紅花黃酮類生物合成途徑查爾酮合酶基因的功能研究
發(fā)布時間：2018-08-03 13:45
【摘要】：黃酮類化合物作為一類重要的次生代謝產(chǎn)物,具有廣泛的藥理活性,決定著許多中藥材的品質(zhì)。因此,研究黃酮類生物合成途徑關(guān)鍵酶基因的功能對于闡明中藥品質(zhì)形成的分子機制具有重要意義。紅花(Carthamus tinctorius L.)是著名的藥用植物,其花活血通經(jīng)、散瘀止痛,具有抗凝血、抗氧化、耐缺氧、防治血栓形成等多種藥理作用,是活血埅瘀類的代表中藥。紅花中含有的查爾酮類成分和多種黃酮醇化合物如羥基紅花黃色素A、槲皮素及其苷類、山柰酚及其苷類等是紅花發(fā)揮活血化瘀活性的重要物質(zhì)基礎(chǔ)。但是,由于植物次生代謝生物合成途徑的極其復(fù)雜性,加之紅花的品質(zhì)成分羥基紅花黃色素A(HSYA)僅在花中特異性積累、紅花本身的組織培養(yǎng)再生率低以及生根困難等問題,一直以來對紅花黃酮生物合成途徑的研究進展相當緩慢,制約著通過植物基因工程手段提高紅花品質(zhì)的步伐。大量研究表明,查爾酮合酶是黃酮類化合物形成的入口酶,調(diào)控著黃酮類成分的合成,因此,深入研究紅花查爾酮合酶基因的功能,對于闡釋紅花品質(zhì)形成的分子機制、進而從基因水平定向調(diào)控紅花的品質(zhì)具有重要意義。課題組前期在構(gòu)建紅花花冠cDNA文庫、進行基因測序和注釋以及定制紅花原位合成基因芯片比較不同開花時間點花冠基因表達差異的基礎(chǔ)上,獲得了紅花中HSYA時序性差異表達的基因,并通過KOBAS分析,獲得了KEGG數(shù)據(jù)庫中黃酮代謝途徑通路上顯著性差異表達的基因,如:肉桂酸4-羥基化酶(C4H)、查爾酮合酶(CHS)、查爾酮異構(gòu)酶(CHI)、黃烷酮2-羥基化酶(F2H)、AT4G33360基因等。并克隆了查爾酮合酶(chalcone synthase,CHS)基因CtCHS533等黃酮代謝途徑的多條關(guān)鍵酶基因。本論文在上述研究工作的基礎(chǔ)上,應(yīng)用cDNA末端快速擴增(rapid amplification of cDNA ends,RACE)技術(shù),繼續(xù)克隆紅花的CHS基因,獲得2條紅花CHS基因全長:CtCHS3720、CtCHS1515。生物信息學(xué)分析顯示CtCHS3720氨基酸序列與菊花(Chryscanthemum boreale)CHS氨基酸序列(AGU91424.1)的同源性最高,覆蓋度達94%,匹配度達到91%。依據(jù)protparam預(yù)測CtCHS3720全長序列編碼的蛋白質(zhì)分子量為43667.25Da,預(yù)測理論等電點為5.72。Ct CHS1515氨基酸序列與毛果楊(Populus trichocarpa)CHS氨基酸序列(XP006375238.1)的同源性最高,覆蓋度達93%,匹配度達到82%。依據(jù)protparam預(yù)測CtCHS1515全長序列編碼的蛋白質(zhì)分子量為43016.36Da,預(yù)測理論等電點為5.67。依據(jù)系統(tǒng)進化樹圖譜分析,CtCHS3720、ctchs533和ctchs1515在聚類分支上距離較遠,推測3個chs基因在氨基酸活性催化功能上存在較大差異性。為了深入研究紅花的3個chs基因,本論文以課題組前期研究發(fā)現(xiàn)并經(jīng)多代純化選擇獲得的紅花不同化學(xué)型(即以hsya為主成分的hsya型和以山柰酚及其苷類為主的kaeg型,云南巍山紅花和新紅花7號)為材料,均勻種植于溫室,以茉莉酸甲酯(meja,100μm)分別刺激開花當天的花冠,采用qpcr法,于刺激后0.1h,3h,6h,12h分析3個基因ctchs3720、ctchs1515、ctchs533的相對表達量。結(jié)果表明,3個基因響應(yīng)meja誘導(dǎo)的模式因品系不同而不同。ctchs3720基因的表達量在誘導(dǎo)云南巍山紅花的0.1h、3h和6h明顯升高,特別在誘導(dǎo)6h,表達量提高了2.3倍,誘導(dǎo)12h,則沒有明顯變化;而meja誘導(dǎo)新紅花7號后,ctchs3720基因的表達量則有所降低,在誘導(dǎo)后的6h和12h降低最明顯。ctchs1515基因的表達量在誘導(dǎo)云南巍山紅花0.1h、3h、6h和12h明顯降低;ctchs1515基因在誘導(dǎo)新紅花7號的不同時間點表達量均較低,對meja的誘導(dǎo)響應(yīng)也不明顯;ctchs533基因在云南巍山紅花的3h、6h和12h,表達量均降低,而meja在新紅花7號誘導(dǎo)的0.1h表達量明顯上升,誘導(dǎo)3h、6h和12h則沒有明顯變化。提示ctchs3720、ctchs1515和ctchs533基因?qū)t花黃酮類生物合成途徑的貢獻度因品系不同而不同,可能參與了紅花不同化學(xué)型品系的形成過程。uplc-qtof/ms檢測meja誘導(dǎo)后不同時間點(0.1h、3h、6h、12h)紅花不同品系花冠12個黃酮類成分d-phenylalanine、hydroxysaffloryellowa、rutin、kaempferol-3-o-β-d-glucoside、kaempferol-3-o-β-rutinoside、kaempferol、apigenin、dihydrokaepferol、quercetin3-β-d-glucoside、scutellarin、carthamin、luteolin的積累情況,從含量變化折線圖可以看出,不同品系12個黃酮成分均受到meja的誘導(dǎo),但成分種類和積累模式存在明顯不同。hydroxysaffloryellowa、carthamin、luteolin、kaempferol-3-o-β-d-glucoside僅在云南巍山紅花中有積累,而且hydroxysaffloryellowa的積累量遠遠高于其他黃酮成分,特別在誘導(dǎo)的3h、6h,d-phenylalanine、kaempferol-3-o-β-rutinoside、carthamin的積累量均高于對照組,kaempferol、kaempferol-3-o-β-d-glucoside、luteolin、quercetin-3-β-d-glucoside的積累則受到不同程度的抑制。dihydrokaepferol、apigenin、scutellarin僅在新紅花7號中有積累,特別在在誘導(dǎo)的0.1h、3h、6h,apigenin、d-phenylalanine、dihydrokaepferol、kaempferol、quercetin-3-β-d-glucoside、的積累被抑制,而kaempferol-3-o-β-rutinoside、rutin則持續(xù)升高,而scutellarin在4個時間點積累均受到抑制,提示紅花不同品系黃酮成分的種類與積累模式的不同可能是導(dǎo)致不同化學(xué)型形成的重要原因。整合分析CtCHS3720、CtCHS1515和CtCHS533基因響應(yīng)MeJA誘導(dǎo)的表達量與黃酮類化合物積累量的關(guān)聯(lián)分析顯示,云南巍山紅花Hydroxysafflor yellow A、D-Phenylalanine、Carthamin的積累量與CtCHS3720的表達均成正相關(guān)(r=0.92、0.88、0.76);提示CtCHS3720、CtCHS533可能是云南巍山紅花形成Hydroxysafflor yellow A和Carthamin的關(guān)鍵基因;CtCHS1515在新紅花7號中與Scutellarin的積累有正相關(guān)關(guān)系(r=0.64),可能是新紅花7號形成Scutellarin的關(guān)鍵基因。采用無縫克隆技術(shù)構(gòu)建了CtCHS3720與真核表達載體pMT39的重組質(zhì)粒并轉(zhuǎn)化了LBA4404農(nóng)桿菌。洋蔥表皮的亞細胞定位顯示,CtCHS3720基因在細胞膜和細胞核表達。將構(gòu)建的原核表達重組載體質(zhì)粒轉(zhuǎn)入原核表達宿主菌體BL21(DE3)pLys S中,加入IPTG誘導(dǎo)表達融合蛋白。CtCHS3720和CtCHS1515分別編碼分子量為43.6kD和43kD的蛋白質(zhì)。體外構(gòu)建酶促反應(yīng)體系驗證CtCHS3720所編碼酶可催化1分子的香豆酰COA(p-coumaryl-COA)和3分子的丙二酰COA(malonyl-COA)生成柚皮素,證明CtCHS3720所編碼的酶具有體外催化活性。
