苔类植物多酚及萜类化合物生物合成途径相关基因的克隆与功能研究
发布时间：2018-08-25 17:49
【摘要】：苔藓植物在植物界中介于藻类与蕨类植物之间,是仅次于种子植物的第二大植物种群,在自然界中分布广泛。苔类植物富含大量的次生代谢产物,主要是萜类以及多酚类化合物,化学结构复杂多样,具有抗肿瘤、抗真菌、抗氧化以及昆虫拒食等广泛的生物活性。但是由于苔藓植物本身个体较小以及生长环境复杂多样,对其进行采集困难重重,对其中分离得到的活性较好的化合物,在短期内难以进行大量的富集,用于进一步的研究,这极大地限制了新药研发的步伐。随着生物技术的不断进步,代谢工程以及合成生物学的应用,为解决该问题提供了一个途径。苔类植物中富含大量比较独特的的双联苄类化合物以及特殊结构的萜类化合物,然而对于其生物合成反应催化的关键酶研究还比较少,因此,探寻苔类植物中催化产生次级代谢产物的关键酶,阐明一些重要次级代谢产物的生物合成途径,通过组合生物合成的方法得到目标化合物或者通过基因改造的手段来提高目标化合物的产量,已经成为解决活性化合物来源的一个重要途径。本文通过从儿种苔类植物转录组测序以及cDNA文库中,筛选出了儿个与萜类以及多酚类生物合成相关的基因进行研究。包括二萜合酶(diTPS),从萜类含量丰富的蛇苔中克隆获得两个二萜合酶,并对其功能进行了初步的研究;酚酸脱羧酶(PAD),从小蛇苔中克隆得到了一个PAD基因,对其体外功能以及基因定位进行了研究;对羟基肉桂酰辅酶A连接酶(4CL),从钝鳞紫背苔以及粗裂地钱中总共克隆得到了 7个4CL基因,对其体外功能以及非生物胁迫下表达量随时间的变化关系进行了研究,同时挑选了活性较好的两个基因转入拟南芥中,对转基因植株中黄酮以及木质素的含量进行了分析,对其在植物体内的功能进行了研究。主要的研究内容以及结果如下:1.蛇苔中二萜合酶基因的克隆和功能研究本文从蛇苔(Conocephalum conicum)的转录组数据库中筛选到两个二萜合酶,通过NCBI数据库进行分析发现,他们分别注释为焦磷酸古巴酯合酶(CPS)以及对映贝壳杉烯合酶(KS),分别命名为CcCPS和CcSS。CcCPS与拟南芥的AtCPS以及小立碗藓的PpCPS/KS具有较高同源性,而且都能够找到CPSs保守的功能域DXDD,属于(Class Ⅱ型二萜合酶。CcSS具有保守的功能域DDXXD,属于Class Ⅰ型二萜合酶。对其进行进化地位分析,发现CcCPS和CcSS都与已报道的其他三种苔藓植物的二萜合酶归为一簇。将它们N端的转运肽去掉,并构建到原核表达载体上,在大肠杆菌中进行表达,得到重组蛋白。以焦磷酸香叶基香叶酯(geranylgeranyl diphosphate,GGPP)为底物,对目的蛋白进行体外酶活测定,结果表明,CcCPS能够催化GGPP生成对映-柯杷酰焦磷酸(ent-CPP),CcSS则催化ent-CPP生成对映海松烷型二萜(ent-sandaracopimaradiene)以及对映贝壳杉烷型二萜(ent-kaurene)两种不同结构类型的二萜化合物,其中以对映海松烷型二萜化合物为主要的酶活产物。2.苔类植物中对羟基肉桂酰辅酶A连接酶(4CL)基因的克隆和功能研究4CL是苯丙烷代谢途径上的第三个酶,也是调控苯丙烷代谢途径方向的关键酶。本文从苔类植物cDNA文库以及转录组测序中,发现几个注释为对羟基肉桂酰辅酶A连接酶(4CL)的序列,将其进行克隆得到全长。其中钝鳞紫背苔(Plagiochasma appendiculatum)中有 3 个,分别命名为Pa4CL1-3 粗裂地钱(Marchantiapaleacea)中克隆得到 4 个基因,命名为Mp4CL1-4。将这些基因分别构建到大肠杆菌中进行蛋白表达,得到重组蛋白,并进行体外酶活反应,测定其功能。研究发现Pa4CL1、Pa4CL2、Mp4CL1以及Mp4CL2都能够以对羟基肉桂酸为最佳底物,生成对羟基肉桂酰辅酶A,同时能够催化二氢对羟基肉桂酸生成双联苄类化合物的前体dihydro-p-coumaryo-CoA,但是其催化活性以及底物的选择性有所差异,Pa4CL3以及Mp4CL3、Mp4CL4则没有检测到催化活性。同时为了探究关键氨基酸对底物选择性以及活性的影响,我们对Pa4CL1进行了点突变,发现氨基酸Met-247以及Ala-251参与底物的催化,与蛋白对底物的结合能力有关,跟底物的选择性没有必然关系。将Pa4CL2以及Mp4CL1转入模式植物拟南芥中,对转基因植株中木质素以及黄酮类化合物的含量进行了测定,发现Pa4CL2以及MP4CL1转基因拟南芥植株中木质素的含量明显增加,黄酮类化合物的含量则有所减少。对Pa4CL1以及Mp4CL1用茉莉酸甲酯、水杨酸以及脱落酸等进行处理,发现它们的表达量受到非生物胁迫因子的诱导。Pa4CL1、Pa4CL2、Mp4CL1以及Mp4CL2定位在细胞质以及细胞核中。3.小蛇苔中酚酸脱羧酶(PAD)基因的克隆和功能鉴定本文从小蛇苔(Conocephalum japonicum)的转录组测序结果中,发现了一个与微生物中酚酸脱羧酶同源性较高的序列,将其克隆并命名为CjPAD,这是首次从植物当中克隆得到的酚酸脱羧酶基因。该基因序列长度大约是微生物酚酸脱羧酶基因序列的二倍,同时具有4个酚酸脱羧酶保守的催化位点:Tyr-60,Tyr-62,Arg-90以及Glu-114。为了进一步研究其功能,我们将该基因分别在N端和C端进行了截短,并与原长序列一起分别构建到原核表达载体上,在大肠杆菌中进行蛋白表达得到了相应的重组蛋白。以酚酸类化合物为底物对其进行了体外酶活功能研究,发现CjPAD能够催化对羟基肉桂酸、阿魏酸、咖啡酸以及芥子酸等,生成相应的乙烯基化合物,截短后的蛋白则失去了活性,不能够催化这些酚酸化合物。同时构建了全长GFP-CjPAD和N端截短的GFP-CjPAD-Tr定位表达载体,将其分别在烟草叶片中进行定位观察,发现都同时定位在细胞质和细胞核中,说明CjPAD的N端不具备转运信号肽,对于基因定位不具备关键作用。
[Abstract]:Bryophytes are the second largest plant population in the plant kingdom, which lies between algae and ferns. bryophytes are widely distributed in nature. bryophytes are rich in secondary metabolites, mainly terpenoids and polyphenols, with complex chemical structures, antitumor, antifungal, antioxidant and insect resistance. Bryophytes have a wide range of bioactivities, such as food. However, it is difficult to collect these compounds because of their small size and complex growing environment. It is difficult to enrich the compounds with better bioactivities in a short period of time for further research, which greatly limits the pace of new drug development. The advances in biotechnology, metabolic engineering and the application of synthetic biology provide a way to solve this problem. There are abundant bibenzyl compounds and terpenoids with special structures in mosses. However, few studies have been done on the key enzymes involved in