基于轉(zhuǎn)錄組測(cè)序?qū)V-B輻射下甘遂乳汁的比較蛋白質(zhì)組學(xué)研究
發(fā)布時(shí)間：2018-07-27 16:21
【摘要】：甘遂(Euphorbia kansui L.)是大戟科(Euphorbiaceae)大戟屬(Euphorbia)的多年生草本植物,為中國特有植物。其干燥塊根是傳統(tǒng)中藥,歷代《中國藥典》皆有收載。甘遂全株含有白色乳汁,乳汁是乳汁管的原生質(zhì)體。乳汁中含有許多具有生理活性的蛋白質(zhì),這些蛋白質(zhì)與乳汁管的發(fā)育和乳汁管中次生代謝產(chǎn)物的合成密切相關(guān)。我們先前研究了甘遂乳汁管的分布、發(fā)育、乳汁管的超微結(jié)構(gòu)變化以及初步的乳汁蛋白質(zhì)組學(xué),但是,甘遂基因組信息的缺乏限制了其乳汁蛋白質(zhì)的鑒定及其分子生物學(xué)的研究。第二代轉(zhuǎn)錄組測(cè)序以及蛋白質(zhì)組學(xué)技術(shù)的發(fā)展,極大的推動(dòng)了非模式生物以及缺乏基因組參考數(shù)據(jù)的植物的分子生物學(xué)研究。本研究采用Illumina雙端測(cè)序技術(shù)對(duì)甘遂轉(zhuǎn)錄組進(jìn)行測(cè)序,得到大量的獨(dú)立基因。并且在轉(zhuǎn)錄組數(shù)據(jù)庫的基礎(chǔ)上,利用iTRAQ標(biāo)記以及質(zhì)譜技術(shù)研究甘遂乳汁全蛋白以及UV-B輻射對(duì)甘遂乳汁蛋白質(zhì)的影響,為深入探討甘遂乳汁管細(xì)胞的發(fā)育過程以及萜類物質(zhì)的合成和調(diào)控提供理論基礎(chǔ)。主要研究結(jié)果如下：1.甘遂轉(zhuǎn)錄組測(cè)序以及開發(fā)SSR標(biāo)記1.1甘遂轉(zhuǎn)錄組序列組裝及數(shù)據(jù)分析在本研究中,采用Illumina雙端測(cè)序技術(shù)對(duì)甘遂進(jìn)行轉(zhuǎn)錄組測(cè)序,共獲得43211690個(gè)高質(zhì)量的reads。.將得到的reads進(jìn)行組裝得到58362個(gè)unigenes,且unigenes的平均長度和N50長度均為1683 bp。將得到的unigenes在Nr、Swiss-Prot、KEGG、COG和GO數(shù)據(jù)庫中進(jìn)行比對(duì)和注釋,其中36396(62.36%)個(gè)unigenes成功注釋。其中有36318個(gè)unigenes注釋到Nr數(shù)據(jù)庫中,26640個(gè)注釋到Swiss-Prot數(shù)據(jù)庫中,13528個(gè)注釋到COG數(shù)據(jù)庫中,9562個(gè)注釋到KEGG數(shù)據(jù)庫中,15506個(gè)注釋到GO數(shù)據(jù)庫中。1.2甘遂萜類化合物合成相關(guān)基因的鑒定及分析萜類化合物是甘遂主要的生物活性成分。基于KEGG數(shù)據(jù)庫分析,在甘遂轉(zhuǎn)錄組中鑒定出了萜類骨架合成相關(guān)酶的nigenes,包括3-羥基-3-甲基戊二酰輔酶A合酶以及甲羥戊酸焦磷酸脫羧酶等的unigenes。此外,還發(fā)現(xiàn)了催化其活性成分二萜類化合物合成的重要酶——蓖麻烯合酶的unigene。1.3 SSR位點(diǎn)的檢測(cè)和驗(yàn)證為了開發(fā)分子標(biāo)記,在6150個(gè)unigenes中檢測(cè)到7016個(gè)候選的SSR位點(diǎn)并隨機(jī)選取40對(duì)引物在兩個(gè)居群中進(jìn)行多態(tài)性檢測(cè),28對(duì)引物成功地?cái)U(kuò)增出預(yù)期大小的條帶并且23對(duì)引物存在多態(tài)性,5對(duì)引物存在單態(tài)性。多態(tài)性SSR位點(diǎn)的等位基因有2-8個(gè),平均等位基因數(shù)為3.391。期望雜合度和實(shí)際雜合度分別為0.099至0.809及0.100至1.000。