| Ligusticum chuanxiong Hort was a well-known medicinal herb in china, which belonged to Umbelliferae plant. The dry rhizome of L. chuanxiong, also known as chuanxiong, has been widely used as traditional medicine for the treatment of headache, rheumatic arthralgia, menstrual disorder, swelling pain, cardiovascular and cerebrovascular diseases in China, Japan and Korea for thousands of years. Meanwhile, the rhizome of L. chuanxiong has also been used as the components of cosmetics for improving the skin color and fragrance. There were a variety of significant pharmacological compounds, including ferulic acid, essential oil, terpenoids and alkaloids, reported in the rhizome of L. chuanxiong. It was important to cognize metabolic pathways of active components, development and resistance mechanism by transcriptome sequencing of L.chuanxiong. In this study, It was obtained transcriptome data of the rhizome and leaf in L. chuanxiong by Illumina high-throughput sequencing, to complete the analysis and verification, which provided genetic background for exploring key genes of development, stress resistance, active ingredient and metabolic pathways. The main results of this paper were as follows:1) The rhizome and leaf of L. chuanxiong was firstly subjected to transcriptome sequencing and obtained raw data respectively (a total of 5.36 Gb).13,717,234 and 12,823,395 clean reads were generated from the rhizome and leaf (average length was 101 nt), respectively. It eventually won 57,705 unigenes from the rihzome (average length 713 nt) and 50,326 unigenes from leaf (average length 798 nt) by the Trinity software assembly, and then the unigenes were subjected to functional classification and annotation by Nr, Swiss-Prot, GO, COG and KEGG,35,952 and 31,421 unigenes got functional annotation, respectively. In KEGG metabolic pathways,700,659 unigenes was found to involve in secondary metabolic pathways, such as phenylpropanoid biosynthesis, phenylalanine metabolism, glycerophospholipid metabolism, propanoate metabolism, glyoxylate and dicarboxylate metabolism. Some genes had been cloned, which was clear about molecular function in biosynthesis pathway, and some genes were potential candidate genes. It was very helpful to improve the related metabolism by genetic engineering and molecular biology methods.2) To comparative transcriptome of rhizome and leaf in L. chuanxiong, there were totally 4,680 differentially expressed genes between the rhizome and leaf, including 1,714 up-regulated unigenes in rhizome and 2,966 up-regulated unigenes in leaf, meanwhile, to analyze them about GO analysis, COG analysis, transcription factors and so on.3) The suitable reference genes of L. chuanxiong in different tissues were firstly selected and evaluated. Seven conventional were chosen as candidate reference genes to do quantitative analysis from the transcriptome database, like RPLll, ATC7, RPS18, BTF3, CYB5, GAPDH and UBC. By the geNorm, NormFinder software analysing and valuing, RPLll was the most stable genes which would be used as reference genes in L. chuanxiong. And the same time, RPL11 and BTF3 were considered as the the most stable gene combination by geNorm calculating. The results provided a reliable standard for gene expression analysis of L. chuanxiong.4) Expression patterns of some unigenes were analyzed in rhizome and leaf of L.chuanxiong, such as BDP, LealP, ECP40, C3HC4, PAL, COMT-1 COMT-2. The results showed that PAL was up-regulated in leaf, nevertheless, the other genes were up-regulated in rhizome, for instance, BDP, Lea1P, ECP40, C3HC4 and COMT-1, the results of qRT-PCR seemed to be in tune with reaults of transcriptome data analysis. Unigenes related to ferulic acid biosynthesis pathway in L. chuanxiong, because the expression level of COMT-1 (c55080.graph_c0) is higher in rhizome, which was differentially expressed gene, to deduce that COMT-1 could belong to the key enzymes in the biosynthesis of ferulic acid. |
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