# Triterpennoids Biosynthesis Regulation for Leaf Coloring of Wheel Wingnut (_Cyclocarya paliurus_)
## 3 Results
### RNA-seq and Data Analysis  
The differentially expressed genes (DEGs) were analyzed by the transcriptome method. Nine leaf samples in total yielded 64.36 Gb clean data with 93% of bases scoring Q30, and 26,4908 unique genes were expressed. According to the principal component analysis (PCA) of FPKM values, nine samples (3 samples with 3 biological replicates) were clearly separated into three growing stages, respectively, exhibiting accuracy and reliability of our experiment data (Figure 2A). DEGs from A, B, and C stages were screened, annotated, and clustered (Figure 2). According to KEGG enrichment (Figure 2E), it was found that the metabolites and secondary metabolites biosynthesis-related genes occupied most DEGs. Among secondary metabolites biosynthesis, DEGs were found in sesquiterpenoids and triterpenoids biosynthesis along with flavonoids biosynthesis and isoflavbiosis. In total, 852 DEGs (Figure 2D) overlapping expression in CvsA, CvsB, and BvsA was screened, meaning they might be key DEGs regulated in the leaf development stage. Among these conservative genes, triterpenoids biosynthesis-related unigenes were found including 1-deoxy-D-xylulose-5-phosphate synthase (Cluster-7239.2898, Cluster-7239.130383), alpha-farnesene synthase (Cluster-7239.77779), (3S,6E)-merololol synthase (Cluster-3255.0), (-)-germacrene D synthase (Cluster-7239.155003, Cluster-7239.161173), and lupeol synthase (Cluster-7239.65073, Cluster-7239.59075, Cluster-7239.80579, Cluster-7239.144814, Cluster-7239.77098). All DEGs were then clustered by the K-means method and separated into six clusters according to expression differences during three leaf development stages (Figure 2C). Among them, 5528 unigenes in sub-class 6 exhibited gradient high expression levels in the old green leaf stage, moderate levels in the half red leaf stage, and low levels in the red new leaf stage. Unigenes regulating triterpenoid biosynthesis were also found in sub-class 6 including hydroxymethylglutaryl-CoA reductase, farnesyl diphosphate synthase, farnesyl diphosphate farnesyltransferase, diphosphomevalonate decarboxylase, hydroxymethylglutaryl-CoA synthase, isopentenyl-diphosphate Delta-isomerase, isopentenyl phosphate kinase, alpha-farnesene synthase, (3S,6E)-nerolidol synthase, (-)-germacrene D synthase, beta-amyrin synthase, NAD +-dependent farnesol dehydrogenase, and lupeol synthase. On the contrary, 2794 unigenes in sub-class 2 exhibited down-regulation during the C to A leaf development stage, also containing triterpenoid biosynthesis-related unigenes: hydroxymethylglutaryl-CoA reductase (NADPH), squalene monooxygenase, 1-deoxy-D-xylulose-5-phosphate synthase, and 1-deoxy-D-xylulose-5-phosphate synthase. In sub-class 3, 2299 unigenes exhibited a random variation trend, meaning they might not be correlated with triterpenoid accumulation. UDP-glucosyltransferase is also considered a regulated factor in triterpenoid biosynthesis [11; 12; 13]. According to our analysis, related UGT unigenes were also identified including UDP-glucosyltransferase 73C (5 unigenes), UDP-glucosyltransferase 75B1 (2 unigenes), UDP-glucosyltransferase 73B2 (2 unigenes), cyanohydrin UDP-glucosyltransferase-UGT85K (3 unigenes), and UDP-glucosyltransferase 85A (1 unigenes). Notably, UGT73B2 (Cluster-7239.138070, Cluster-7239.150441), UGT85K (Cluster-7239.198214, Cluster-7239.80990), and UGT85A (Cluster-7239.41467) were clustered into sub-class 6 according to the K-means method, meaning they are highly correlated with triterpenoid accumulation; while UDP-glucosyltransferase 75B1 unigenes (Cluster-7239.124835, Cluster-7239.26044) were clustered into sub-class 2, implying a negative regulation. Despite being clustered into sub-class 5, UDP-glycostransferase 73C3 unigenes (Cluster-7239.140635, Cluster-7239.50887, Cluster-7239.106623, Cluster-7239.90121) still showed significantly higher expression in the A stage than C stage, showing positive effects as UGT73B2, UGT85K, and UGT85A [14].