biosynthetic reactions. Therefore, the exploration of mosses is necessary. The key enzymes that catalyze the production of secondary metabolites in plant-like organisms, elucidate the biosynthetic pathways of some important secondary metabolites, obtain target compounds by combinatorial biosynthetic methods or increase the yield of target compounds by genetic modification, have become an important way to solve the source of active compounds. In this paper, two diterpenoid synthases (DTS) have been cloned and their functions have been preliminarily studied, including diterpenoid synthase (diTPS) and phenolic decarboxylase (PAD). A PAD gene was cloned from the lichen, and its in vitro function and gene localization were studied. Hydroxycinnamyl CoA ligase (4CL), a total of 7 4CL genes were cloned from the lichen and Dioscorea rugosa, and their in vitro function and expression under abiotic stress with time were studied. At the same time, two genes with better activity were selected and transfected into Arabidopsis. The contents of flavonoids and lignin in transgenic plants were analyzed, and their functions in plants were studied. Two diterpenoid synthases were screened from the transcriptome database of lum conicum and analyzed by NCBI database. They were annotated as Cuba pyrophosphate synthase (CPS) and kaempferene synthase (KS), named CcCPS and CCSS. The conserved domain of CPSs, DXDD, was found to be (Class II diterpenoid synthase. CcSS has a conserved domain of DDXXD, belonging to Class I diterpenoid synthase). Evolutionary status analysis showed that both CcCPS and CCSS were clustered with the other three reported bryophyte diterpenoid synthases. The recombinant protein was expressed in E. coli on the prokaryotic expression vector. The enzyme activity of the target protein was determined in vitro using geranyl diphosphate (GGPP) as substrate. The results showed that CcCPS could catalyze the formation of enantiomeric-coxobyl pyrophosphate (ent-CPP) and CcSS could catalyze the formation of enantiomeric-CPP. Two different types of diterpenoids, ent-sandaracopimaradine and ent-kaurene, were identified. Among them, enantiomeric diterpenoids were the main enzyme active products. 2. Cloning and functional characterization of 4-hydroxycinnamic coenzyme A ligase (4CL) gene from mosses 4CL was phenylpropane. The third enzyme in the metabolic pathway is also the key enzyme that regulates the direction of phenylpropane metabolism. In this paper, several annotated sequences of 4-hydroxycinnylcoenzyme A ligase (4CL) were found in the cDNA library and transcriptome sequencing of bryophytes, and cloned into full length. Among them, 3 were found in Plagiochasma appendiculatum. Four genes, named Mp4CL1-4, were cloned from Pa4CL1-3 Marchantia paleacea. The recombinant proteins were constructed into E. Cinnamic acid is the best substrate to produce p-hydroxycinnamic coenzyme A and dihydro-p-coumaryo-CoA, the precursor of dihydro-p-hydroxycinnamic acid to bibenzyl compounds, but its catalytic activity and substrate selectivity are different. Pa4CL3, Mp4CL3, Mp4CL4 have not detected the catalytic activity. Point mutation of Pa4CL1 showed that amino acid Met-247 and Ala-251 were involved in the catalysis of substrate, which was related to the binding ability of protein to substrate, but not to the selectivity of substrate. The content of lignin and flavonoids in Pa4CL2 and MP4CL1 transgenic Arabidopsis thaliana plants increased significantly, but the content of flavonoids decreased. Pa4CL1 and Mp4CL1 were treated with methyl jasmonate, salicylic acid and abscisic acid, and their expression was found to be under abiotic stress. Induction of factors. Location of Pa4CL1, Pa4CL2, Mp4CL1 and Mp4CL2 in cytoplasm and nucleus. 