金荞麦[Fagopyrum dibotrys （D.Don） Hara]类黄酮生物合成途径重要功能基因的克隆、功能验证及表达特性分析
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摘要
金荞麦[Fagopyrum dibotrys(D.Don)Hara]是原产于我国的一种蓼科(Polygonaceae)荞麦属(Fagopyrum Mill.)多年生草本植物。因其根状茎中活性物质(主要为类黄酮次生代谢产物)所具有的抗肿瘤、抗炎等多种药效学功能,而成为了多种重要的预癌、抗癌药物的主要成分。近年来备受关注的同时也使得金荞麦野生资源遭到了极大的破坏而濒临灭绝,被国家列为二级保护植物,这就使野生金荞麦生物活性物的工厂化生产显得十分迫切。多年来,国内外关于金荞麦资源收集、多样性、生理特性、育种、药效学以及生态环境等方面的研究已有大量报道,但基于与活性成分合成相关的功能基因的分子生物学和基因工程方面的研究,国内外还鲜有报道。
本研究旨在通过对金荞麦类黄酮生物合成途径中重要基因(FdLAR、FdDFR、FdCHS、 FdMYBP1)的克隆和功能的初步分析,阐明金荞麦类黄酮生物合成途径的分子调控机制,为下一步利用基因工程手段,通过调控野生金荞麦次生代谢产物合成途径而实现药用活性物质的工厂化生产提供理论依据,主要结果如下：
(1)金荞麦无色花色素还原酶基因(FdLAR)的克隆及功能初步分析
采用简并PCR结合RACE技术,从金荞麦中分离得到了一个无色花色素还原酶LAR(leucoanthcyanidin4-reductase,LAR)基因FdLAR的全长cDNA序列(GenBank accession: JN793953).序列分析表明,FdLAR基因cDNA全长1158bp,含有1个1176bp的最大开放阅读框,无内含子,编码一个长391个aa的蚩白质。同源比对和系统进化分析表明FdLAR基因的编码蛋白与其他已知物种中的LAR蛋白具有较高的同源性,其中与草莓(Fragaria chiloensis,ADP37950.1)的LAR蛋白相似性最高,为75%,而与大戟科的蓖麻(Ricinus communi)的LAR蛋白遗传距离最近。生物信息学分析结果显示金荞麦FdLAR蛋白具有植物LAR蛋白的保守结构域,在N端具有NADPH结合位点和底物特异结合位点,氨基酸序列中无信号肽,属于亲水性蛋白,可能定位于线粒体基质中,具有酶的典型三级结构特征。
构建了FdLAR和F'dDFR基因的原核表达载体,并在大肠杆菌表达菌株Transetta(DE3)中诱导表达重组蛋白,以FdDFR/FdLAR酶催化反应体系为整体,在NADPH存在的反应体系中,测定两种重组蛋白的酶活,结果显示FdDFR/FdLAR体外重组蛋白能够催化起始底物二氢槲皮素(Dihydroquercetin、DHQ)向下游终产物儿茶素[(+)catechin]转化,具有相应的生物酶活,且具有底物特异性。
构建了FdLAR基因的植物超表达载体,通过农杆菌介导的叶盘法转化烟草,通过分析转基因植株中目的基因表达水平与类黄酮积累之间的关系,对FdLAR基因的功能进行了初步验证。结果表明,通过GUS组织化学染色法、基因组DNA的PCR检测及荧光定量PCR检测,获得了阳性转基因烟草15株。15株转基因烟草叶片中类黄酮含量均有不同程度的提高,其中以T2株最为明显,是野生型的7倍。进一步检测转基因烟草中目的基因的表达发现,FdLAR基因的表达水平与其类黄酮含量成正相关关系,说明克隆得到的FdLAR基因是金荞麦类黄酮合成途径后期的一个关键基因。
(2)金荞麦查尔酮合成酶基因(FdCHS)的克隆与功能初步分析
根据已在NCBI登录的金荞麦查尔酮合成酶(Chalcone synthase, CHS)基因FdCHS的核苷酸序列,克隆其编码框全长并构建原核表达载体和植物超表达载体。在大肠杆菌Transetta(DE3)中诱导表达重组蛋白,以香豆酰-CoA (4-Coumaroyl-CoA)和丙二酰CoA (Malonyl-CoA)作为底物,测定其酶活,结果显示获得的重组蛋白具有正常的生物酶活,能够将其特异底物转化为下游产物。利用农杆菌介导的叶盘法获得转基因烟草,通过分析转基因植株中目的基因的表达水平与类黄酮积累之间的关系,对FdCHS基因的功能进行了初步验证。结果表明,获得的11株转基因烟草中,有10株叶片中类黄酮含量有不同程度的提高,其中以T3株最为明显,是野生型的3.4倍。而T10株类黄酮含量没有显著提高,进一步检测转基因烟草中目的基因的表达发现,FdCHS基因的表达水平与其类黄酮含量总体上成正相关关系,而T10株中没有检测到目的基因的表达,可能是发生了同源基因的共抑制现象。说明FdCHS基因是金荞麦类黄酮合成途径中的一个关键基因,其表达水平直接影响植株中类黄酮类物质的积累。
(3)金荞麦类黄酮的生物合成与金荞麦FdCHS, FdDFR、FdLAR及FdMYBPl基因的表达特征分析
研究了金荞麦在不同发育时期各个组织中类黄酮含量和相应组织中与类黄酮生物合成相关的3个重要结构基因FdCHS、FdDFR、FdLAR和1个转录调控因子MYBPl的表达水平之间的关系。结果表明金荞麦不同组织中的类黄酮含量在不同发育时期,呈现出不同的变化规律,在幼苗的营养生长阶段,不同组织中类黄酮含量总体情况是叶>根>茎,在生殖生长阶段的总体变化规律为根状茎>花>叶>茎。3个结构基因在金荞麦不同生育期的表达变化趋势却基本保持一致,都呈现出“两头高,中间低”的表达模式。根据4个基因在茎段中高表达和根状茎中的低表达,对应其茎段中的低类黄酮含量和根状茎中的高类黄酮含量结果推测：金荞麦类黄酮合成和积累的部位存在差异,茎段可能是类黄酮合成的重要部位,但是类黄酮并不同在茎段中持续的大量积累,而是转移至根状茎积累。进一步对生殖生长期不同组织中与类黄酮合成紧密相关的DFR/LAR酶活进行测定,发现茎段中的酶活均高于其他组织,说明其中发生着大量与类黄酮合成相关的酶催化反应,进一步证明了金荞麦类黄酮合成和积累的组织特异性,其类黄酮合成的主要部位是茎段,并向下运输至根状茎大量积累。
