红花黄酮类生物合成途径查尔酮合酶基因的功能研究
发布时间：2018-08-03 13:45
【摘要】：黄酮类化合物作为一类重要的次生代谢产物,具有广泛的药理活性,决定着许多中药材的品质。因此,研究黄酮类生物合成途径关键酶基因的功能对于阐明中药品质形成的分子机制具有重要意义。红花(Carthamus tinctorius L.)是著名的药用植物,其花活血通经、散瘀止痛,具有抗凝血、抗氧化、耐缺氧、防治血栓形成等多种药理作用,是活血埅瘀类的代表中药。红花中含有的查尔酮类成分和多种黄酮醇化合物如羟基红花黄色素A、槲皮素及其苷类、山柰酚及其苷类等是红花发挥活血化瘀活性的重要物质基础。但是,由于植物次生代谢生物合成途径的极其复杂性,加之红花的品质成分羟基红花黄色素A(HSYA)仅在花中特异性积累、红花本身的组织培养再生率低以及生根困难等问题,一直以来对红花黄酮生物合成途径的研究进展相当缓慢,制约着通过植物基因工程手段提高红花品质的步伐。大量研究表明,查尔酮合酶是黄酮类化合物形成的入口酶,调控着黄酮类成分的合成,因此,深入研究红花查尔酮合酶基因的功能,对于阐释红花品质形成的分子机制、进而从基因水平定向调控红花的品质具有重要意义。课题组前期在构建红花花冠cDNA文库、进行基因测序和注释以及定制红花原位合成基因芯片比较不同开花时间点花冠基因表达差异的基础上,获得了红花中HSYA时序性差异表达的基因,并通过KOBAS分析,获得了KEGG数据库中黄酮代谢途径通路上显著性差异表达的基因,如:肉桂酸4-羟基化酶(C4H)、查尔酮合酶(CHS)、查尔酮异构酶(CHI)、黄烷酮2-羟基化酶(F2H)、AT4G33360基因等。并克隆了查尔酮合酶(chalcone synthase,CHS)基因CtCHS533等黄酮代谢途径的多条关键酶基因。本论文在上述研究工作的基础上,应用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术,继续克隆红花的CHS基因,获得2条红花CHS基因全长:CtCHS3720、CtCHS1515。生物信息学分析显示CtCHS3720氨基酸序列与菊花(Chryscanthemum boreale)CHS氨基酸序列(AGU91424.1)的同源性最高,覆盖度达94%,匹配度达到91%。依据protparam预测CtCHS3720全长序列编码的蛋白质分子量为43667.25Da,预测理论等电点为5.72。Ct CHS1515氨基酸序列与毛果杨(Populus trichocarpa)CHS氨基酸序列(XP006375238.1)的同源性最高,覆盖度达93%,匹配度达到82%。依据protparam预测CtCHS1515全长序列编码的蛋白质分子量为43016.36Da,预测理论等电点为5.67。依据系统进化树图谱分析,CtCHS3720、ctchs533和ctchs1515在聚类分支上距离较远,推测3个chs基因在氨基酸活性催化功能上存在较大差异性。为了深入研究红花的3个chs基因,本论文以课题组前期研究发现并经多代纯化选择获得的红花不同化学型(即以hsya为主成分的hsya型和以山柰酚及其苷类为主的kaeg型,云南巍山红花和新红花7号)为材料,均匀种植于温室,以茉莉酸甲酯(meja,100μm)分别刺激开花当天的花冠,采用qpcr法,于刺激后0.1h,3h,6h,12h分析3个基因ctchs3720、ctchs1515、ctchs533的相对表达量。结果表明,3个基因响应meja诱导的模式因品系不同而不同。ctchs3720基因的表达量在诱导云南巍山红花的0.1h、3h和6h明显升高,特别在诱导6h,表达量提高了2.3倍,诱导12h,则没有明显变化;而meja诱导新红花7号后,ctchs3720基因的表达量则有所降低,在诱导后的6h和12h降低最明显。ctchs1515基因的表达量在诱导云南巍山红花0.1h、3h、6h和12h明显降低;ctchs1515基因在诱导新红花7号的不同时间点表达量均较低,对meja的诱导响应也不明显;ctchs533基因在云南巍山红花的3h、6h和12h,表达量均降低,而meja在新红花7号诱导的0.1h表达量明显上升,诱导3h、6h和12h则没有明显变化。提示ctchs3720、ctchs1515和ctchs533基因对红花黄酮类生物合成途径的贡献度因品系不同而不同,可能参与了红花不同化学型品系的形成过程。uplc-qtof/ms检测meja诱导后不同时间点(0.1h、3h、6h、12h)红花不同品系花冠12个黄酮类成分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的积累情况,从含量变化折线图可以看出,不同品系12个黄酮成分均受到meja的诱导,但成分种类和积累模式存在明显不同。hydroxysaffloryellowa、carthamin、luteolin、kaempferol-3-o-β-d-glucoside仅在云南巍山红花中有积累,而且hydroxysaffloryellowa的积累量远远高于其他黄酮成分,特别在诱导的3h、6h,d-phenylalanine、kaempferol-3-o-β-rutinoside、carthamin的积累量均高于对照组,kaempferol、kaempferol-3-o-β-d-glucoside、luteolin、quercetin-3-β-d-glucoside的积累则受到不同程度的抑制。dihydrokaepferol、apigenin、scutellarin仅在新红花7号中有积累,特别在在诱导的0.1h、3h、6h,apigenin、d-phenylalanine、dihydrokaepferol、kaempferol、quercetin-3-β-d-glucoside、的积累被抑制,而kaempferol-3-o-β-rutinoside、rutin则持续升高,而scutellarin在4个时间点积累均受到抑制,提示红花不同品系黄酮成分的种类与积累模式的不同可能是导致不同化学型形成的重要原因。整合分析CtCHS3720、CtCHS1515和CtCHS533基因响应MeJA诱导的表达量与黄酮类化合物积累量的关联分析显示,云南巍山红花Hydroxysafflor yellow A、D-Phenylalanine、Carthamin的积累量与CtCHS3720的表达均成正相关(r=0.92、0.88、0.76);提示CtCHS3720、CtCHS533可能是云南巍山红花形成Hydroxysafflor yellow A和Carthamin的关键基因;CtCHS1515在新红花7号中与Scutellarin的积累有正相关关系(r=0.64),可能是新红花7号形成Scutellarin的关键基因。采用无缝克隆技术构建了CtCHS3720与真核表达载体pMT39的重组质粒并转化了LBA4404农杆菌。洋葱表皮的亚细胞定位显示,CtCHS3720基因在细胞膜和细胞核表达。将构建的原核表达重组载体质粒转入原核表达宿主菌体BL21(DE3)pLys S中,加入IPTG诱导表达融合蛋白。CtCHS3720和CtCHS1515分别编码分子量为43.6kD和43kD的蛋白质。体外构建酶促反应体系验证CtCHS3720所编码酶可催化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,证明CtCHS3720所编码的酶具有体外催化活性。
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S567.219
本文编号：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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S567.219
【参考文献】
相关期刊论文 前1条
1 廖海;周嘉裕;张超;贺葵邦;;何首乌查尔酮合成酶基因的克隆及序列分析[J];安徽农业科学;2009年15期
，本文编号：2161929
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