基于转录组测序对UV-B辐射下甘遂乳汁的比较蛋白质组学研究

发布时间：2018-07-27 16:21

【摘要】：甘遂(Euphorbia kansui L.)是大戟科(Euphorbiaceae)大戟属(Euphorbia)的多年生草本植物,为中国特有植物。其干燥块根是传统中药,历代《中国药典》皆有收载。甘遂全株含有白色乳汁,乳汁是乳汁管的原生质体。乳汁中含有许多具有生理活性的蛋白质,这些蛋白质与乳汁管的发育和乳汁管中次生代谢产物的合成密切相关。我们先前研究了甘遂乳汁管的分布、发育、乳汁管的超微结构变化以及初步的乳汁蛋白质组学,但是,甘遂基因组信息的缺乏限制了其乳汁蛋白质的鉴定及其分子生物学的研究。第二代转录组测序以及蛋白质组学技术的发展,极大的推动了非模式生物以及缺乏基因组参考数据的植物的分子生物学研究。本研究采用Illumina双端测序技术对甘遂转录组进行测序,得到大量的独立基因。并且在转录组数据库的基础上,利用iTRAQ标记以及质谱技术研究甘遂乳汁全蛋白以及UV-B辐射对甘遂乳汁蛋白质的影响,为深入探讨甘遂乳汁管细胞的发育过程以及萜类物质的合成和调控提供理论基础。主要研究结果如下：1.甘遂转录组测序以及开发SSR标记1.1甘遂转录组序列组装及数据分析在本研究中,采用Illumina双端测序技术对甘遂进行转录组测序,共获得43211690个高质量的reads。.将得到的reads进行组装得到58362个unigenes,且unigenes的平均长度和N50长度均为1683 bp。将得到的unigenes在Nr、Swiss-Prot、KEGG、COG和GO数据库中进行比对和注释,其中36396(62.36%)个unigenes成功注释。其中有36318个unigenes注释到Nr数据库中,26640个注释到Swiss-Prot数据库中,13528个注释到COG数据库中,9562个注释到KEGG数据库中,15506个注释到GO数据库中。1.2甘遂萜类化合物合成相关基因的鉴定及分析萜类化合物是甘遂主要的生物活性成分。基于KEGG数据库分析,在甘遂转录组中鉴定出了萜类骨架合成相关酶的nigenes,包括3-羟基-3-甲基戊二酰辅酶A合酶以及甲羟戊酸焦磷酸脱羧酶等的unigenes。此外,还发现了催化其活性成分二萜类化合物合成的重要酶——蓖麻烯合酶的unigene。1.3 SSR位点的检测和验证为了开发分子标记,在6150个unigenes中检测到7016个候选的SSR位点并随机选取40对引物在两个居群中进行多态性检测,28对引物成功地扩增出预期大小的条带并且23对引物存在多态性,5对引物存在单态性。多态性SSR位点的等位基因有2-8个,平均等位基因数为3.391。期望杂合度和实际杂合度分别为0.099至0.809及0.100至1.000。2.甘遂乳汁蛋白质的鉴定基于甘遂转录组数据库和大戟科蛋白数据库,利用iTRAQ标记和质谱技术在甘遂乳汁中共鉴定出584个蛋白质。2.1甘遂乳汁管发育相关的蛋白质本研究中,营养生长时期的乳汁管处于乳汁管发育后期以前的阶段,而生殖生长时期的乳汁管多为发育末期和成熟的乳汁管。对处于营养生长时期和生殖生长时期乳汁管的乳汁进行差异蛋白质组学分析,发现蛋白酶体蛋白的丰度下调,即其在营养生长时期的乳汁管中的含量较高。抗泛素抗体的免疫印迹结果也发现营养生长时期的乳汁管的乳汁中泛素化蛋白的含量较生殖生长时期稍多,尤其是分子量约为35 kDa处的条带。在乳汁管发育过程中,降解错误折叠蛋白质的内质网相关降解途径中的一些蛋白质的含量也发生改变。这表明在乳汁管发育过程中泛素-蛋白酶体途径参与了乳汁管细胞中细胞质以及内质网上相关蛋白的调控或降解。此外,在甘遂乳汁中鉴定到了一些溶酶体酶,其中,随着甘遂乳汁管的发育V-ATPase的含量下调,故推测自噬途径也可能参与了甘遂乳汁管的发育过程。2.2乳汁管中萜类化合物骨架合成相关的蛋白质在甘遂乳汁中鉴定到了萜类化合物骨架合成相关的蛋白质,蛋白质定量分析发现,异戊烯焦磷酸异构酶及法呢基焦磷酸合酶在营养生长时期的乳汁管的乳汁中的含量较高。随着乳汁管细胞的生长发育,这些酶的含量下调。2.3 UV-B辐射下的差异表达蛋白分析通过差异蛋白质组学研究发现UV-B辐射处理后,乳汁中14-3-3蛋白、V-ATPase、溶酶体酶和cathepsin B的含量上调,UV-B辐射可能影响甘遂乳汁管的发育。此外,UV-B处理后,乳汁中甲羟戊酸焦磷酸脱羧酶的含量上调,并得到western blot结果的验证,表明UV-B辐射也影响乳汁管中萜类化合物的合成。综上所述,本研究首先利用第二代高通量测序技术对甘遂进行大规模转录组测序,得到大量独立基因,并对得到的独立基因进行注释和分析。此外,还鉴定到了参与萜类骨架合成以及二萜类化合物合成的候选基因,并且开发和验证一些SSR引物。在转录组数据库的基础上,利用iTRAQ标记和质谱技术鉴定甘遂乳汁蛋白。通过差异蛋白质组学分析,鉴定到多个参与甘遂乳汁管细胞发育和乳汁管中萜类化合物合成相关的蛋白,为进一步探讨乳汁管细胞的发育及萜类化合物的合成和调控提供理论基础。
[Abstract]:Euphorbia kansui L. is a perennial herb of euphorbifamily (Euphorbiaceae) genus of Euphorbia (Euphorbia). It is a Chinese endemic plant. Its dry root is a traditional Chinese medicine, the Chinese Pharmacopoeia (Chinese Pharmacopoeia) has been loaded. The whole plant contains white milk and milk is a protoplast of the milk tube. Milk contains many proteins with physiological activity. These proteins are closely related to the development of the milk tube and the synthesis of secondary metabolites in the milk tube. We previously studied the distribution, development, ultrastructural changes of the milk tube and the primary milk proteomics. However, the lack of genomic information of the radix euphorbium restricts the identification and classification of the milk protein. The study of subbiology. The second generation transcriptional group sequencing and the development of proteomics technology have greatly promoted the molecular biology research of non pattern organisms and plants lacking the reference data of the genome. This study uses Illumina double end sequencing technology to order the sequence of the radix Euphorbia transcriptome and obtain a large number of independent genes. On the basis of the group database, iTRAQ markers and mass spectrometry were used to study the effect of total protein and UV-B radiation on the protein of Euphorbia Euphorbia milk. It provides a theoretical basis for the in-depth study of the development process and the synthesis and regulation of terpenoids. The main results are as follows: 1. In this study, the sequence assembly and data analysis of the SSR tagged 1.1 Tagus transcriptional group were sequenced by Illumina double end sequencing technology. A total of 43211690 high quality reads.. Would be assembled to get 58362 unigenes, and the average length of unigenes and the length of N50 were 1683 bp.. NES performs comparison and annotations in the Nr, Swiss-Prot, KEGG, COG, and GO databases, in which 36396 (62.36%) unigenes are successfully annotated. There are 36318 unigenes annotations to the Nr database, 26640 annotations to the Swiss-Prot database, 13528 annotations to the COG database, 9562 annotations to the KEGG database, and 