[Abstract]:As a class of important secondary metabolites, flavonoids have extensive pharmacological activity and determine the quality of many Chinese medicinal materials. Therefore, it is of great significance to study the function of the key enzyme genes of the flavonoid biosynthesis pathway to elucidate the molecular mechanism of the quality formation of traditional Chinese medicine. Carthamus tinctorius L. is a famous medicine. Plants have a variety of pharmacological effects, such as activating blood circulation, dispersing blood stasis and relieving pain, having anticoagulant, antioxidation, anoxia resistance, preventing thrombosis and so on. It is a representative Chinese medicine for blood stasis and blood stasis. The chalcone components and a variety of flavonol compounds in safflower, such as hydroxysafflower yellow A, quercetin and its glycosides, kaempferol and its glycosides, are used as safflower flowers. However, due to the complexity of the biosynthetic pathway of plant secondary metabolism, and the specific accumulation of safflower A (HSYA) in the flower, and the low regeneration rate of safflower itself and the difficulty of rooting difficulty, the biosynthesis of safflower flavonoids has always been applied to the biosynthesis of safflower flavonoids. The research progress of the pathway is rather slow and restricts the step of improving the quality of safflower through plant genetic engineering. A large number of studies have shown that chalcone synthase is an entry enzyme of flavonoids and regulates the synthesis of flavonoids. Therefore, the function of chalcone synthase gene in safflower is deeply studied and the quality of safflower is explained. The molecular mechanism is of great significance to regulate the quality of safflower flower from the gene level. In the earlier period, the sequence of HSYA in safflower was obtained by constructing the cDNA Library of the safflower corolla, sequencing and annotating the gene, and comparing the differences in the gene expression of the corolla at different flowering time points. By KOBAS analysis, the genes expressed in the pathway pathway of flavonoid metabolism in the KEGG database, such as cinnamic acid 4- hydroxylase (C4H), chalcone synthase (CHS), chalcone isomerase (CHI), xanone 2- hydroxylase (F2H) and AT4G33360 gene, were obtained, and the chalcone synthase (chalcone synthase, CHS) base was cloned. Based on the above research work, we continue to clone the CHS gene of safflower by rapid amplification of cDNA terminal (rapid amplification of cDNA ends, RACE) on the basis of the above research work, and obtain 2 full length of safflower CHS gene: CtCHS3720, CtCHS1515. bioinformatics analysis shows the amino group. The homology of acid sequence and chrysanthemum (Chryscanthemum Boreale) CHS amino acid sequence (AGU91424.1) has the highest homology, the coverage reaches 94%, the matching degree is 91%. based on protparam prediction that the protein