本文選題：山雞椒 + IPS　； 參考：《中國林業(yè)科學研究院》2017年碩士論文

【摘要】：山雞椒(Litsea cubeba(Lour.)Pers)是我國重要的天然香料樹種,以果實為主要原料提取的山雞椒精油在化工業(yè)、農(nóng)業(yè)、制藥業(yè)等行業(yè)有廣泛應用,供不應求。山雞椒精油主要成分萜類物質(zhì)的合成途徑及關(guān)鍵基因暫不清楚,萜類合成通路關(guān)鍵酶的研究可以為品種的遺傳改良提供基因資源。山雞椒精油以單萜與倍半萜為主要活性物質(zhì),目前在山雞椒萜類合成通路上已陸續(xù)有關(guān)鍵酶基因被克隆鑒定,但其中具有競爭性關(guān)鍵調(diào)控作用的異戊烯基轉(zhuǎn)移酶(IPS)尚缺乏了解。本研究基于山雞椒果實發(fā)育過程的轉(zhuǎn)錄組數(shù)據(jù),鑒定了來自LcIPS三個基因家族共14條基因,對這14條基因進行了表達模式分析;在此基礎上,挑選來自山雞椒香葉基二磷酸合酶(LcGPPS)家族與山雞椒香葉基香葉基二磷酸合酶(LcGGPPS)家族的4條基因進行了序列分析與蛋白表達分析;并對來自兩個家族的蛋白之間的互作關(guān)系進行了預測與驗證,研究結(jié)果為深入闡明山雞椒精油合成的調(diào)控機制,提高品質(zhì)和產(chǎn)量提供了理論基礎,主要研究結(jié)果如下:(1)山雞椒LcIPS三個家族基因的鑒定與表達模式分析。依據(jù)山雞椒果實發(fā)育過程的轉(zhuǎn)錄組數(shù)據(jù),尋找到分別來自香葉基焦磷酸合酶基因(GPPS)、法尼基焦磷酸合酶基因(FPPS)與香葉基香葉基焦磷酸合酶基因(GGPPS)家族共14條基因,以山雞椒各組織及不同發(fā)育時期的果實RNA反轉(zhuǎn)錄的cDNA為模板分析這14條基因的表達模式,經(jīng)分析,14條LcIPSs都有其獨特的表達模式,每個家族都有至少一個基因發(fā)揮著主要作用。在LcFPPS家族里,LcFPPS2在果實發(fā)育過程中呈現(xiàn)出顯著的表達高峰。在LcGPPS家族里,我們發(fā)現(xiàn)了同質(zhì)體與異質(zhì)體兩類蛋白,其中屬于異質(zhì)體小亞基的LcGPPS.SSU1在果實發(fā)育過程中出現(xiàn)顯著的表達高峰。在LcGGPPS家族中,LcGGPPS1參與較多組織中萜類物質(zhì)的生成,LcGGPPS2的作用更具特異性,LcGGPPS3-7在花和果實的發(fā)育過程中萜類物質(zhì)的合成都起著作用,LcGGPPS6在果實發(fā)育過程中的表達趨勢并沒有明顯的規(guī)律,其余6條基因在果實發(fā)育過程中都展現(xiàn)出特異的作用時期。(2)分離LcGPPS1、LcGPPS.SSU1、LcGGPPS1與LcGGPPS3基因,進行序列分析和理化性質(zhì)預測。LcGPPS1的ORF為966bp,編碼321個氨基酸;LcGPPS.SSU1的ORF包含897bp,編碼298個氨基酸;LcGGPPS1和LcGGPPS3基因的ORF均為1152bp,編碼383個氨基酸。ExPASy預測顯示LcGPPS1、LcGPPS.SSU1、LcGGPPS1、LcGGPPS3蛋白的相對分子量分別為34.97、32.21、41.39、41.49 kDa,理論等電點pI分別為5.43、5.33、5.86、5.87,蛋白的分子式分別為C1535H2499N421O477S15、C1413H2279N395O431S16、C1828H2960N504O551S18、C1820H2926N510O554S21。對4個蛋白進行亞細胞定位預測顯示,LcGPPS1定位于細胞核中,LcGPPS.SSU1可能定位于葉綠體或細胞質(zhì)中,LcGGPPS1和LcGGPPS3都有最高的可能性位于線粒體內(nèi)。(3)通過真核與原核外源表達系統(tǒng)進行LcGPPS1、LcGPPS.SSU1、LcGGPPS1與LcGGPPS3重組蛋白表達分析。通過構(gòu)建真核分泌表達載體轉(zhuǎn)化畢赤酵母,甲醇誘導表達后,發(fā)現(xiàn)4個重組蛋白都以單體和二聚體的形式主要存在于菌體中,較少分泌到細胞外。對表達產(chǎn)物進行鎳柱純化時,得到LcGGPPS3在畢赤酵母里表達的重組蛋白。進一步構(gòu)建原核表達載體pET28a-LcGGPPS1與pET28a-LcGPPS.SSU1,分別轉(zhuǎn)化大腸桿菌BL21(DE3)與Rosetta菌株,IPTG誘導表達后經(jīng)螯合SFF(Ni)柱純化得到LcGGPPS1重組蛋白。(4)通過三維結(jié)構(gòu)預測和酵母雙雜驗證LcGPPS.SSU1分別與LcGGPPS1和LcGGPPS3的互作關(guān)系。進化關(guān)系分析發(fā)現(xiàn)LcGGPPS1與LcGGPPS3都沒有聚類到LSU類別,且LcGGPPS3與大部分GGPPS都相距較遠。在LcGPPS.SSU1、LcGGPPS1與LcGGPPS3的三維結(jié)構(gòu)中都包含一個GPPS核心保守區(qū)域,GPPS核心組成了LcGPPS.SSU1大部分結(jié)構(gòu)。且根據(jù)預測LcGGPPS1與LcGGPPS3都可以與LcGPPS.SSU1進行互作。酵母雙雜實驗表明,LcGGPPS3能夠與LcGPPS.SSU1進行蛋白互作,但顯示互作較弱;LcGGPPS1則不能與LcGPPS.SSU1互作。
[Abstract]:Litsea cubeba (Lour.) Pers is an important natural spice tree in China. The essential oil extracted from fruit is widely used in chemical industry, agriculture, pharmaceutical industry and other industries. The main components of terpenoids in the main components of the essential oil are not clear, and the key enzymes in the terpenoid synthesis pathway The study can provide genetic resources for genetic improvement of varieties. The essential oil of the essential oil is monoterpene and sesquiterpene as the main active substance. At present, the key enzyme genes have been cloned and identified in the synthesis pathway of terpenoid terpenoids, but the competitive key regulatory effect of Isoamyl transferase (IPS) is still short of understanding. A total of 14 genes from three LcIPS genes were identified and the expression patterns were analyzed. On this basis, 4 genes from the two phosphate synthase (LcGPPS) family and the fragrant leaf based two phosphate synthase (LcGGPPS) family of the fragrant leaf base of Zanthoxylum chilli were selected. Sequence analysis and protein expression analysis, and the interaction between the proteins from two families were predicted and verified. The results provide a theoretical basis for further clarifying the regulation mechanism of essential oil synthesis and improving quality and yield. The main results are as