| Eucommia ulmoides belonging to Eucommia, Eucommiaceae is a famous medicinal woody species as well as a rare species with high-quality rubber distributed in subtropical and temperate area. Eucommia contents plentiful terpenoids, among which at least 15 iridoids were isolated. Most of the iridoids have anti-cancer, anti-oxidation, anti-aging and antihypertensive effect. Another important terpene-gutta rubber, its "rubber-plastic duality" opens up the application of gutta rubber. Its comprehensive development and utilization has aroused great attention of relevant state departments as the greatest potential for development of high-quality rubber tree. But with the low content of gutta rubber, high cost for glue and little research about the biosynthesis of terpenoids and rubber, it is important to study the biosynthesis and accumulation mechanisms of Eucommia terpenoids and rubber on molecular level, identify the gene involved in rubber biosynthesis, combine molecular and traditional breeding to improve the quantity and quality of terpenoids. This study firstly incorporated the genomic and transcriptome data, systematically studied the genes involved in terpenoids biosynthesis and identified the genes related to rubber biosynthesis, offering a theoretical basis for elucidating the molecular mechanism of terpene synthesis and gutta-percha mechanisms and molecular breeding.(1) Based on Eucommia genomic data, we identified 70 genes involved in terpenoids biosynthesis, among which 13 involved in MEP pathway,15 involved in MVA pathway and 42 involved in the downstream of terpenoids synthesis.(2) The range of effective number of codon, amino acid length, protein isoelectric point and molecular weight are 48.64～61.00,69 aa～846 aa,4.61～10.04, 7.8-9.17 kDa, respectively. Proteins encoded by EuHMGRl, EuHMGR2, EuREF1, EuREF4, EuREF8, EuREF10 and EuREF12 have transmembrane helix structures. Most genes’ 5’flanking region have a lot of cis-acting elements related to stress, such as hypoxic induction, MeJa response and so on, among which light response elements are the most, occupying 31%-72%.(3) Subcellular localization found that most proteins encoded by genes involved in MEP were located in chloroplast, while MVA in mitochondria, downstream in chloroplast, mitochondria and secretory, indicating their complex function.(4) Phylogenetic analysis showed that REFs and MLPs were clustered in two groups respectively. EuREF1 and EuREF11 which were related to rubber synthesis belong to clade I of REFs while EuMLP1, EuMLP3 and EuMLPIO belong to clade I of MLPs and EuMLP6 belongs to clade Ⅱ of MLPs. There were three clades of DXS genes in Eucommia genome, playing different roles in terpenoides biosynthesis. Proteins encoded by EuGPPS1 and EuGPPS2 are homodimer.(5)EuDXS3,EuDXS4,EuHDR2,EuHMGS1,EuHMGS2,EuHMGR4,EuHMGR5,Eu MDC1,EuFPPS3,EuFPPS4,EuGPPS1,EuGGPPS2,EuGGPPS3,EuGGPPS4,EuREF4, EuREF7,EuREF8,EuREF9,EuREF11,EuMLP6,EuMLP7,EuMLP8 and EuMLPl were specific to Eucommia, among which EuHMGSl,EuHMGS2,EuMDC1,EuREF11and EuMLP6 were related to rubber biosynthesis.(6) Gene expression patterns showed that MVA pathway may be the main pathway that produced the precursor IPP for rubber synthesis. Expression patterns of genes involved in MVA pathway and downstream of terpenoids synthesis such as EuAACT1,EuHMGS1,EuHMGS2,EuHMGR1,EuMK,EuPMK,EuMDC1,EuMDC2, EuIDI,EuFPPS6,EuGGPPS7,EuREF1,EuREF11,EuMLP1,EuMLP3,EuMLP6 and EuMLP10 were in accordance with gutta rubber accumulation regularity. EuREF1, EuREF11 and EuMLP1 have a very large expression and they may play an important role in rubber biosynthesis. EuFPPS6, EuGGPPS7, EuREF11 and EuMLP6 may specifically participated in rubber synthesis in fruits while EuREF1 0 specifically participated in rubber synthesis in leaves. At the same period, expression of genes involved in MEP pathway in leaves were larger than that in fruits, while MVA pathway on the contrary. |
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