3. Cloning and functional characterization of phenolic acid decarboxylase (PAD) gene from the snake moss Conocephalum japonicum The gene is about twice the length of the microbial phenolic acid decarboxylase gene and has four conserved catalytic sites for phenolic acid decarboxylase: Tyr-60, Tyr-62, Arg-90 and Glu-114. The recombinant protein was expressed in E. coli and truncated at N-terminal and C-terminal, respectively. Phenolic acid compounds were used as substrates to study the enzymatic activity in vitro. It was found that CjPAD could catalyze p-hydroxycinnamic acid, ferulic acid and caffeic acid. At the same time, the full-length GFP-CjPAD and N-terminal truncated GFP-CjPAD-Tr expression vectors were constructed and localized in the cytoplasm and nucleus of tobacco leaves. It is indicated that the N end of CjPAD does not have the signal peptide, which does not play a key role in gene mapping.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2
本文编号：2203642
[Abstract]:Bryophytes are the second largest plant population in the plant kingdom, which lies between algae and ferns. bryophytes are widely distributed in nature. bryophytes are rich in secondary metabolites, mainly terpenoids and polyphenols, with complex chemical structures, antitumor, antifungal, antioxidant and insect resistance. Bryophytes have a wide range of bioactivities, such as food. However, it is difficult to collect these compounds because of their small size and complex growing environment. It is difficult to enrich the compounds with better bioactivities in a short period of time for further research, which greatly limits the pace of new drug development. The advances in biotechnology, metabolic engineering and the application of synthetic biology provide a way to solve this problem. There are abundant bibenzyl compounds and terpenoids with special structures in mosses. However, few studies have been done on the key enzymes involved in biosynthetic reactions. Therefore, the exploration of mosses is necessary. The key enzymes that catalyze the production of secondary metabolites in plant-like organisms, elucidate the biosynthetic pathways of some important secondary metabolites, obtain target compounds by combinatorial biosynthetic methods or increase the yield of target compounds by genetic modification, have become an important way to solve the source of active compounds. In this paper, two diterpenoid synthases (DTS) have been cloned and their functions have been preliminarily studied, including diterpenoid synthase (diTPS) and phenolic decarboxylase (PAD). A PAD gene was cloned from the lichen, and its in vitro function and gene localization were studied. Hydroxycinnamyl CoA ligase (4CL), a total of 7 4CL genes were cloned from the lichen and Dioscorea rugosa, and their in vitro function and expression under abiotic stress with time were studied. At the same time, two genes with better activity were selected and transfected into Arabidopsis. The contents of flavonoids and lignin in transgenic plants were analyzed, and their functions in plants were studied. Two diterpenoid synthases were screened from the transcriptome database of lum conicum and analyzed by NCBI database. They were annotated as