molecular weight of CtCHS3720 full length sequence is 43667.25Da, and the prediction theory is 5.72.Ct CHS1515 amino acid sequence and pilocarpine (Populus trichocarpa). The amino acid sequence (XP006375238.1) has the highest homology, the coverage is 93%, the matching degree is 82%. according to protparam, the protein molecular weight of the CtCHS1515 full length sequence is 43016.36Da. The prediction theory is 5.67. based on the phylogenetic tree analysis. CtCHS3720, ctchs533 and ctchs1515 are far away from the cluster branch, and 3 In order to study the 3 CHS genes of safflower, the different chemical types of safflower (Hsya type with Hsya as the main component and kaeg with kaempferol and its glycosides, Weishan Safflower in Yunnan, Yunnan, and safflower in Yunnan, Weishan and safflower) were found in this paper. New Safflower 7) was planted in the greenhouse, and the Corolla was stimulated with methyl jasmonate (MeJA, 100 mu m) respectively. The relative expression of 3 genes ctchs3720, ctchs1515, ctchs533 was analyzed by qPCR method after stimulation with 0.1h, 3h, 6h, 12h. The results showed that the 3 genes responded to MeJA induced patterns and different.Ctchs3720 bases. The expression of 0.1h, 3H and 6h in the inducement of Weishan Safflower in Yunnan was significantly increased, especially in the induction of 6h, the expression amount increased by 2.3 times, and the induction of 12h was not obviously changed. While MeJA induced the New Safflower 7, the expression of ctchs3720 gene decreased, and the most obvious expression of.Ctchs1515 gene in the induced 6h and 12h was induced in the induction of Yunnan. The 0.1h, 3h, 6h and 12h decreased significantly in Weishan safflower, and the expression of ctchs1515 gene in the induction of New Safflower 7 at different time points was lower and the induction response to MeJA was not obvious. The ctchs533 gene was reduced in 3h, 6h and 12h in Weishan Safflower in Yunnan, while MeJA in the New Safflower 7 The contribution of ctchs3720, ctchs1515 and ctchs533 genes to the biosynthesis pathway of safflower flavonoids is different, which may be involved in the formation process of different chemical strains of safflower,.Uplc-qtof/ms detection of 12 flavonoids in different time points (0.1h, 3h, 6h, 12h) of safflower (0.1h, 3h, 6h, 12h). -phenylalanine, hydroxysaffloryellowa, rutin, kaempferol-3-o- beta -d-glucoside, kaempferol-3-o- beta -rutinoside, kaempferol, apigenin, dihydrokaepferol, quercetin3- beta -d-glucoside. But the species and accumulation patterns are obviously different.Hydroxysaffloryellowa, carthamin, luteolin, kaempferol-3-o- beta -d-glucoside are accumulated only in the Weishan Safflower in Yunnan, and the accumulation of hydroxysaffloryellowa is much higher than that of other flavonoids, especially in the induced 3h, 6h, D-phenylalanine, kaempferol-3-o- beta -rutinoside, car. The accumulation of thamin was higher than that of the control group, while the accumulation of kaempferol, kaempferol-3-o- beta -d-glucoside, luteolin, quercetin-3- beta -d-glucoside was inhibited to varying degrees of.Dihydrokaepferol, apigenin, and scutellarin only accumulated in the New Safflower 7, especially in the induced 0.1h. The accumulation of mpferol, quercetin-3- beta -d-glucoside was inhibited, while kaempferol-3-o- beta -rutinoside and rutin continued to