基于轉錄組測序對UV-B輻射下甘遂乳汁的比較蛋白質組學研究
發(fā)布時間：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體。乳汁中含有許多具有生理活性的蛋白質,這些蛋白質與乳汁管的發(fā)育和乳汁管中次生代謝產物的合成密切相關。我們先前研究了甘遂乳汁管的分布、發(fā)育、乳汁管的超微結構變化以及初步的乳汁蛋白質組學,但是,甘遂基因組信息的缺乏限制了其乳汁蛋白質的鑒定及其分子生物學的研究。第二代轉錄組測序以及蛋白質組學技術的發(fā)展,極大的推動了非模式生物以及缺乏基因組參考數據的植物的分子生物學研究。本研究采用Illumina雙端測序技術對甘遂轉錄組進行測序,得到大量的獨立基因。并且在轉錄組數據庫的基礎上,利用iTRAQ標記以及質譜技術研究甘遂乳汁全蛋白以及UV-B輻射對甘遂乳汁蛋白質的影響,為深入探討甘遂乳汁管細胞的發(fā)育過程以及萜類物質的合成和調控提供理論基礎。主要研究結果如下：1.甘遂轉錄組測序以及開發(fā)SSR標記1.1甘遂轉錄組序列組裝及數據分析在本研究中,采用Illumina雙端測序技術對甘遂進行轉錄組測序,共獲得43211690個高質量的reads。.將得到的reads進行組裝得到58362個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數據庫中進行比對和注釋,其中36396(62.36%)個unigenes成功注釋。其中有36318個unigenes注釋到Nr數據庫中,26640個注釋到Swiss-Prot數據庫中,13528個注釋到COG數據庫中,9562個注釋到KEGG數據庫中,15506個注釋到GO數據庫中。1.2甘遂萜類化合物合成相關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�；贙EGG數據庫分析,在甘遂轉錄組中鑒定出了萜類骨架合成相關酶的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位點的檢測和驗證為了開發(fā)分子標記,在6150個unigenes中檢測到7016個候選的SSR位點并隨機選取40對引物在兩個居群中進行多態(tài)性檢測,28對引物成功地擴增出預期大小的條帶并且23對引物存在多態(tài)性,5對引物存在單態(tài)性。多態(tài)性SSR位點的等位基因有2-8個,平均等位基因數為3.391。期望雜合度和實際雜合度分別為0.099至0.809及0.100至1.000。2.甘遂乳汁蛋白質的鑒定基于甘遂轉錄組數據庫和大戟科蛋白數據庫,利用iTRAQ標記和質譜技術在甘遂乳汁中共鑒定出584個蛋白質。2.1甘遂乳汁管發(fā)育相關的蛋白質本研究中,營養(yǎng)生長時期的乳汁管處于乳汁管發(fā)育后期以前的階段,而生殖生長時期的乳汁管多為發(fā)育末期和成熟的乳汁管。對處于營養(yǎng)生長時期和生殖生長時期乳汁管的乳汁進行差異蛋白質組學分析,發(fā)現蛋白酶體蛋白的豐度下調,即其在營養(yǎng)生長時期的乳汁管中的含量較高�？狗核乜贵w的免疫印跡結果也發(fā)現營養(yǎng)生長時期的乳汁管的乳汁中泛素化蛋白的含量較生殖生長時期稍多,尤其是分子量約為35 kDa處的條帶。在乳汁管發(fā)育過程中,降解錯誤折疊蛋白質的內質網相關降解途徑中的一些蛋白質的含量也發(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胞中細胞質以及內質網上相關蛋白的調控或降解。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,故推測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的蛋白質在甘遂乳汁中鑒定到了萜類化合物骨架合成相關的蛋白質,蛋白質定量分析發(fā)現,異戊烯焦磷酸異構酶及法呢基焦磷酸合酶在營養(yǎng)生長時期的乳汁管的乳汁中的含量較高。隨著乳汁管細胞的生長發(fā)育,這些酶的含量下調。2.3 UV-B輻射下的差異表達蛋白分析通過差異蛋白質組學研究發(fā)現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,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,并得到western blot結果的驗證,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序技術對甘遂進行大規(guī)模轉錄組測序,得到大量獨立基因,并對得到的獨立基因進行注釋和分析。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)和驗證一些SSR引物。在轉錄組數據庫的基礎上,利用iTRAQ標記和質譜技術鑒定甘遂乳汁蛋白。通過差異蛋白質組學分析,鑒定到多個參與甘遂乳汁管細胞發(fā)育和乳汁管中萜類化合物合成相關的蛋白,為進一步探討乳汁管細胞的發(fā)育及萜類化合物的合成和調控提供理論基礎。
[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.
【學位授予單位】：西北大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q946;Q943.2
本文編號：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.
【學位授予單位】：西北大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q946;Q943.2
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