in the xylem ray of JX in which the viable cells were few with high lignification of the cell wall.Meanwhile,the storage substance changed from starch grain to polysaccharide in the progress of Jiexiang.2.The volatile metabolome analysis of JX and WJXVolatile metabolome was performed to analyze the metabolites in JX and WJX,and they were different significantly.The importance of sesquiterpenes in JX was identified because of the significant upregulation of all 37 sesquiterpenes in JX compared to WJX,so that the accumulation of sesquiterpenes would be a chemical characterization.3.The transcriptome analysis of JX and WJXUsing transcriptome technology,the comparison between JX and WJX was performed.It was found that many pathways about the biosynthesis of terpenes including sequiterpenes and others upregulated in JX.In this section,the change of gene expression in the progress of Jiexiang was discussed and explored from a molecular perspective.4.Clone and analysis of sesquiterpene synthase genesThe most efficient formula of hormones for the induction of callus from the leaves of S.pinnatifolia was screened,and a stable calli culture system was set up.On the basis of the culture system,the stable reference genes for the normalization of gene expression were screened from the differentiated tissues and calli with different treatments.Forthemore,two sesquiterpene synthase genes including Sp TPS1 and Sp TPS2 were collected and cloned according to the transcriptome database.Analyzing the selected genes,some differences were revealed: Sp TPS1 expressed highly in root while the expression of Sp TPS2 was high in leaf,as well as the latter lacked a DDX8 W motif,and they belong to different terpene subfamilies.5.Expression and functional verification of sesquiterpene synthase genesThe expression vector and prokaryotic expression of Sp TPS1 and Sp TPS2 were performed using two vectors including p ET32 a and p MAL-c2 x,and they were suited in low temperature according to the experiment.Based on the result of the functional verification of the synthases through GC-MS,eremophilene could be produced by Sp TPS1 and trans-nerolidol formed by the catalysis of Sp TPS2.Regarding eremophilene which is eremophilane-type sesquiterpenes was one of the types of sesquiterpenes accumulating more in JX compared to WJX,and the corresponding gene was similar to the change fold of expression in transcriptome database,it was speculated that Sp TPS1 was one of the key genes in the mechanism of Jiexiang in SCX.