rise, while scutellarin accumulation at 4 time points was inhibited. It suggested that the different types of Flavonoids from different strains of safflower and accumulation patterns may be important reasons for the formation of different chemical forms. Integrated analysis of CtCHS37 20, the correlation between CtCHS1515 and CtCHS533 genes in response to MeJA induced expression and the accumulation of flavonoids showed that the accumulation of Hydroxysafflor yellow A, D-Phenylalanine, Carthamin in Weishan, Yunnan, was positively correlated with the expression of CtCHS3720 (r=0.92,0.88,0.76), suggesting that CtCHS533 may be the red flower shape of Weishan in Yunnan. The key gene of Hydroxysafflor yellow A and Carthamin; CtCHS1515 has a positive correlation with the accumulation of Scutellarin in the New Safflower 7 (r=0.64). It may be the key gene for the formation of Scutellarin in the New Safflower 7. The recombinant plasmid of CtCHS3720 and eukaryotic expression carrier pMT39 was constructed by seamless cloning technology and transformed into LBA4404 Agrobacterium. The subcellular localization of the onion epidermis shows that the CtCHS3720 gene is expressed in the cell membrane and the nucleus. The recombinant plasmid of the prokaryotic expression vector is transferred into the prokaryotic expression host strain BL21 (DE3) pLys S, and IPTG induced expression fusion protein.CtCHS3720 and CtCHS1515 encodes a protein of 43.6kD and 43kD respectively. The reaction system verified that the enzyme encoded by CtCHS3720 can catalyze the 1 molecule coumaryl COA (p-coumaryl-COA) and the 3 molecule COA (malonyl-COA) to produce naringin. It is proved that the enzyme encoded by CtCHS3720 has the catalytic activity in vitro.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;S567.219
本文編號：2161929
[Abstract]:As a class of important secondary metabolites, flavonoids have extensive pharmacological activity and determine the quality of many Chinese medicinal materials. Therefore, it is of great significance to study the function of the key enzyme genes of the flavonoid biosynthesis pathway to elucidate the molecular mechanism of the quality formation of traditional Chinese medicine. Carthamus tinctorius L. is a famous medicine. Plants have a variety of pharmacological effects, such as activating blood circulation, dispersing blood stasis and relieving pain, having anticoagulant, antioxidation, anoxia resistance, preventing thrombosis and so on. It is a representative Chinese medicine for blood stasis and blood stasis. The chalcone components and a variety of flavonol compounds in safflower, such as hydroxysafflower yellow A, quercetin and its glycosides, kaempferol and its glycosides, are used as safflower flowers. However, due to the complexity of the biosynthetic pathway of plant secondary metabolism, and the specific accumulation of safflower A (HSYA) in the flower, and the low regeneration rate of safflower itself and the difficulty of rooting difficulty, the biosynthesis of safflower flavonoids has always been applied to the biosynthesis of safflower flavonoids. The research progress of the pathway is rather slow and restricts the step of improving the quality of safflower through plant genetic engineering. A large number of studies have shown that chalcone synthase is an entry enzyme of flavonoids and regulates the