| Eucommia ulmoides Oliv. is a famous traditional Chinese medicine tree for being rich of valuable ingredients including phenyl propanoids, unsaturated fatty acids, flavonoids, iridoids, lignins in fruits,leaves,bark and stamens. These active components are provided with evident function of nourishing liver and kidney, improving bones and muscles, tocolysis, antibacterial, antihypertensive and hypolipidemic, which are widely used in medicine, health, food and cosmetics industries. In order to achieve the current goal of making a substantial increase in the content of the active ingredients in Eucommia, the gene diversity and expression mechanism of key enzyme gene should be cleared out at first. By taking fruit and leaves of Eucommia fine varieties ’Huazhong No.6’ as experimental materials, analysis of the transcriptome sequencing were completed. The genetic diversity and the expression mechanism referring to 4 active ingredients of chlorogenic acid, alpha-linolenic acid, quercetin and kaempferol were revealed by using bioinformatics methods, and the physical and chemical properties as well as to structural features of the full-length cDNA of the key gene family members were predicted, which would provide an important theoretical basis of efficient accumulation mechanism of the active ingredients.1. Transcriptome sequencing,assembly and functional annotation were studied on Eucommia fine varieties ’Huazhong No.6’. Total of 106 264 108 reads,7491046 464 nucleotides,1 316 317 contigs,292 591 scaffolds and 185403 unigene fragments were obtained. Unigenes of 67119 CDS,67119 protein, and 4948 EST were obtained after functional annotation of all-unigene, and 72893 GO unigene,42359 COG unigene,43715 KEGG unigene were obtained after annotation classification.2. We obtained 27 enzyme genes of Eucommia chlorogenic acid synthesis pathway, 20 of which gene expression displayed significant differences. Gene diversity of PAL gene family was observed extraordinary high, which included 12 members (EuPALl-EuPAL12) and expressed both in young fruits and mature leaves. The gene expressive level of EuPAL gene family including EuPAL3-EuPAL12 in fruit was significantly higher than in the leaves, while EuPAL1 and EuPAL2 were expressed dominant in leaves. EuPAL genes were aboundant expressed in Eucommia fruits and the expression mechanism was relativel complex. With full length cDNA of 2133 bp and encoding 711 amino acids, the molecular weight and theoretical isoelectric point of EuPAL1 was 77.14 kD and 6.24.EuPAL1 were classified as stable hydrophobic proteins and lyase-I-like superfamily, the secondary protein structure of which were dominant in alpha helix and random coil.Two members (EuC4H1,EuC4H2) were observed in cinnamic acid 4-hydroxylase (C4H) gene family, the gene expressive level of which was displayed no significant difference.However, the expressive level of EuC4H1 and EuC4H2 in fruits were 105.25 times and 6.32 times higher than in the leaves, respectively, and it is deduced that EuC4H1 play important role in fruits and leaves. With full length cDNA of 1641 bp and encoding 547 amino acids, the molecular weight and theoretical isoelectric point of EuC4H1 was 62.94 kD and 9.34, while EuC4H2 was of 1611 bp, encoded 537 amino acids, molecular weight of 60.96 kD and theoretical isoelectric point of 8.85.EuC4H1 and EuC4H1 were classified as stable hydrophobic proteins and Cypx superfamily, the secondary protein structure of which were dominant in a-helix,β-turn and random coil.Including 11 members, the gene diversity of 4CL gene family was observed relatively complex, in which the expressive level of 5 members referring to Eu4CL1、Eu4CL4-Eu4CL6、Eu4CL8 in young fruits and leaves were observed in significant differences, 3 members referring to Eu4CL9-Eu4CL11 were specific expressed in young fruits,6 members referring to Eu4CL1～Eu4CL6 were highly expressed in young fruits and leaves.Including 3 members, the HCT genes expressed both in leaves and fruits, in which the expressive level of EuHCT1 was observed significant differences, and it is deduced that EuHCT1 play important role in young fruits, EuHCT2 in leaves. With full length cDNA of 1692 bp and encoding 564 amino acids, the molecular weight and theoretical isoelectric point of EuHCT1 was 61.94 kD and 6.17. EuHCT1 were classified as stable hydrophilic proteins and AFD-class-I superfamily, the