Research On Volatile Terpenoids And Transcriptome Changes Of MeJA-induced Amomum Villosum Lour.

The dried ripe fruit of Amomum villosum Lour. known as Fructus Amomi,is one of the mainstream varieties of traditional Chinese medicine Amomum villosum, and has important medicinal and economic values. However, Factors such as special growth environment and low seed setting rate of Amomum villosum Lour.lead to the low yield of Fructus Amomi and cause the shortage of resources. MeJA (methyl jasmonate) as a kind of abiotic inducer, can effectively reglulate the biosynthesis of secondary metabolites in medicinal plants. Essential oil is the main active ingredients for dissipating dampness in Amomum villosum L.,which is composed of volatile terpenoids.Therefore, using MeJA treating the plants of Amomum villosum L. would be a reliable means for improving the content of active ingredients in Amomum villosum L.. At the same time, we could preliminarily explore the molecular mechanisms of MeJA regulating terpenoids metabolism in Amomum villosum L. with the use of high-throughput sequencing technology.1 ObjectiveBy using different concentrations of MeJA solution inducing the fruits and leaves of Amomum villosum Lour., we analyzed the changes of volatile terpenoids in peels and seeds of Amomum villosum L..Then through taking advantage of high-throughput sequen-cing technology, we got the transcriptome data of MeJA-induced Amomum villosum L. At the same time, the gene expression profiles of Amomum villosum L. (divided into seeds and peels) induced by different concentrations of MeJA were obtained, facilitating the analysis of different expression genes(DEGs) and the preliminary exploration on the molecular mechanisms of the biosynthesis of volatile terpenoids in Amomum villosum L. induced by MeJA.2 Methods2.1 The induction of different concentrations of MeJA solution impacts the biosynthesis of volatile terpenoidsFive different concentrations of MeJA solution was used to spray the leaves of Amomum villosum L.,then the leaves induced by MeJA were sampled at different time points, respectively. Using the microwave method,we extracted the volatile terpenoids in leaves.After GC-MS detection and analysis of the results,we found out the suitable concentrations of MeJA and sampling time points for further study.Then we used 200 μmol/L and 600 μmol/L MeJA solution to spray the fruits and leaves of Amomum villosum L. on the stage of fruit maturation, detecting the variation of volatile terpenoids in seeds and peels and analyzing the different regulating roles of different concenrations of MeJA solution on the biosynthesis of volatile terpenoids.2.2 Transcriptome sequencing of MeJA-induced Amomum villosum L. and data analysisThe RNA was extracted from the peels,seeds and leaves of Amomum villosum L.induced by different concentrations of MeJA, respectively. Then the equal amounts of RNA from different samples were mixed and reversed in order to build the cDNA library using Illumina HiSeq 2000 sequencing platform. The high quality reads was assembled by de novo and further generated hundred thous- ands of unigenes.The unigenes obtained functional annotation information by blastx with four public protein databases. What’s more, the indepth analysis was performed on terpenoids metabolism, JA biosynthesis and signal transduction pathways.2.3 Gene expression profile sequencing of MeJA-induced Amomum villosum L. and screening of differentially expressed genesThe method of obtaining and mixing of RNA from the peels and seeds of Amomum villosum L.induced by MeJA was same as above.Then using transcriptome data as reference sequences, the gene expression profile sequencing was performed successfully by RNA-Seq.After GO and pathway enrichment analyses, the differentially expressed genes were analyzed through the expression trends and the comparison between multiple samples,especially the DEGs related to terpenoids metabolism and JA signal transduction pathways.2.4 Expression level and correlation analysis of several genes in Amomum villosum L.after induction of MeJACombining the annotation information from transcriptome and gene expression profilings,the candidate functional genes which have a close relationship with terpenoids metabolism and JA biosynthesis were selected out for RT-qPCR.Using the selected unigenes