Cuba pyrophosphate synthase (CPS) and kaempferene synthase (KS), named CcCPS and CCSS. The conserved domain of CPSs, DXDD, was found to be (Class II diterpenoid synthase. CcSS has a conserved domain of DDXXD, belonging to Class I diterpenoid synthase). Evolutionary status analysis showed that both CcCPS and CCSS were clustered with the other three reported bryophyte diterpenoid synthases. The recombinant protein was expressed in E. coli on the prokaryotic expression vector. The enzyme activity of the target protein was determined in vitro using geranyl diphosphate (GGPP) as substrate. The results showed that CcCPS could catalyze the formation of enantiomeric-coxobyl pyrophosphate (ent-CPP) and CcSS could catalyze the formation of enantiomeric-CPP. Two different types of diterpenoids, ent-sandaracopimaradine and ent-kaurene, were identified. Among them, enantiomeric diterpenoids were the main enzyme active products. 2. Cloning and functional characterization of 4-hydroxycinnamic coenzyme A ligase (4CL) gene from mosses 4CL was phenylpropane. The third enzyme in the metabolic pathway is also the key enzyme that regulates the direction of phenylpropane metabolism. In this paper, several annotated sequences of 4-hydroxycinnylcoenzyme A ligase (4CL) were found in the cDNA library and transcriptome sequencing of bryophytes, and cloned into full length. Among them, 3 were found in Plagiochasma appendiculatum. Four genes, named Mp4CL1-4, were cloned from Pa4CL1-3 Marchantia paleacea. The recombinant proteins were constructed into E. Cinnamic acid is the best substrate to produce p-hydroxycinnamic coenzyme A and dihydro-p-coumaryo-CoA, the precursor of dihydro-p-hydroxycinnamic acid to bibenzyl compounds, but its catalytic activity and substrate selectivity are different. Pa4CL3, Mp4CL3, Mp4CL4 have not detected the catalytic activity. Point mutation of Pa4CL1 showed that amino acid Met-247 and Ala-251 were involved in the catalysis of substrate, which was related to the binding ability of protein to substrate, but not to the selectivity of substrate. The content of lignin and flavonoids in Pa4CL2 and MP4CL1 transgenic Arabidopsis thaliana plants increased significantly, but the content of flavonoids decreased. Pa4CL1 and Mp4CL1 were treated with methyl jasmonate, salicylic acid and abscisic acid, and their expression was found to be under abiotic stress. Induction of factors. Location of Pa4CL1, Pa4CL2, Mp4CL1 and Mp4CL2 in cytoplasm and nucleus. 3. Cloning and functional characterization of phenolic acid decarboxylase (PAD) gene from the snake moss Conocephalum japonicum The gene is about twice the length of the microbial phenolic acid decarboxylase gene and has four conserved catalytic sites for phenolic acid decarboxylase: Tyr-60, Tyr-62, Arg-90 and Glu-114. The recombinant protein was expressed in E. coli and truncated at N-terminal and C-terminal, respectively. Phenolic acid compounds were used as substrates to study the enzymatic activity in vitro. It was found that CjPAD could catalyze p-hydroxycinnamic acid, ferulic acid and caffeic acid. At the same time, the full-length GFP-CjPAD and N-terminal truncated GFP-CjPAD-Tr expression vectors were constructed and localized in the cytoplasm and nucleus of tobacco leaves. It is indicated that the N end of CjPAD does not have the signal peptide, which does not play a key role in gene mapping.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2
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