secondary protein structure of which were dominant in a-helix, β-turn and random coil.3. We obtained 13 enzyme genes of Eucommia a-linolenic acid synthesis pathway in young fruit and mature fruit, 6 of which were different expressed gene and 3 of which were different expressed in leaves and young fruits. The gene expressive level of FatA gene in young fruit and mature fruit showed no significant difference. With full length cDNA of 1338 bp and encoding 446 amino acids, the molecular weight and theoretical isoelectric point of EuFatA was 50.59 kD and 8.07. EuFatA were classified as stable hydrophobic proteins and CAP-ED superfamily, the secondary protein structure of which were dominant in a-helix,β-turn and random coil.Including 5 members,, the gene diversity of FAD2 gene family was observed relatively complex. The expressive level of EuFAD2-1 and EuFAD2-2 in young fruits and mature fruits reached to significant standard, EuFAD2-1 and EuFAD2-3 in fruits expressed higher than in leaves while EuFAD2-4 and EuFAD2-5 in young fruits expressed higher than in mature fruits. With full length cDNA of 993 bp and encoding 331 amino acids, the molecular weight and theoretical isoelectric point of EuFAD2 was 38.04 kD and 9.26. EuFAD2 were classified as unstable hydrophobic proteins and FAD enzyme family, the secondary protein structure of which were dominant in a-helix,β-turn and random coil.Including 6 members, the gene expressive level of EuFAD3-1～EuFAD3-5 in young fruit and mature fruit were not reached to significant standard except EuFAD3-6, the expressive level of more than 2/3 EuFAD members in mature fruits expressed higher than in young fruits. With full length cDNA of 1278 bp and encoding 426 amino acids, the molecular weight and theoretical isoelectric point of EuFAD3 was 49.18 kD and 7.43. EuFAD3 were classified as stable hydrophobic proteins and FAD enzyme family, the secondary protein structure of which were dominant in a-helix,p-turn and random coil.4. We obtained 11 enzyme genes of Eucommia quercetin and kaempferol synthesis pathway in young fruit and mature leaves, 8 of which were different expressed gene.Including 3 members, the gene expressive levels of EuCHS1 and EuCHS2 of EuCHS gene family in leaves and young fruit were reached to significant standard, in which EuCHS1 in leaves expressed 7.88 times higher than in fruits while EuCHS2 in fruits expressed 6.56 times higher than in leaves. It is deduced that EuCHSl play important role leaves and EuCHS2 in fruits. With full length cDNA of 1191 bp and encoding 397 amino acids, the molecular weight and theoretical isoelectric point of EuCHS was 43.33 kD and 6.23 respectively. EuCHS were classified as stable hydrophobic proteins and cond enzymes superfamily, the secondary protein structure of which were dominant in a-helix,p-turn and random coil.EuCHI expressed both in leaves and young fruits, but the expressive level did not reach to significant level. An CHI-like protein was obtained and its expressive level arrived at significant standard. With full length cDNA of 777 bp and encoding 259 amino acids, the molecular weight and theoretical isoelectric point of EuCHI was 27.63 kD and 4.81. EuCHI were classified as stable hydrophobic proteins and chalone superfamily, the secondary protein structure of which were dominant in a-helix,p-turn and random coil.EuF3H expressed both in leaves and young fruits but the expressive level did not reached to significant level.EuF3’H expressed both in leaves and young fruits but the expressive level did not reach to significant level. With full length cDNA of 1551 bp and encoding 517 amino acids, the molecular weight and theoretical isoelectric point of EuF3’H was 56.71 kD and 8.72. EuF3’Hwere classified as stable hydrophobic proteins and Cypx superfamily, the secondary protein structure of which were dominant in a-helix and random coil.Including 4 members, the gene expressive levels of EuFLS1～Eu FLS4 of EuFLS gene family in leaves and young fruit were reached to significant standard, in which EuFLS3 and EuFLS4 in leaves expressed 12.98 times and 44.24 times higher than in fruits, respectively, and in which EuFLSl and EuFLS2 in fruits expressed 15.38 times and 17.65 times higher than in leaves.lt is deduced that EuFLS3 and EuFLS4 play key roles in leaves and EuFLS1 and EuFLS2 in fruits. |
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