as reference sequences,the primers were designed and their effectiveness and specificity were verified. After that,optimizing primer annealing temperature was performed. The relative expression level of target genes was compared with RPKM values in gene expression profilings in order to verify the transcriptome data.Besides, pearson correlation analysis was applied to investigate the relationships between JA signal transduction and terpenoids biosynthesis.3 Results3.1 Induction of MeJA could affect the biosynthesis of volatile terpenoids in Amomum villosum L.After induction of different concentrations of MeJA solution, there were 7 kinds of volatile terpenoids detected in all the treatment groups, inclu- ding three mono terpenoids, three sesquiterpenoids and one diterpenoid. The obtained data was analyzed from the perspective of different concentrations of MeJA solution and different sampling points, the results showed that changes of after induction of MeJA was quite complicated, finally we chose 200μmol/L and 600μmol/L MeJA as the appropriate concentrations to spray Amomum villosum L. for 24h and 48h, respectively.Using 200μmol/L and 600μmol/L MeJA to induce Amomum villosum L.on fruit riping stage, there were 20 kinds of volatile terpenoids in peels, containing monoterpenes, sesquiterpenes and their derivatives. While there were 33 kinds of volatile terpenoids in seeds.600μmol/L MeJA inducing for 24h can significantly promoted the synthesis of volatile terpenoids in peels, while 200μmol/L MeJA inhibited the synthe- sis of volatile terpenoids in peels. On the contrary,200μmol/L MeJA affected the sesquiterpenoids in seeds more significantly, while 600μmol/L MeJA whose role of inhibitation faded out over time inhibited the biosynthesis of volatile terpenoids.In addition,200 μmol/L MeJA which induced the leaves of Amomum villosum L.could improve the content of camphor and borneol significantly, while 200 μmol/L MeJA which induced the fruits of Amomum villosum L.could significantly improve the content of linalool, α-pinene,β-pinene and sabinene in peels, and increase the content of most volatile terpenods in seeds.3.2 Induction of MeJA could affect the transcriptome of Amomum villosum L.We obtained the transcriptome data of Amomum villosum L. induced by MeJA by Illumina sequencing. There were 138,679 unigenes in total,and 70,323 unigenes got annotations. Among the data,there were 27,382 unigenes classified into 25 COG functional categories. KEGG annotation results showed that there were 21,503 unigenes classified into 125 metabolic pathways.Among them,there were 21 pathways related to secondary metabolism, containing 3,398 unigenes (about 16%). Terpenoids metabolism pathways annotated 299 unigenes, including three monoterpene synthase genes:myrcene /ocimene synthase, (3S)-linalool synthase and (+)-neomenthol dehydrogenase, and two sesquiterpene synthase genes:farnesyl-diphosphate farnesyl transferase and squalene monooxygenase.In addition, through using PBI-BLAST,we found out 9 monoterpene synthase genes and 7 sesquiterpene synthase genes. On the other side, there were 61 unigenes in JA signal transduction pathway, including JAR], JAZ, COI1 and MYC2. What’s more, there were 180 unigenes in JA biosynthesis pathway.These unigenes would provide resources for deeper understanding of the molecular mechanism on terpenoids metabolism as well as JA biosynthesis and signal transduction.3.3 Induction of MeJA could affect the differentially expression of genes of Amomum villosum L. in expression profilesComparison between two different profiles showed that different genes differentially expressed presented different sensitivity to the induction of MeJA solution. There were more differentially expressed genes in MEP pathway,including DXS and HDS which differentially expressed more signifi- cantly.At the same time,MeJA could promote JAZ in peels up-regulated expression,and the higher concentration of MeJA,the more significant the promoting effect was.In addition, there were more DEGs in JA signal transdu-ction in seeds,indicating that unigenes in peels could response the induction of MeJA more positively.Comparison among three different profiles which had a progressive relationship showed that HMGR,MVK,DXS,DXR,HDR and GGPPS in terpenoid biosynthesis pathway and JAR1 and JAZ in JA signal