Golden-buckwheat[Fagopyrum dibotrys (D. Don) Hara)]is one of perennial herbs of the genus Polygonaceae in the Fagopyrum Mill family, origins from southwest of China.It is an important resource plant with rich nutrition and great medicinal values, and contains a large number of excellent genes. As an important traditional Chinese medicinal materials, the active extract of rhizome(flavonoids secondary metabolites) of Fagopyrum dibotrys is one of main constituents of many efficient drugs, and has prominent functions in anti-cancer, controlling tumour cell affect and transfering to lung, and anti-inflammatony antibacteria ect. At present, cultivated yield of Fagopyrum dibotrys is restricted and cultivated species are lesser than wild type in nutrition value, efficient constituents and quantity for medicine, which leads to requesting for Fagopyrum dibotrys largely, so that wild resource is destroyed greatly. In view of the conditions, it is ranked into the second degree of national protected plants. Domestically and abroad resource collection, diversity, physiological characteristic, breeding, pharmacodynamics and ecology environment and other aspects are outspread, however, in the light of functional genes research relating to active components of medicine were barely reported at home and abroad.This thesis aims to elucidate biosynthetic pathway of flavonoids through cloning and characterizing key genes,thus provides theory foundation for laying the groundwork for future producing medicinal second metabolic products on a large scale by genetic engineering.The main results in this rasearch are as follows:
1.Cloning and functional analysis of leucoanthcyanidin4-reductase(FdLAR)gene from Fagopyrum dibotrys
Based on the methods of degemerate PCR and RACE technique,the full length cDNA of leucoanthcyanidin4-reductase named as FdLAR was cloned from Fagopyrum dibotrys(GenBank accession:JN793953).The sequence analysis revealed that the full-length cDNA of FdLAR is1158 bp long fragment containing a1176bp open reading frame(ORF) encoding a polypeptide of391amino acids.Blastn and blastp analysis on NCBI showed that the amino acid sequence of the protein encoding by FdLAR gene shares a high similarity with other reported LAR proteins,especially with LAR from strawberry (Fragaria chiloensis,ADP37950.1)(75%identity).The phylogenetic analysis indicated that the genetic diatance between FdLAR and LAR from castor (Ricinus communi) is the nearest.The bioinformatics analysis showed that the FdLAR protein contains highly conserved NADPH and substrate specific binding site at N-terminal.There is no signal peptide in the deduced amino acid sequence and the FdLAR protein which is the hydrophilic protein with the typical tertiary tructure of enzyme is localized in motochondrial matrix predicted by the bioinformatics software.
The prokarytic expression vector of FdLAR and FdDFR genes were constructed and transformed into Escherichia coli Transetta(DE3),also the FdDFR and FdLAR proteins expressed and purified.The FdDFR/FdLAR-NADPH enzymatic reaction system was recongnized as a whole to determine the enzyme activity.The results showed that the recombinant protein of FdDFR/FdLAR could catalyze (+)-DHQ(Dihydroquercetin)as an initial sunbstrate to change into downstream end-product (+)-catechin which demonstrated that both of the recombinant proteins FdDFR and FdLAR had enzymatic activity.