synthesis of flavonoids. Therefore, the function of chalcone synthase gene in safflower is deeply studied and the quality of safflower is explained. The molecular mechanism is of great significance to regulate the quality of safflower flower from the gene level. In the earlier period, the sequence of HSYA in safflower was obtained by constructing the cDNA Library of the safflower corolla, sequencing and annotating the gene, and comparing the differences in the gene expression of the corolla at different flowering time points. By KOBAS analysis, the genes expressed in the pathway pathway of flavonoid metabolism in the KEGG database, such as cinnamic acid 4- hydroxylase (C4H), chalcone synthase (CHS), chalcone isomerase (CHI), xanone 2- hydroxylase (F2H) and AT4G33360 gene, were obtained, and the chalcone synthase (chalcone synthase, CHS) base was cloned. Based on the above research work, we continue to clone the CHS gene of safflower by rapid amplification of cDNA terminal (rapid amplification of cDNA ends, RACE) on the basis of the above research work, and obtain 2 full length of safflower CHS gene: CtCHS3720, CtCHS1515. bioinformatics analysis shows the amino group. The homology of acid sequence and chrysanthemum (Chryscanthemum Boreale) CHS amino acid sequence (AGU91424.1) has the highest homology, the coverage reaches 94%, the matching degree is 91%. based on protparam prediction that the protein molecular weight of CtCHS3720 full length sequence is 43667.25Da, and the prediction theory is 5.72.Ct CHS1515 amino acid sequence and pilocarpine (Populus trichocarpa). The amino acid sequence (XP006375238.1) has the highest homology, the coverage is 93%, the matching degree is 82%. according to protparam, the protein molecular weight of the CtCHS1515 full length sequence is 43016.36Da. The prediction theory is 5.67. based on the phylogenetic tree analysis. CtCHS3720, ctchs533 and ctchs1515 are far away from the cluster branch, and 3 In order to study the 3 CHS genes of safflower, the different chemical types of safflower (Hsya type with Hsya as the main component and kaeg with kaempferol and its glycosides, Weishan Safflower in Yunnan, Yunnan, and safflower in Yunnan, Weishan and safflower) were found in this paper. New Safflower 7) was planted in the greenhouse, and the Corolla was stimulated with methyl jasmonate (MeJA, 100 mu m) respectively. The relative expression of 3 genes ctchs3720, ctchs1515, ctchs533 was analyzed by qPCR method after stimulation with 0.1h, 3h, 6h, 12h. The results showed that the 3 genes responded to MeJA induced patterns and different.Ctchs3720 bases. The expression of 0.1h, 3H and 6h in the inducement of Weishan Safflower in Yunnan was significantly increased, especially in the induction of 6h, the expression amount increased by 2.3 times, and the induction of 12h was not obviously changed. While MeJA induced the New Safflower 7, the expression of ctchs3720 gene decreased, and the most obvious expression of.Ctchs1515 gene in the induced 6h and 12h was induced in the induction of Yunnan. The 0.1h, 3h, 6h and 12h decreased significantly in Weishan safflower, and the expression of ctchs1515 gene in the induction of New Safflower 7 at different time points was lower and the induction response to MeJA was not obvious. The ctchs533 gene was reduced in 3h, 6h and 12h in Weishan Safflower in