Studies On Transcriptome And Digital Gene Expression Of Chinese Agarwood By RNA-Seq

Chinese agarwood is a traditional Chinese rare medicinal herb, and Aquilaria sinensisis the only plant resources. Health A.sinensis could not produce agarwood. In this reason,many peoples researched the artificial induction of agarwood and found that the agarwoodcould be induced by all damages to the A.sinensis trunk. Even thought, what’s themechanism of inductions, how does it produce agarwood, hardly any reports right now.This project studied the total RNA isolation of A.sinensis, and made a transcriptomesequencing to the Chinese agarwood by next-generation sequencing technology, and thenmade a DGE analysis to each tissues of Chinese agarwood, sifted out the differentexpression genes, and revealed the A.sinensis production agarwood mechanism fromsesquiterpene biosynthesis and damage defense reaction both sides. The results obtainedare as follows:(1) It was determined the best method to extracting A.sinensis total RNAs wasmodified guanidine isothiocyanate-CTAB method and successfully extracted the totalRNAs from all kinds of A.sinensis tissues for transcriptome sequencing by this method.(2) In this study, the A.sinensis transcriptome sequencing result was high ideal, theQ20valued up to97.45%, this score was high enough and the sequencing quality wasgreat. After assembled, there were83,467unigenes obtained, their average length was702nt, and the N50valued up to1120. These transcriptome sequences enriched thetranscriptome information in the process that A.sinensis producing agarwood. About50,565unigenes were annotated by containing Nr, SwissProt, KEGG, COG, GO, and Ntdatabases, accounting for60.58%of the total unigenes. Among these sequences,171,33and1352unigenes were respectively annotated by Terpenoid backbone biosynthesis(ko00900), Sesquiterpenoid and triterpenoid biosynthesis (ko00909), and Plant-pathogeninteraction (ko03040) pathway. These sequences were conducive to research thesesquiterpene biosynthesis and injury prevention reactive in A.sinensis.(3) Through digital gene expression analysis for Chinese agarwood by RNA-Seq,every tissues was acquired about7M clean reads, up to80%clean reads could becompared with the reference transcriptome database. After quantitated by RPKM methodand sifted the different expression genes out of those compared sequences by limited term,there were4784unigenes in agarwood tissue expressed higher than those expression inwhite wood tissue. On the contrary, there were3603unigenes expressed lower. Thedifferent expression in agarwood sample and white wood sample was significant, and thesedifferentially expressed genes were likely to take on important tasks in the process thatA.sinensis producing agarwood.(4) The expression conditions of enzymes used in sesquiterpene biosynthesispathway were acquired. It were found that HMGS and HMGR expression level wassignificantly higher than that of other enzymes, and the expression of these two enzymes inagarwood sample to be significantly higher than the other samples, indicating these twoenzymes, especially HMGR, is extremely important enzymes in the A.sinensissesquiterpene biosynthesis metabolism; Defense-related transcription factor, WRKY,MYC2and JAZ factors were differentially expressed among all kinds of Chineseagarwood tissues. The expression patterns of WRKY transcription factor were divided intothree classes. On the other hand, the MYC2transcription factor might be involved in thestress response of A.sinensis or secondary metabolic processes.(5) Successfully amplified the putative sesquiterpene synthase As-SesTPS fromChinese agarwood cDNA. This sequence coding the protein that was highly similar to theVitis vinifera germacrene-D synthase after similarity comparison and homology analysis,the similarity value was up to68%, the identity value was up to50%. By bioinformatics analysis, the coding protein was analyzed for all kinds of physical and chemical properties.It was confirmed the molecular weight of the coding protein was62.80kD, the isoelectricpoint was5.26. The coding protein was a acidic protein, and the total average hydrophobicindex was-0.303. It was predicted as a hydrophilic protein.In summary, there were a large number of Chinese agarwood transcriptomeinformations acquired by transcriptome sequencing. The informations provided a basisdatabase to the Chinese agarwood transcriptome analysis. DGE analyzed to the Chineseagarwood, different expression genes in all tissues had been researched. According to thedifferent expression genes, it could be infered the key gene in the process that A.sinensisproducing agarwood. After that, Successfully cloned the highly expressed sequencesesquiterpene synthase gene As-SesTPS in agarwood sample, this result could be as areference to the A.sinensis produced agarwood study.