Excavation And Analysis Of Key Enzymes And MiRNAs Of Notoginsenoside Biosynthetic Pathway

The root of Panax notoginseng （Buek） F. H. Chen, also called Radix Notoginseng or Sanchi, is a well-known traditional Chinese medicine, belonging to the genus Panax of the family Araliaceae, has been cultivated and used in medicine since ancient times for its remarkable hemostatic and analgesia effect. P. notoginseng recorded in past Chinese Pharmacopoeia was warm in nature, sweet and taste bitter. The principal bioactive constituents of the herb are triterpene saponins （Rg1,Rb1 and R1）, which belong to dammarane-rype glycosides. Evidences from laboratory and clinical trials show that Sanchi saponins possessed prevention of cardiovascular disease, anti-hyperlipidemia, antidiabetics, anti-hypertensive, anti-inflammatory, antifatigue, anti-oxidant, anti-thrombotic, anti-aterosclerotic, hepatoprotective and neuroprotective activities.Presently, the annual yield of P. notoginseng is behind the requirement, resulting in prices continue to soar. The methods of traditional cultivation and breeding can’t fundamentally improve Sanchi saponins contents. Therefore, molecule mechanism of notoginsenosides biosynthesis should be carried out right now. After that, single or multiple enzyme reactions will be improved, then notoginsenosides contents will be increased too. In this paper, key enzyme genes and miRNA in notoginsenosides biosynthesis will be excavated, aiming to analysis its molecular mechanism and provide basis for genetic improvement.1. Saponins contents of Rg1, Rb1, R1 and Panax Notoginseng Saponins （PNS） in roots enhanced with the increase of growth age, and their contents between different years showed highly significant difference, except for R1 between 2a and la roots..2. With Solexa high-throughput sequencing technology for Sanchi transcriptome studying,4.62 Gb of the raw data is obtained, and 96,704 Unigenes were obtained after filtering. There are 2,517 Unigenes, of which 194 Unigenes involve in the synthesis of terpenoid skeleton, associated with secondary metabolism. With the usage of transcriptome data, almost all genes related to the saponins biosynthesis pathway have been found. Additionally,384 CYP450s,120 glycosyltransferases and 219 glucosyltransferases are also discovered. These Unigenes are important genetic resources in the regulation for notoginsenosides biosynthesis in the future.3. In the la,2a and 3a of mRNA expression profiles, more than five million clean tags were obtained and more than 157,000 different tags after removing the redundancy were obtained in each sample. In 2a₁a, the expression pattern of 1,008 genes were up-regulated and 535 genes were down-regulated; in 3a₁a,1,841 genes were up-regulated and 1,891 genes were down-regulated; in 3a₂a,1,387 genes were up-regulated and 1,841 genes were down-regulated. Those significant differentially expressed genes may be associated with notoginsenosides skeleton biosynthesis.4. After filtering, nine candidate CYP450 genes and eight candidate UDPG genes may be involved in notoginsenosides skeleton biosynthesis.5. The Open Reading Frame （ORF） sequences of PnFPS、PnSS, PnSE, PnCAS were obtained using RT-PCR method, then the protein structure and functional site of these genes were predictively analyzed by means of the bioinformatics methods. The results will provide a foundation for exploring the function of these genes and studying the molecular mechanism of notoginsenosides biosynthesis.6. In the 1a,2a and 3a of miRNA expression profiles, more than twenty million clean reads were obtained form each sRNA, which length distribution was mainly in 21 nt and 24 nt. In total, there were 154 conserved miRNAs belonged to 116 genes families and 77 novel miRNAs.7. There were 118,121, and 143 conserved miRNAs with significantly differentially expressed in 2a₁a,3a₁a and 3a₂a, respectively, in which 55,42 and 101 significantly down-regulated. The significant differentially expressed novel miRNAs in 2a₁a,3a₁a and 3a₂a were 13,10 and 14, respectively, and significant down-regulation genes were 7,4 and 6, respectively. Those significant differentially expressed miRNAs may be associated with notoginsenosides skeleton biosynthesis.8. Eight miRNA and WRKY transcription factor were found and may be involved in notoginsenosides contents. Twenty one miRNAs were found involved in plant-pathogen interaction.The aims of the excavation of key enzyme genes and miRNA involved in notoginsenosides biosynthesis were to provide a foundation for analysis its molecular mechanism, study unctional genomics and post-genomics of P. notoginseng, and provide theoretical basis for its genetic improvement.