2.甘遂乳汁蛋白質(zhì)的鑒定基于甘遂轉(zhuǎn)錄組數(shù)據(jù)庫和大戟科蛋白數(shù)據(jù)庫,利用iTRAQ標(biāo)記和質(zhì)譜技術(shù)在甘遂乳汁中共鑒定出584個(gè)蛋白質(zhì)。2.1甘遂乳汁管發(fā)育相關(guān)的蛋白質(zhì)本研究中,營養(yǎng)生長時(shí)期的乳汁管處于乳汁管發(fā)育后期以前的階段,而生殖生長時(shí)期的乳汁管多為發(fā)育末期和成熟的乳汁管。對(duì)處于營養(yǎng)生長時(shí)期和生殖生長時(shí)期乳汁管的乳汁進(jìn)行差異蛋白質(zhì)組學(xué)分析,發(fā)現(xiàn)蛋白酶體蛋白的豐度下調(diào),即其在營養(yǎng)生長時(shí)期的乳汁管中的含量較高。抗泛素抗體的免疫印跡結(jié)果也發(fā)現(xiàn)營養(yǎng)生長時(shí)期的乳汁管的乳汁中泛素化蛋白的含量較生殖生長時(shí)期稍多,尤其是分子量約為35 kDa處的條帶。在乳汁管發(fā)育過程中,降解錯(cuò)誤折疊蛋白質(zhì)的內(nèi)質(zhì)網(wǎng)相關(guān)降解途徑中的一些蛋白質(zhì)的含量也發(fā)生改變。這表明在乳汁管發(fā)育過程中泛素-蛋白酶體途徑參與了乳汁管細(xì)胞中細(xì)胞質(zhì)以及內(nèi)質(zhì)網(wǎng)上相關(guān)蛋白的調(diào)控或降解。此外,在甘遂乳汁中鑒定到了一些溶酶體酶,其中,隨著甘遂乳汁管的發(fā)育V-ATPase的含量下調(diào),故推測(cè)自噬途徑也可能參與了甘遂乳汁管的發(fā)育過程。2.2乳汁管中萜類化合物骨架合成相關(guān)的蛋白質(zhì)在甘遂乳汁中鑒定到了萜類化合物骨架合成相關(guān)的蛋白質(zhì),蛋白質(zhì)定量分析發(fā)現(xiàn),異戊烯焦磷酸異構(gòu)酶及法呢基焦磷酸合酶在營養(yǎng)生長時(shí)期的乳汁管的乳汁中的含量較高。隨著乳汁管細(xì)胞的生長發(fā)育,這些酶的含量下調(diào)。2.3 UV-B輻射下的差異表達(dá)蛋白分析通過差異蛋白質(zhì)組學(xué)研究發(fā)現(xiàn)UV-B輻射處理后,乳汁中14-3-3蛋白、V-ATPase、溶酶體酶和cathepsin B的含量上調(diào),UV-B輻射可能影響甘遂乳汁管的發(fā)育。此外,UV-B處理后,乳汁中甲羥戊酸焦磷酸脫羧酶的含量上調(diào),并得到western blot結(jié)果的驗(yàn)證,表明UV-B輻射也影響乳汁管中萜類化合物的合成。綜上所述,本研究首先利用第二代高通量測(cè)序技術(shù)對(duì)甘遂進(jìn)行大規(guī)模轉(zhuǎn)錄組測(cè)序,得到大量獨(dú)立基因,并對(duì)得到的獨(dú)立基因進(jìn)行注釋和分析。此外,還鑒定到了參與萜類骨架合成以及二萜類化合物合成的候選基因,并且開發(fā)和驗(yàn)證一些SSR引物。在轉(zhuǎn)錄組數(shù)據(jù)庫的基礎(chǔ)上,利用iTRAQ標(biāo)記和質(zhì)譜技術(shù)鑒定甘遂乳汁蛋白。通過差異蛋白質(zhì)組學(xué)分析,鑒定到多個(gè)參與甘遂乳汁管細(xì)胞發(fā)育和乳汁管中萜類化合物合成相關(guān)的蛋白,為進(jìn)一步探討乳汁管細(xì)胞的發(fā)育及萜類化合物的合成和調(diào)控提供理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q946;Q943.2
本文編號(hào)：2148401
[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.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q946;Q943.2
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