The plant over-expression vector of FdLAR named FdLAR-2301G was consrructed and transformed into tobacco using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens GV3101(including FdLAR-2301G recombinant plasmid).15transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS(β-glucuronidase)histochemical staining and PCR and real-time quantitative PCR(qPCR) analysis.In15lines of transgenic plants, it was variable of total flavonoids contents in different lines and all of them were improved in varied degree. The content of total flavonoids in leaves of the number T2transgenic tobacco plant increased significantly, which is the highest and being up to7folds of the wild type tobacco.In subsequent molecular detection by qPCR,the high expression level of FdLAR in transgenic line number T2proved that improved flavonoids content was positively co-related with the expression of FdLAR.
2. Cloning and functional analysis of chalcone synthase (FdCHS)gene from Fagopyrum dibotrys
Based on the nuclectide sequence submitted on the NCBI(GenBank accession number:GU169470),the FdCHS gene was cloned by reverse transcription PCR(RT-PCR)from the first strand of cDNA of the Fagopyrum dibotrys. The prokarytic expression vector and plant over-expression vector were constructed respectively in order to reveal the preliminary charecterization of the FdCHS gene. the FdCHS protein was expressed in Escherichia coli Transetta(DE3) and then was purified by the His Bind Purification Kit..Took the4-Coumaroyl-CoA and Malonyl-CoA as substrates to determine the enzyme activity,and the enzyme activity determination of the recombinant protein showed that the prokaryotic expressed reconmbinant protein of FdCHS could catalyze4-Coumaroyl-CoA and Malonyl-CoA to change into downstream substances,which demonstrated that the recombinant protein had enzymatic activity. The plant over-expression vector FdCHS-2301G was constructed and transformed into tobacco plants using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens GV3101.11transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS(β-glucuronidase)histochemical staining and PCR and real-time quantitative PCR(qPCR) analysis.In11lines of transgenic plants, it was variable of total flavonoids contents in different lines and10of them were improved in varied degree. The content of total flavonoids in leaves of the number T3transgenic tobacco plant increased significantly, which is the highest and being up to3.4folds of the wild type tobacco.In subsequent molecular detection by qPCR,the high expression level of FdCHS in transgenic line number T3proved that improved flavonoids contene was positively co-related with the expression of FdCHS.However,positive transgenic line number T10did not show FdCHS function,which may be resulted from the gene silence of introduced FdCHS by co-suppression.The q-PCR also did not detect the expression the FdCHS in the number T10tobacco plant.
