苔類植物多酚及萜類化合物生物合成途徑相關(guān)基因的克隆與功能研究
發(fā)布時間：2018-08-25 17:49
【摘要】：苔蘚植物在植物界中介于藻類與蕨類植物之間,是僅次于種子植物的第二大植物種群,在自然界中分布廣泛。苔類植物富含大量的次生代謝產(chǎn)物,主要是萜類以及多酚類化合物,化學(xué)結(jié)構(gòu)復(fù)雜多樣,具有抗腫瘤、抗真菌、抗氧化以及昆蟲拒食等廣泛的生物活性。但是由于苔蘚植物本身個體較小以及生長環(huán)境復(fù)雜多樣,對其進行采集困難重重,對其中分離得到的活性較好的化合物,在短期內(nèi)難以進行大量的富集,用于進一步的研究,這極大地限制了新藥研發(fā)的步伐。隨著生物技術(shù)的不斷進步,代謝工程以及合成生物學(xué)的應(yīng)用,為解決該問題提供了一個途徑。苔類植物中富含大量比較獨特的的雙聯(lián)芐類化合物以及特殊結(jié)構(gòu)的萜類化合物,然而對于其生物合成反應(yīng)催化的關(guān)鍵酶研究還比較少,因此,探尋苔類植物中催化產(chǎn)生次級代謝產(chǎn)物的關(guān)鍵酶,闡明一些重要次級代謝產(chǎn)物的生物合成途徑,通過組合生物合成的方法得到目標化合物或者通過基因改造的手段來提高目標化合物的產(chǎn)量,已經(jīng)成為解決活性化合物來源的一個重要途徑。本文通過從兒種苔類植物轉(zhuǎn)錄組測序以及cDNA文庫中,篩選出了兒個與萜類以及多酚類生物合成相關(guān)的基因進行研究。包括二萜合酶(diTPS),從萜類含量豐富的蛇苔中克隆獲得兩個二萜合酶,并對其功能進行了初步的研究;酚酸脫羧酶(PAD),從小蛇苔中克隆得到了一個PAD基因,對其體外功能以及基因定位進行了研究;對羥基肉桂酰輔酶A連接酶(4CL),從鈍鱗紫背苔以及粗裂地錢中總共克隆得到了 7個4CL基因,對其體外功能以及非生物脅迫下表達量隨時間的變化關(guān)系進行了研究,同時挑選了活性較好的兩個基因轉(zhuǎn)入擬南芥中,對轉(zhuǎn)基因植株中黃酮以及木質(zhì)素的含量進行了分析,對其在植物體內(nèi)的功能進行了研究。主要的研究內(nèi)容以及結(jié)果如下:1.蛇苔中二萜合酶基因的克隆和功能研究本文從蛇苔(Conocephalum conicum)的轉(zhuǎn)錄組數(shù)據(jù)庫中篩選到兩個二萜合酶,通過NCBI數(shù)據(jù)庫進行分析發(fā)現(xiàn),他們分別注釋為焦磷酸古巴酯合酶(CPS)以及對映貝殼杉烯合酶(KS),分別命名為CcCPS和CcSS。CcCPS與擬南芥的AtCPS以及小立碗蘚的PpCPS/KS具有較高同源性,而且都能夠找到CPSs保守的功能域DXDD,屬于(Class Ⅱ型二萜合酶。CcSS具有保守的功能域DDXXD,屬于Class Ⅰ型二萜合酶。對其進行進化地位分析,發(fā)現(xiàn)CcCPS和CcSS都與已報道的其他三種苔蘚植物的二萜合酶歸為一簇。將它們N端的轉(zhuǎn)運肽去掉,并構(gòu)建到原核表達載體上,在大腸桿菌中進行表達,得到重組蛋白。以焦磷酸香葉基香葉酯(geranylgeranyl diphosphate,GGPP)為底物,對目的蛋白進行體外酶活測定,結(jié)果表明,CcCPS能夠催化GGPP生成對映-柯杷酰焦磷酸(ent-CPP),CcSS則催化ent-CPP生成對映海松烷型二萜(ent-sandaracopimaradiene)以及對映貝殼杉烷型二萜(ent-kaurene)兩種不同結(jié)構(gòu)類型的二萜化合物,其中以對映海松烷型二萜化合物為主要的酶活產(chǎn)物。2.苔類植物中對羥基肉桂酰輔酶A連接酶(4CL)基因的克隆和功能研究4CL是苯丙烷代謝途徑上的第三個酶,也是調(diào)控苯丙烷代謝途徑方向的關(guān)鍵酶。