transduction pathway in peels differentially expressed under the regulation of MeJA.While DXS,CMK, MVK,HDS,GGPPS,DHDDS,and CHLP in terpenoid biosynthesis pathway, and CYP71 and ALDH in limonene and pinene degradation pathway and SQLE in sesquiter- penoid biosynthesis pathway, and the JAR1,JAZ and MYC2 in JA signal transdu- ction pathway in seeds differentially expressed under the regulation of MeJA as well.However,induced by MeJA and differentially expressed.However,HMGR in peels and DXS,CMK,JAR1 JAZ and MYC2 in seeds were the only unigenes not sensitive to the concentration of MeJA solution.In addition, analysis of gene expression trends showed that the regulation of different concentrations of MeJA solution played different roles on unigenes in terpenoid backbone biosynthesis and JA signal transduction pathways in different tissues.3.4 Induction of MeJA could affect the expression level of several genes in Amomum villosum L.The expression level of JAR1,CHLP, HMGR and GGPPS were low in seeds, while the expression level of the OPR, HDR, ISPD and DXR were high. Treatment of solvent and 600μmol/L MeJA improved the expression of DXR in seeds significantly, and 200μmol/L MeJA increased significantly the expression of OPR in seeds. With the increase of MeJA concentrations, the expression of HMGR in seeds was always rising, and the expression of OPR was down after rising first, while CHLP, HDR, ISPD, GGPPS and DXR were up after declining first, JAR1 was declined significantly from the beginning to the end,indicating that MeJA may have negative effect on the expression of JAR1.On the other side,the expression level of JAR1, HMGR, GGPPS and OPR were low in peels, and the expression level of CHLP, HDR, ISPD and DXR were high. Treatment of solvent,200μmol/L MeJA and 600μmol/L MeJA could significantly improve the expression of DXR in seeds, and the solvent could reglulate the expre- ssion of DXR most greatly. With the increase of MeJA concentration, the expression of JAR1 had a significant rise, HMGR, OPR, ISPD and HDR were down after rising first, while CHLP, GGPPS and DXR were always declined, indicating that MeJA may play a negative role in the expression of CHLP, GGPPS and DXR. The expression trends of RT-qPCR results were mostly consistent with those in expression profiles. However, the expression trend of CHLP was reduced after rising first in expression profiles, while it was up after declined first in RT-qPCR results.In addition, terpenoid backbone and volatile terpenods biosynthesis had a closely positive relationship with JA signal transduction.Among them,AACT and FDFT was closely related to JAZ, MVD was closely related to the MYC2. And there were more unigenes which had strong correlation with JA signal transduction in peels than those in seeds.At the same time,most volatile terpenoids in peels had strong correlation with MYC2,While most volatile terpenoids in seeds had strong correlation with JAZ. Among them, bicyclo-elemene and germacra-1,6-dien-5-ol was closely related to JAZ,α-copaene was closely related to the MYC2.Besides,bornyl acetate had strong correla- tion with MYC2 in peels and JAZ in seeds, indicating that certain relevance between JA signal transduction and bornyl acetate biosynthesis did exist.4 ConclusionsThis research obtained the content changing information of volatile terpenoids in leaves and fruits of Amomum villosum L.induced by different concentrations of MeJA. Then we got the transcriptome and expression profiles of Amomum villosum L. induced by MeJA, and analyzed unigenes differentially expressed in terpenoids metabolism and JA signal transduction pathways.The results showed that MeJA can effectively regulate the content changes of volatile terpenoids and the differentially expression of terpenoids biosynthesis genes in Amomum villosum L..What’s more, JA signal transduction had strong correlation with part of the terpenoids biosynthesis genes and volatile terpenoids, indicating that MeJA could regulate the JA signal transduction pathway by means of regulating the expression of key genes, which further regulated the expression of terpenoids biosynthesis genes and volatile terpenoids biosynthesis and accumulation. These results laid foundation for further research on molecular mechanism of how MeJA regulated the terpenoids metabolism of Amomum villosum L.