15506 annotations to the database. The identification and analysis of the related genes of 2 terpenoids synthesis and analysis of terpenoids are the main bioactive components of the terpenoid. Based on the KEGG database analysis, the nigenes of the terpene skeleton synthesis related enzymes was identified in the KEGG transcriptome, including the unige of the 3- hydroxyl -3- methylglutaryl coenzyme A synthase and the metholopate pyrophosphate decarboxylase. Nes. also found that the unigene.1.3 SSR site of the ricinene synthase, an important enzyme that catalyzes the synthesis of two terpenoids, has been detected and verified in order to develop molecular markers, to detect 7016 candidate SSR loci in 6150 unigenes and randomly select 40 pairs of primers for polymorphism detection in two populations and 28 pairs of primers. The expected size was amplified and 23 pairs of primers were polymorphic and 5 pairs of primers were singlet. There were 2-8 alleles of the polymorphic SSR loci. The average allele number was 3.391. expected heterozygosity and the actual heterozygosity was 0.099 to 0.809 and 0.100 to 1.000.2. respectively. In the database and Euphorbia protein database, iTRAQ markers and mass spectrometric techniques have been used to identify 584 proteins related to the development of protein.2.1 in the milk tube. The milk tube in the vegetative period is in the stage of the late stage of the late development of the milk tube, and the milk tube in the growing period is mostly at the end of the development and in the stage of growth. A mature milk tube. A differential proteomic analysis of milk in the vegetative and reproductive period of the milk tube showed that the abundance of the proteasome was down, that is, the content of the protein in the milk tube was higher in the vegetative period. The immunoblotting of anti ubiquitin antibody also found milk tube milk during the vegetative growth period. The content of ubiquitin protein in the juice is a little more than that of the reproductive period, especially the band of about 35 kDa. During the development of the milk tube, the content of some proteins in the endoplasmic reticulum degradation pathway that degrades the wrong folding protein is also changed. This indicates that the ubiquitin proteasome pathway is involved in the development of the milk tube. The control or degradation of cytoplasm and endoplasmic reticulum related proteins in the milk tube cells. In addition, some lysosomal enzymes have been identified in the milk juice of Euphorbia Euphorbia. With the downregulation of V-ATPase in the development of the Euphorbia Euphorbia milk tube, it is presumed that the autophagy pathway may also be involved in the terpenoid bone in the.2.2 milk tube. The proteins related to the framework of terpenoids were identified by the protein of the frame synthesis. Quantitative analysis of protein found that the content of Isoamyl pyrophosphate isomerase and pyrophosphoric acid synthase in the milk tube of the vegetative period was higher. With the growth and development of the milk tube cells, these enzymes were contained. Differential expression protein analysis under.2.3 UV-B radiation, the content of 14-3-3 protein, V-ATPase, lysosomal enzyme and cathepsin B in milk was up-regulated after UV-B radiation treatment, and UV-B radiation might affect the development of Euphorbia Euphorbia decarboxylase after UV-B treatment. The amount is up, and the results of Western blot verify that UV-B radiation also affects the synthesis of terpenoids in the milk tube. To sum up, this study first uses second generation high throughput sequencing technology to sequence the large scale transcriptional group, and annotate and analyze the obtained independent genes. The candidate genes involved in terpene skeleton synthesis and the synthesis of two terpenoids were identified, and some SSR primers were developed and verified. On the basis of the transcriptional database, iTRAQ markers and mass spectrometry were used to identify the milk protein. The proteins related to the synthesis of terpenoids in juice tubes provide a theoretical basis for further exploration of the development of milk duct cells and the synthesis and regulation of terpenoids.
【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q946;Q943.2

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