Yunnan, while MeJA in the New Safflower 7 The contribution of ctchs3720, ctchs1515 and ctchs533 genes to the biosynthesis pathway of safflower flavonoids is different, which may be involved in the formation process of different chemical strains of safflower,.Uplc-qtof/ms detection of 12 flavonoids in different time points (0.1h, 3h, 6h, 12h) of safflower (0.1h, 3h, 6h, 12h). -phenylalanine, hydroxysaffloryellowa, rutin, kaempferol-3-o- beta -d-glucoside, kaempferol-3-o- beta -rutinoside, kaempferol, apigenin, dihydrokaepferol, quercetin3- beta -d-glucoside. But the species and accumulation patterns are obviously different.Hydroxysaffloryellowa, carthamin, luteolin, kaempferol-3-o- beta -d-glucoside are accumulated only in the Weishan Safflower in Yunnan, and the accumulation of hydroxysaffloryellowa is much higher than that of other flavonoids, especially in the induced 3h, 6h, D-phenylalanine, kaempferol-3-o- beta -rutinoside, car. The accumulation of thamin was higher than that of the control group, while the accumulation of kaempferol, kaempferol-3-o- beta -d-glucoside, luteolin, quercetin-3- beta -d-glucoside was inhibited to varying degrees of.Dihydrokaepferol, apigenin, and scutellarin only accumulated in the New Safflower 7, especially in the induced 0.1h. The accumulation of mpferol, quercetin-3- beta -d-glucoside was inhibited, while kaempferol-3-o- beta -rutinoside and rutin continued to rise, while scutellarin accumulation at 4 time points was inhibited. It suggested that the different types of Flavonoids from different strains of safflower and accumulation patterns may be important reasons for the formation of different chemical forms. Integrated analysis of CtCHS37 20, the correlation between CtCHS1515 and CtCHS533 genes in response to MeJA induced expression and the accumulation of flavonoids showed that the accumulation of Hydroxysafflor yellow A, D-Phenylalanine, Carthamin in Weishan, Yunnan, was positively correlated with the expression of CtCHS3720 (r=0.92,0.88,0.76), suggesting that CtCHS533 may be the red flower shape of Weishan in Yunnan. The key gene of Hydroxysafflor yellow A and Carthamin; CtCHS1515 has a positive correlation with the accumulation of Scutellarin in the New Safflower 7 (r=0.64). It may be the key gene for the formation of Scutellarin in the New Safflower 7. The recombinant plasmid of CtCHS3720 and eukaryotic expression carrier pMT39 was constructed by seamless cloning technology and transformed into LBA4404 Agrobacterium. The subcellular localization of the onion epidermis shows that the CtCHS3720 gene is expressed in the cell membrane and the nucleus. The recombinant plasmid of the prokaryotic expression vector is transferred into the prokaryotic expression host strain BL21 (DE3) pLys S, and IPTG induced expression fusion protein.CtCHS3720 and CtCHS1515 encodes a protein of 43.6kD and 43kD respectively. The reaction system verified that the enzyme encoded by CtCHS3720 can catalyze the 1 molecule coumaryl COA (p-coumaryl-COA) and the 3 molecule COA (malonyl-COA) to produce naringin. It is proved that the enzyme encoded by CtCHS3720 has the catalytic activity in vitro.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;S567.219
【參考文獻】
相關(guān)期刊論文 前1條
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，本文編號：2161929
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