Cloning And Expression Analysis Of Genes Involved In Dendrobium Flavonoids Biosynthesis Based On RNA-seq

Dendrobium catenatum Lindley is a spiece of Dendrobium SW., belongs to perennial herbs of the Orchidaceae family. It is one of the genuine Dendrobium which are collected by "Chinese Pharmacopoeia". As the secondary metabolism of D. catenatum, flavonoids is the primary bioactive component which has the effect of antioxidation, anti-aging and anti virus and so on, and it also as the main component which can resist diseases and stresses for the plant itself. Currently the study of flavonoids in D. catenatum mainly concentrated on component identification and species analysis.However, the details of flavonoids biosynthesis have not been effectively explored. This study respectively from three aspects to provide basis for the study of flavonoids metabolic pathways in Dendrobium SW.. The main results of the study include the following three aspects:1. The RNA-seq of Dendrobium catenatumRNA-seq analysis of the leaves of common and purple plants in artificial cultivation. In this study, we analyzed RNA isolated from D.catenatum using Hiseq 2000 Illumina platform to generate64,195 unigenes, the total length, average length, N50, and GC content of unigenes are 66,479,063 bp, 1,035 bp, 1,742 bp, and 42.47 %, respectively. A total of 35.92% of the unigenes were annotated with Kyoto Encyclopedia of Genes and Genomes(KEGG) terms, and a detailed view of flavonoids biosynthesis was obtained. Differential expression analysis results under the control of common leaves showed that there were 7177 differentially expressed genes, the number of up-regulated expression genes is 3155, and down-regulated is 4022, and there were 13 differentially expressed genes related to the biosynthesis of flavonoids. We selected the Chalcone synthase(CHS),Flavanone 3-dioxygenase(F3H) and Flavanone 4-reductase(DFR) gene which are up-regulated expression obviously for this study.2. The chalcone synthase gene cloning and prokaryotic expression analysis in Dendrobium catenatumTranscriptome sequencing results as the reference, selecting the first key chalcone synthase genes which is differentially expressed in two samples, combining homologous cloning and RACEtechnique, we obtained the chalcone synthase gene c DNA sequence, named Dc CHS gene(Gen Bank accession number is KR078267). The full-length of Dc CHS gene is 1311 bp, and the ORF is 1188 bp encoding 395 amino acids. The predicted molecular weight is 43.14 Kda. BLAST and multiple alignment of the amino acid sequence showed that Dc CHS gene has higher similarities with other species. Phylogenetic analysis indicated that Dc CHS gene is more closely related to Orchid CHS group 1 from orchidaceae plants than to those of other plants, and it’s in the high evolutionary position. We have constructed the prokaryotic expression vector p ET32a-Dc CHS successfully, and SDS-PAGE analysis showed that the fusion protein was highly expressed in E. coli BL21 with isopropyl-D-thiogalactoside(IPTG) induction, and it produced a protein about 43 k D, a size matching that of the predicted by bioinformatic analysis. Vitro enzyme activeity test by high performance liquid chromatography showed that the prokaryotic expression product has the activity of chalcone synthase.3. Quantitative expression analysis of Dc CHS, Dc F3 H and Dc DFR genesThe conserved sequences of Dc F3 H and Dc DFR genes were cloned, and the Dc CHS, Dc F3 H and Dc DFR genes expression were analyzed by fluorescence quantitative PCR analysis. We found that the expression of three genes in purple plants is higher than common plants, and it was expressed higher in leaves than in stems, in addition, the expression quantity in the protocorm and seedlings is very low, this is consistent with the transcriptome sequencing results.