The Mechanism Of Diterpenoid Biosynthetic In Coleus Forskohlii And The Differential Gene Expression Between Leaf And Rhizome In Atractylodes Lancea

This thesis consists of two parts, the first part is the mechanism of diterpenoid biosynthetic in Coleus forskohlii, the second part is the differential gene expression between leaf and rhizome in Atractylodes lancea.Part I:Coleus forskohlii(Wild) Briq belongs to Lamiaceae, Coleus genus. Wild Coleus forskolin originates from the Indian subcontinent, mainly in India,Nepal, Myanmar, Sri Lanka, Thailand and other subtropical countries. It is also found in Huize, Dongcuan of Yunnan province in small amount. In traditional Indian herbal therapy, Coleus is used to treat eczema, psoriasis,asthma, hypertension and cardiovascular disease. These diseases usually occur because of the decrease of the intracellular c AMP levels and diterpene constituents forskolin originating from Coleus is an activator of adenylate cyclase. It has an significant treatment effect by direct activating of adenylate cyclase to increase cyclic adenosine monophosphate(c AMP) levels.Unfortunately, the content of the active ingredient is low, the thesis was to study Coleus diterpene synthases associated CPS and KS gene cloning,specific expression in order to elevate the content of the active compound in Coleus.1 Coleus seed germination seedling about two weeks was as the test material.Extracte RNA, removed g DNA, reverse transcribed into c DNA, and then cloned as the template. Primers were designed in diterpene synthesis pathway related genes on different CPS and KS conserved region.Fragment in conserved region was cloned by specific primers PCR, and amplified through 3’RACE and 5’RACE. The sequencing results were assembled, using splicing primer design primers to amplify the full-length gene, again specific primers PCR, Finally, we could obtain full-length sequence of the gene.2 Full-length sequence bioinformatics analysis was done. BlastX aligment on NCBI, Cf TPS 14 refers to ent-kaurene synthase, Cf TPS15 to ent-Copalyl diphosphate synthase. Cf TPS14 isoelectric point of 5.38;formula C3987H6231N1073O1194S42; coefficient of fat 91.35; average major hydrophobic properties(GRAVY) is-0.189, showing hydrophilic,cellular localization revealed its distribution out of the cell membrane.Cf TPS15 isoelectric point of 6.86; formula C3633H5629N993O1046S27;coefficient of fat 87.65; average main hydrophobic properties(GRAVY) of-0.371, showing hydrophilicity, positioned on the extracellular membraneane.3 CfTPS1 is a heterogeneous copalyl diphosphate synthase. CfTPS3 and Cf TPS4 are tanshinone diene and manoyl oxide(13R) synthase; Cf TPS14 is an ent-kaurene synthase; Cf TPS15 is a ent-copalyl diphosphate synthase.Cf TPS1 and Cf TPS3 are involved in the synthase of ferruginol precursor tanshinones, tanshinone mainly generated in root, so Cf TPS1 and Cf TPS3 mainly express in the root and rhizome, and less in stem; Cf TPS3 and Cf TPS4 are involved in the synthase of forskolin precursor manoyl oxide(13R), so Cf TPS3 is mainly expressed in root and rhizome. The stem was less, Cf TPS4 may be involved in other catalytic process, therefore its content in the flowers and leaves is higher; Cf TPS15 and Cf TPS14 in mildew element precursor reflect the type of ENT kaurene synthesis, so in the flowers and leaves are higher, in the root content is low. QPCR results with the gene specific expression trend are consistent.Part II:Atractylodes lancea(Thunb.) DC.(Compositae), also called Cangzhu in Chinese, is a well-known and widely prescribed traditional Chinese herb. The rhizome of A. In this study, we performed high-throughput Illumina sequencing to comprehensively characterize the transcriptome of A. lancea,and reveal differential gene expression profiles between rhizome and leaf,which would facilitate uncovering the molecular mechanisms of regulating rhizome formation and growth of the most important medicinal plants in genus Atractylodes.1 Leaves and rhizomes of A. lancea were collected from Mao Mountain,Jiangsu Province, China Total RNA from each tissue was isolated using the RNAprep Pure Plant Kit. c DNA library construction and normalization were performed according to published protocols. Five c DNA libraries(2for leaves and 3 for rhizomes) were finally sequenced using an Illumina Hi Seq2000 platform, and paired-end reads were generated. Clean reads were obtained by removing the adapter sequences, low quality sequences,and sequences shorter than 35 bases. The remaining high-quality reads were de novo assembled into candidate unigenes using the Trinity program.After the removal of adaptor sequences and low-quality reads,approximately 118.4 and 152.7 million clean reads were acquired for the leaf and rhizome transcriptomes, respectively. Thus, a total of 33,885 Mb valid data were acquired with an average length of 125 bp. The unigenes were aligned against public protein databases(NR, Swiss-Prot, GO, and KEGG) using BLAST with a cut-off E-value of 1.0e-5. A total of 39,664unigenes(42.90% of the total assembled unigenes) had a match in the NR database, and 38,699(41.19%), 26,159(28.32%), and 10,508(11.38%)unigenes showed significant similarity to sequences in the Swiss-Prot, GO,and KEGG databases, respectively. KEGG pathway analysis was performed to identify the biochemical pathways active in the leaf and rhizome of A. lancea. A total of 10,504 unigenes were annotated and assigned to 289 KEGG pathways. A total of 10,103 SSRs were identified in 92,366 unigenes, 1,074 of which contained more than one SSR, and 406 SSRs were present in compound form.2 Highly expressed and tissue-specific genes of A. lancea.We identified 227 transcripts in the leaf and 105 in the rhizome with an FPKM value greater than 1,000, of these, 49 were in both tissues. In order to further understand the biological functions of the DEGs, enrichment analyses based on GO and KEGG pathways were performed. When the 4,982 DEGs were checked against the GO database, 262 GO terms were significantly enriched. In the KEGG analysis, the 1,518 up-regulated unigenes were linked to 159 KEGG pathways. The pathway assigned the largest number of unigenes(29) was “plant hormone signal transduction”(ko04075),followed by “starch and sucrose metabolism”(ko00500), “protein processing in endoplasmic reticulum”(ko04141), and “biosynthesis of amino acids”(ko01230). 39 unigenes were mapped to secondary metabolism pathways, including 18 unigenes which might be involved in“metabolism of terpenoids and polyketides”. A total of 42 TF families were identified when aligning the annotated A. lancea transcripts to the AGRIS database. There were 60 TFs up-regulated in the leaf, and 67 TFs were up-regulated in the rhizome.3 The RNA-Seq and computational results were verified by q PCR using 20 selected DEGs. The expression patterns of all the selected genes show the same trend in the transcriptome analysis and the q RT-PCR. We also tested the correlations of these genes and found a significant positive correlation between them, with the correlation coefficient reaching 0.81.4 DEGs were further analyzed to screen candidate genes involved in rhizome formation and development. A total of 104 genes involved in organ development, hormone biosynthesis, and hormone signal transduction were identified, including some transcription factors(e.g., 2MADS-box proteins, 12 AP2-like transcription factors).