本文從苔類植物cDNA文庫以及轉(zhuǎn)錄組測序中,發(fā)現(xiàn)幾個注釋為對羥基肉桂酰輔酶A連接酶(4CL)的序列,將其進行克隆得到全長。其中鈍鱗紫背苔(Plagiochasma appendiculatum)中有 3 個,分別命名為Pa4CL1-3 粗裂地錢(Marchantiapaleacea)中克隆得到 4 個基因,命名為Mp4CL1-4。將這些基因分別構(gòu)建到大腸桿菌中進行蛋白表達,得到重組蛋白,并進行體外酶活反應(yīng),測定其功能。研究發(fā)現(xiàn)Pa4CL1、Pa4CL2、Mp4CL1以及Mp4CL2都能夠以對羥基肉桂酸為最佳底物,生成對羥基肉桂酰輔酶A,同時能夠催化二氫對羥基肉桂酸生成雙聯(lián)芐類化合物的前體dihydro-p-coumaryo-CoA,但是其催化活性以及底物的選擇性有所差異,Pa4CL3以及Mp4CL3、Mp4CL4則沒有檢測到催化活性。同時為了探究關(guān)鍵氨基酸對底物選擇性以及活性的影響,我們對Pa4CL1進行了點突變,發(fā)現(xiàn)氨基酸Met-247以及Ala-251參與底物的催化,與蛋白對底物的結(jié)合能力有關(guān),跟底物的選擇性沒有必然關(guān)系。將Pa4CL2以及Mp4CL1轉(zhuǎn)入模式植物擬南芥中,對轉(zhuǎn)基因植株中木質(zhì)素以及黃酮類化合物的含量進行了測定,發(fā)現(xiàn)Pa4CL2以及MP4CL1轉(zhuǎn)基因擬南芥植株中木質(zhì)素的含量明顯增加,黃酮類化合物的含量則有所減少。對Pa4CL1以及Mp4CL1用茉莉酸甲酯、水楊酸以及脫落酸等進行處理,發(fā)現(xiàn)它們的表達量受到非生物脅迫因子的誘導(dǎo)。Pa4CL1、Pa4CL2、Mp4CL1以及Mp4CL2定位在細胞質(zhì)以及細胞核中。3.小蛇苔中酚酸脫羧酶(PAD)基因的克隆和功能鑒定本文從小蛇苔(Conocephalum japonicum)的轉(zhuǎn)錄組測序結(jié)果中,發(fā)現(xiàn)了一個與微生物中酚酸脫羧酶同源性較高的序列,將其克隆并命名為CjPAD,這是首次從植物當中克隆得到的酚酸脫羧酶基因。該基因序列長度大約是微生物酚酸脫羧酶基因序列的二倍,同時具有4個酚酸脫羧酶保守的催化位點:Tyr-60,Tyr-62,Arg-90以及Glu-114。為了進一步研究其功能,我們將該基因分別在N端和C端進行了截短,并與原長序列一起分別構(gòu)建到原核表達載體上,在大腸桿菌中進行蛋白表達得到了相應(yīng)的重組蛋白。以酚酸類化合物為底物對其進行了體外酶活功能研究,發(fā)現(xiàn)CjPAD能夠催化對羥基肉桂酸、阿魏酸、咖啡酸以及芥子酸等,生成相應(yīng)的乙烯基化合物,截短后的蛋白則失去了活性,不能夠催化這些酚酸化合物。同時構(gòu)建了全長GFP-CjPAD和N端截短的GFP-CjPAD-Tr定位表達載體,將其分別在煙草葉片中進行定位觀察,發(fā)現(xiàn)都同時定位在細胞質(zhì)和細胞核中,說明CjPAD的N端不具備轉(zhuǎn)運信號肽,對于基因定位不具備關(guān)鍵作用。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q943.2
本文編號：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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q943.2
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