3The biosynthesis of flavonoids in Fagopyrum dibotrys and expression analysis of FdCHS、 FdDFR、FdLAR and transcription regulator FdMYBPl genes.
The paper analysed the relationship between the flavonoids contents in different tissues of Fagopyrum dibotrys at different stages and the three important structural genes FdCHS、FdDFR、 FdLAR and one transcription regulator FdMYBPl gene.The results showed that the the flavonoids contents display the virous patterns during different development stages.During the vegetatibe multiplication,the overall condition of the flavonoids contents was leaf>root>item and rhizome>flower>leaf>leaf during reproductive growth.The expression levels of the three structural genes in the root or rhizome approximately kept consistency with each other which was high at both ends and low in the middle during the whole development stages.Although the four genes had the high expression in item and low expression in rhizome,the flavonoids contents were high in rhizome and low in item. We sepculated that the flavonoids synthesis and accumulation occurred at different parts of Fagopyrum dibotrys plant.The item may be the main synthesis site of flavonoids synthesised and transport to the rhizome which may be the main accumulation site of flavonoids.In order to verify the assumption furthmore,we detemined the enzyme activity of the key enzyme system DFR/LAR which was closely related to the flavonoids biosynthesis in Fagopyrum dibotrys.The results showed that the enzyme activity of DFR/LAR in item are higher than other tissues during reproductive growth which suggested that there were massive enzyme reaction occurring in item. Summarization above results,the flavonoids synthesis and accumulation had the tissue specificity.
本研究旨在通过对金荞麦类黄酮生物合成途径中重要基因(FdLAR、FdDFR、FdCHS、 FdMYBP1)的克隆和功能的初步分析,阐明金荞麦类黄酮生物合成途径的分子调控机制,为下一步利用基因工程手段,通过调控野生金荞麦次生代谢产物合成途径而实现药用活性物质的工厂化生产提供理论依据,主要结果如下：
(1)金荞麦无色花色素还原酶基因(FdLAR)的克隆及功能初步分析
采用简并PCR结合RACE技术,从金荞麦中分离得到了一个无色花色素还原酶LAR(leucoanthcyanidin4-reductase,LAR)基因FdLAR的全长cDNA序列(GenBank accession: JN793953).序列分析表明,FdLAR基因cDNA全长1158bp,含有1个1176bp的最大开放阅读框,无内含子,编码一个长391个aa的蚩白质。同源比对和系统进化分析表明FdLAR基因的编码蛋白与其他已知物种中的LAR蛋白具有较高的同源性,其中与草莓(Fragaria chiloensis,ADP37950.1)的LAR蛋白相似性最高,为75%,而与大戟科的蓖麻(Ricinus communi)的LAR蛋白遗传距离最近。生物信息学分析结果显示金荞麦FdLAR蛋白具有植物LAR蛋白的保守结构域,在N端具有NADPH结合位点和底物特异结合位点,氨基酸序列中无信号肽,属于亲水性蛋白,可能定位于线粒体基质中,具有酶的典型三级结构特征。
