Research On Rhynchophylline And Isorhynchophylline Biosynthetic Pathway

The uncaria (Uncaria rhynchophylla) is traditional medicine. Its active ingredients (rhynchophylline and isocorynoxeine) can ease and resolve Alzheimer’s disease. Rhynchophylline (RIN) and isorhynchophylline (IRN) of natural product were received widespread attention, biosynthetic mechanism of them also was one of the hot research.This study analyzed RIN and IRN accumulation laws in different parts (including different periods capsule) of uncaria by high-performance liquid chromatography. Selecting capsule (including three different developmental stages) as a material, the high-throughput sequencing technology was used to build transcriptome and expression profiling, looking for terpene indole alkaloid biosynthesis genes and differentially expressed genes, understanding furtherly transcriptional gene expression change in RIN and IRN accumulation process, exploring RIN and IRN biosynthesis genes, predicting biosynthetic pathways of RIN and IRN and laying the foundation for RIN and IRN biosynthesis mechanism research.Test results are as follows:1. The uncaria capsules had the highest the RIN and IRN content, followed by the hook stems and stems, and the leaves had the minimum content of the RIN and IRN. Improved CTAB method can separate qualified RNA. Capsules were suitable test materials with construction of transcriptome and expression profiling of uncaria. GAPDH was the most suitable as reference gene for the analysis of differentially expressed genes in different development stages of Uncaria rhynchophylla capsule.2. Transcriptome of Uncaria capsule was sequenced using Illumina HiSeqTM 2000 sequencing platform. More than 50 million high-quality clean reads from a cDNA library were generated, and was assembled into 100,940 transcripts. A total of 28,715 (51.7%) unique sequences were annotated by NCBI non-redundant protein sequences (Nr) and 12,256 (22.1%) of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes Ortholog database (KO).12 unigenes were involved in TIAs synthesis were identified from our library. Additionally, a total of 193 cytochrome P450 (CYP450),280 methyltransferase and 144 isomerase unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the strictosidine into the RIN and IRN.3. We proceeded the capsule DGE sequencing analysis at different developmental stages, and more than 100 million valid data were obtained in each sample of capsule. Capsule 1 annotated 46,785 genes, capsule 2 annotated 47,241 genes and capsule 3 annotated 47,474 genes. Among them, the common genes of three samples reached 38,592, characteristic genes of capusle 1 were 1,687, characteristic genes of capusle 2 were 1,901 and characteristic genes of capusle 3 were 1,986. There were 1,488 differentially expressed genes in capsule 2 vs capsule 1 combinations there were 1,343 differentially expressed genes in capsule 3 vs capsule 1 combinations; there are 1,779 differentially expressed genes in capsule 3 vs capsule 2 combination.4. This study proposed TIAs biosynthetic new branch and RIN and IRN biosynthesis pathway. Loganin and tryptamine synthesized stemmadenine, and then through re-oxidation, methylation and isomerization synthesized RIN and IRN.5. Combined transcriptome data, DGE analysis and cluster analysis of gene expression patterns, we determined the candidate genes of CYP450 had 4 unigenes (comp31075_c0, ;omp31703_c1, comp35950_c0 and comp26673_c0); candidate genes of methyltransferase had unigenes (comp31161_c0, comp33316_c0 and comp34948_c0) and candidate genes of isomerase had 2 unigenes (comp22426_c0 and comp38594_c0), which involved in the late steps of the RIN and IRN biosynthesis.