follows: (1) identification and table of three family genes of LcIPS According to the data of the transcriptional group of the fruit development process, 14 genes from the fragrant leaf based pyrophosphate synthase gene (GPPS), the farnesyl pyrophosphate synthase gene (FPPS) and the fragrant leaf based pyrophosphate synthase gene (GGPPS) family, respectively, were found, and the C of each tissue of the chilli pepper and the RNA reverse transcription of the fruit of different developmental stages was C. DNA was used to analyze the expression patterns of these 14 genes. After analysis, 14 LcIPSs had their unique expression patterns. Each family had at least one gene to play a major role. In the LcFPPS family, LcFPPS2 showed a significant peak in the process of fruit development. In the LcGPPS family, we found homogeneity and heterogeneity two. In the LcGGPPS family, LcGGPPS1 participates in the formation of terpenoids in more tissues, and the effect of LcGGPPS2 is more specific in the LcGPPS.SSU1 family. LcGGPPS3-7 plays a role in the synthesis of terpenoids in the development of flowers and fruits, LcGGPPS6. There is no obvious rule of expression in the process of fruit development. The other 6 genes show a specific period of action in the process of fruit development. (2) separation of LcGPPS1, LcGPPS.SSU1, LcGGPPS1 and LcGGPPS3 genes, sequence analysis and physicochemical properties of.LcGPPS1 ORF, 966bp, 321 amino acids, LcGPPS.SSU1's ORF packet It contains 897bp and encodes 298 amino acids, and the ORF of LcGGPPS1 and LcGGPPS3 is 1152bp. The encoding of 383 amino acids.ExPASy predicts LcGPPS1, LcGPPS.SSU1, LcGGPPS1, and LcGGPPS3 proteins are 34.97,32.21,41.39,41.49 kDa, respectively, and the molecular formula of the protein is respectively 1O477S15, C1413H2279N395O431S16, C1828H2960N504O551S18, C1820H2926N510O554S21. showed subcellular localization of 4 proteins, indicating that LcGPPS1 was located in the nucleus, LcGPPS.SSU1 might be located in the chloroplast or cytoplasm, and both LcGGPPS1 and LcGGPPS3 had the highest potential in mitochondria. (3) through the eukaryotic and prokaryotic exogenous tables The expression analysis of recombinant protein of LcGPPS1, LcGPPS.SSU1, LcGGPPS1 and LcGGPPS3 was carried out. The expression of eukaryotic expression vector was transformed into Pichia pastoris by construction of eukaryotic expression vector. After methanol induction, the 4 recombinant proteins were found mainly in the form of monomers and two polymers in the mycelium, less secreted to the cells. The recombinant protein expressed by LcGGPPS3 in Pichia pastoris was obtained. The prokaryotic expression vector pET28a-LcGGPPS1 and pET28a-LcGPPS.SSU1 were further constructed, and Escherichia coli BL21 (DE3) and Rosetta strain were transformed respectively. The recombinant egg white was purified by the chelated SFF (Ni) column after IPTG induced expression. (4) through three-dimensional structure prediction and yeast heterozygosity validation LcGPPS The relationship between.SSU1 and LcGGPPS1 and LcGGPPS3 respectively. Evolution relationship analysis found that both LcGGPPS1 and LcGGPPS3 did not cluster to LSU category, and LcGGPPS3 was far away from most GGPPS. In LcGPPS.SSU1, LcGGPPS1 and LcGGPPS3 three-dimensional structure contains a GPPS core conservative region, the core consists of most of the structures Both LcGGPPS1 and LcGGPPS3 can be interacted with LcGPPS.SSU1. The yeast double heterozygosity experiment shows that LcGGPPS3 can interact with LcGPPS.SSU1, but the interaction is weak, and LcGGPPS1 can not interact with LcGPPS.SSU1.

【學位授予單位】：中國林業(yè)科學研究院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S793.9

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