| Euphorbia kansui L. which belongs to Euphorbia in Euphorbiaceae is a perennial herbaceous species. E. kansui is an endemic species in China. The dried root of E. kansui is a commonly used and well-known traditional Chinese medicine and is listed in the Chinese Pharmacopoeia. The whole E. kansui plant contains white latex which is the protoplasm constituent of specialized cells known as laticifers. The latex contains lots of latex proteins with various biological activities. These latex proteins are closely related to laticifer development and synthesis of the secondary metabolites. We have studied the distribution, development and ultrastructure of laticifers, and preliminary latex proteomics on E. kansui. However, the genomic information about E. kansui is lacking, and the current lack of genomic sequence data limits the identification of latex proteins and molecular biology researches. The development of next generation sequencing (NGS) and proteomics technologies greatly promoted the molecular biology researches of the species which are non-model plant or lacking of reference genome data information. In the present study, we firstly performed a large-scale transcriptome sequencing using Illumina paired-end sequencing technology to obtain lots of unigenes information. Whereafter, we further identified E. kansui latex proteins and comparatively analyzed differentially expressed latex proteins under UV-B radiation based on the obtained transcriptome database using iTRAQ and mass spectrometry technology. The results will lay a good thesis foundation for further exploration of E. kansui laticifer development and terpenoids synthesis and regulation. The main research results were as follows:1. Transcriptome sequencing and development of SSR markers in Euphorbia kansui1.1 Transcriptome sequences assembly and data analysisIn the present study, a total of 43,211,690 high quality reads were generated using Illumina paired-end sequencing technology. After assembly of the obtained reads,58,362 unigenes were recovered. Both the N50 and average length of unigenes were 1,683 bp. All obtained unigenes were searched against the databases including non-redundant protein (Nr) database, Swiss-Prot protein database, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, Cluster of Orthologous Groups (COG) database and Gene ontology (GO) database with an E-value cut-off of 10'5. A total of 36,396 (62.36%) unigenes had at least one blast hit against the multiple public databases. Among them,36,318 unigenes were annotated in Nr database,26,640 unigenes were homologous with Swiss-Prot database,13,528 unigenes were annotated in COG database,9,562 unigenes were annotated in KEGG database, and 15,506 unigenes were annotated in GO database.1.2 Identification and analysis of candidate genes involved in terpenoid biosynthesisTerpenoid are the major biologically active substances of E. kansui. Based on KEGG pathway analyses, the unigenes which are related to terpenoid backone biosynthesis were discovered, such as the unigenes were encoded 3-hydroxy-3-methylglutaryl coenzyme A synthase and mevalonate disphosphate decarboxylase. In addition, the unigene related to casbene synthase which catalyzed the formation of the major active constituents (diterpenoids) in E. kansui were identified.1.3 SSR discovery and validationFor molecule marker development,7,016 candidate simple sequence repeats (SSR) were identified from 6,150 unigenes. A total of 40 SSR loci were selected randomly and validated in two populations of E. kansui. A total of 28 loci were amplified successfully and 23 loci exhibited polymorphisms,5 loci exhibited monomorphisms. The number of alleles of each polymorphisms SSR locus ranged from 2 to 8 alleles and the mean number of alleles per locus was 3.391. The observed heterozygosity and expected heterozygosity varied from 0.100 to 1.000 and 0.099 to 0.809, respectively.2. Identification of Euphorbia kansui latex proteinsWe identified 584 latex proteins using iTRAQ (isobaric tag for relative and absolute quantitation) and mass spectrometry analysis based on E. kansui transcriptome database and NCBI Euphorbiaceae RefSeq protein database.2.1 The latex proteins related to the development of Euphorbia kansui laticifersIn the present study, the laticifers in the vegetative growth stage were the laticifers before late development stages, and the laticifers in the reproductive growth stage were the laticifers at the end and mature development stages. We analyzed differential proteomics of latex proteins in the vegetative growth stage and reproductive growth stage. Proteasome proteins were down-regulated, and proteasome contents were higher in laticifers at the vegetative growth stage. Western blotting analysis of ubiquitin antibody was also showed that ubiquitinated proteins were more in laticifers at the vegetative growth stage, especially the proteins with molecular weight of about 35 kDa. During development of laticifers, some proteins involved in endoplasmic reticulum associated protein degradation pathway were down-regulated. Misfolded proteins were degraded via endoplasmic reticulum associated protein degradation pathway. These results suggest that ubiquitin-proteasome pathway was involved in the degradation and regulation of laticifers cell cytoplasm and endoplasmic reticulum associated proteins. In addition, some lysosomal enzymes were identified in latex of E. kansui. V-ATPase was down-regulated with the development of laticifers, so autophagy pathway may also be involved in the development of E. kansui laticifers.2.2 The latex proteins related to terpenoid backone biosynthesisThe proteins related to terpenoid backone biosynthesis were identified in E. kansui latex including hydroxymethylglutaryl-CoA synthase, mevalonate disphosphate decarboxylase, isopentenyl-diphosphate isomerase and farnesyl diphosphate synthase. Quantitative analysis revealed that the content of isopentenyl-diphosphate isomerase and farnesyl diphosphate synthase were higher in laticifers at vegetative growth stage. With the development of laticifers, these two enzymes were down-regulated.2.3 Differentially expressed latex proteins analysis under UV-B radiationComparative proteomic research showed that 14-3-3 protein, V-ATPase, lysosomal enzymes and cathepsin B were up-regulated under UV-B treatment. Therefore, UV-B radiation may affect the development of E. kansui laticifers. In addition, mevalonate disphosphate decarboxylase was up-regulated after UV-B treatment, which was tested by western blotting analysis. As a result, UV-B radiation also affects terpenoids synthesis in laticifers.Above all, in present study, a large-scale transcriptome sequencing using the second generation sequencing technology was firstly performed. The obtained unigenes were annotated and analyzed. Secondly, candidate unigenes were discovered to encode known enzymes that catalyze the formation of the terpenoid backone and diterpenoid biosynthesis, and SSR markers were also developed and validated. In addition,584 latex proteins were identified using iTRAQ and mass spectrometry technology based on E. kansui transcriptome database. Lastly, the latex proteins related to development of laticifers and terpenoid biosynthesis were identified and analyzed by differential proteomics research. This study will provide the basis for further researches on the development of laticifers and terpenoids synthesis and regulation in E. kansui. |
PDF Full Text Request