山鸡椒LcIPS家族基因克隆与蛋白表达分析

发布时间：2018-04-21 05:16

本文选题：山鸡椒 + IPS　；参考：《中国林业科学研究院》2017年硕士论文

【摘要】：山鸡椒(Litsea cubeba(Lour.)Pers)是我国重要的天然香料树种,以果实为主要原料提取的山鸡椒精油在化工业、农业、制药业等行业有广泛应用,供不应求。山鸡椒精油主要成分萜类物质的合成途径及关键基因暂不清楚,萜类合成通路关键酶的研究可以为品种的遗传改良提供基因资源。山鸡椒精油以单萜与倍半萜为主要活性物质,目前在山鸡椒萜类合成通路上已陆续有关键酶基因被克隆鉴定,但其中具有竞争性关键调控作用的异戊烯基转移酶(IPS)尚缺乏了解。本研究基于山鸡椒果实发育过程的转录组数据,鉴定了来自LcIPS三个基因家族共14条基因,对这14条基因进行了表达模式分析;在此基础上,挑选来自山鸡椒香叶基二磷酸合酶(LcGPPS)家族与山鸡椒香叶基香叶基二磷酸合酶(LcGGPPS)家族的4条基因进行了序列分析与蛋白表达分析;并对来自两个家族的蛋白之间的互作关系进行了预测与验证,研究结果为深入阐明山鸡椒精油合成的调控机制,提高品质和产量提供了理论基础,主要研究结果如下:(1)山鸡椒LcIPS三个家族基因的鉴定与表达模式分析。依据山鸡椒果实发育过程的转录组数据,寻找到分别来自香叶基焦磷酸合酶基因(GPPS)、法尼基焦磷酸合酶基因(FPPS)与香叶基香叶基焦磷酸合酶基因(GGPPS)家族共14条基因,以山鸡椒各组织及不同发育时期的果实RNA反转录的cDNA为模板分析这14条基因的表达模式,经分析,14条LcIPSs都有其独特的表达模式,每个家族都有至少一个基因发挥着主要作用。在LcFPPS家族里,LcFPPS2在果实发育过程中呈现出显著的表达高峰。在LcGPPS家族里,我们发现了同质体与异质体两类蛋白,其中属于异质体小亚基的LcGPPS.SSU1在果实发育过程中出现显著的表达高峰。在LcGGPPS家族中,LcGGPPS1参与较多组织中萜类物质的生成,LcGGPPS2的作用更具特异性,LcGGPPS3-7在花和果实的发育过程中萜类物质的合成都起着作用,LcGGPPS6在果实发育过程中的表达趋势并没有明显的规律,其余6条基因在果实发育过程中都展现出特异的作用时期。(2)分离LcGPPS1、LcGPPS.SSU1、LcGGPPS1与LcGGPPS3基因,进行序列分析和理化性质预测。LcGPPS1的ORF为966bp,编码321个氨基酸;LcGPPS.SSU1的ORF包含897bp,编码298个氨基酸;LcGGPPS1和LcGGPPS3基因的ORF均为1152bp,编码383个氨基酸。ExPASy预测显示LcGPPS1、LcGPPS.SSU1、LcGGPPS1、LcGGPPS3蛋白的相对分子量分别为34.97、32.21、41.39、41.49 kDa,理论等电点pI分别为5.43、5.33、5.86、5.87,蛋白的分子式分别为C1535H2499N421O477S15、C1413H2279N395O431S16、C1828H2960N504O551S18、C1820H2926N510O554S21。对4个蛋白进行亚细胞定位预测显示,LcGPPS1定位于细胞核中,LcGPPS.SSU1可能定位于叶绿体或细胞质中,LcGGPPS1和LcGGPPS3都有最高的可能性位于线粒体内。(3)通过真核与原核外源表达系统进行LcGPPS1、LcGPPS.SSU1、LcGGPPS1与LcGGPPS3重组蛋白表达分析。通过构建真核分泌表达载体转化毕赤酵母,甲醇诱导表达后,发现4个重组蛋白都以单体和二聚体的形式主要存在于菌体中,较少分泌到细胞外。对表达产物进行镍柱纯化时,得到LcGGPPS3在毕赤酵母里表达的重组蛋白。进一步构建原核表达载体pET28a-LcGGPPS1与pET28a-LcGPPS.SSU1,分别转化大肠杆菌BL21(DE3)与Rosetta菌株,IPTG诱导表达后经螯合SFF(Ni)柱纯化得到LcGGPPS1重组蛋白。(4)通过三维结构预测和酵母双杂验证LcGPPS.SSU1分别与LcGGPPS1和LcGGPPS3的互作关系。进化关系分析发现LcGGPPS1与LcGGPPS3都没有聚类到LSU类别,且LcGGPPS3与大部分GGPPS都相距较远。在LcGPPS.SSU1、LcGGPPS1与LcGGPPS3的三维结构中都包含一个GPPS核心保守区域,GPPS核心组成了LcGPPS.SSU1大部分结构。且根据预测LcGGPPS1与LcGGPPS3都可以与LcGPPS.SSU1进行互作。酵母双杂实验表明,LcGGPPS3能够与LcGPPS.SSU1进行蛋白互作,但显示互作较弱;LcGGPPS1则不能与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.

【学位授予单位】：中国林业科学研究院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S793.9

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