构建了FdLAR和F'dDFR基因的原核表达载体,并在大肠杆菌表达菌株Transetta(DE3)中诱导表达重组蛋白,以FdDFR/FdLAR酶催化反应体系为整体,在NADPH存在的反应体系中,测定两种重组蛋白的酶活,结果显示FdDFR/FdLAR体外重组蛋白能够催化起始底物二氢槲皮素(Dihydroquercetin、DHQ)向下游终产物儿茶素[(+)catechin]转化,具有相应的生物酶活,且具有底物特异性。
构建了FdLAR基因的植物超表达载体,通过农杆菌介导的叶盘法转化烟草,通过分析转基因植株中目的基因表达水平与类黄酮积累之间的关系,对FdLAR基因的功能进行了初步验证。结果表明,通过GUS组织化学染色法、基因组DNA的PCR检测及荧光定量PCR检测,获得了阳性转基因烟草15株。15株转基因烟草叶片中类黄酮含量均有不同程度的提高,其中以T2株最为明显,是野生型的7倍。进一步检测转基因烟草中目的基因的表达发现,FdLAR基因的表达水平与其类黄酮含量成正相关关系,说明克隆得到的FdLAR基因是金荞麦类黄酮合成途径后期的一个关键基因。
(2)金荞麦查尔酮合成酶基因(FdCHS)的克隆与功能初步分析
根据已在NCBI登录的金荞麦查尔酮合成酶(Chalcone synthase, CHS)基因FdCHS的核苷酸序列,克隆其编码框全长并构建原核表达载体和植物超表达载体。在大肠杆菌Transetta(DE3)中诱导表达重组蛋白,以香豆酰-CoA (4-Coumaroyl-CoA)和丙二酰CoA (Malonyl-CoA)作为底物,测定其酶活,结果显示获得的重组蛋白具有正常的生物酶活,能够将其特异底物转化为下游产物。利用农杆菌介导的叶盘法获得转基因烟草,通过分析转基因植株中目的基因的表达水平与类黄酮积累之间的关系,对FdCHS基因的功能进行了初步验证。结果表明,获得的11株转基因烟草中,有10株叶片中类黄酮含量有不同程度的提高,其中以T3株最为明显,是野生型的3.4倍。而T10株类黄酮含量没有显著提高,进一步检测转基因烟草中目的基因的表达发现,FdCHS基因的表达水平与其类黄酮含量总体上成正相关关系,而T10株中没有检测到目的基因的表达,可能是发生了同源基因的共抑制现象。说明FdCHS基因是金荞麦类黄酮合成途径中的一个关键基因,其表达水平直接影响植株中类黄酮类物质的积累。
(3)金荞麦类黄酮的生物合成与金荞麦FdCHS, FdDFR、FdLAR及FdMYBPl基因的表达特征分析
研究了金荞麦在不同发育时期各个组织中类黄酮含量和相应组织中与类黄酮生物合成相关的3个重要结构基因FdCHS、FdDFR、FdLAR和1个转录调控因子MYBPl的表达水平之间的关系。结果表明金荞麦不同组织中的类黄酮含量在不同发育时期,呈现出不同的变化规律,在幼苗的营养生长阶段,不同组织中类黄酮含量总体情况是叶>根>茎,在生殖生长阶段的总体变化规律为根状茎>花>叶>茎。3个结构基因在金荞麦不同生育期的表达变化趋势却基本保持一致,都呈现出“两头高,中间低”的表达模式。根据4个基因在茎段中高表达和根状茎中的低表达,对应其茎段中的低类黄酮含量和根状茎中的高类黄酮含量结果推测：金荞麦类黄酮合成和积累的部位存在差异,茎段可能是类黄酮合成的重要部位,但是类黄酮并不同在茎段中持续的大量积累,而是转移至根状茎积累。进一步对生殖生长期不同组织中与类黄酮合成紧密相关的DFR/LAR酶活进行测定,发现茎段中的酶活均高于其他组织,说明其中发生着大量与类黄酮合成相关的酶催化反应,进一步证明了金荞麦类黄酮合成和积累的组织特异性,其类黄酮合成的主要部位是茎段,并向下运输至根状茎大量积累。
Golden-buckwheat[Fagopyrum dibotrys (D. Don) Hara)]is one of perennial herbs of the genus Polygonaceae in the Fagopyrum Mill family, origins from southwest of China.It is an important resource plant with rich nutrition and great medicinal values, and contains a large number of excellent genes. As an important traditional Chinese medicinal materials, the active extract of rhizome(flavonoids secondary metabolites) of Fagopyrum dibotrys is one of main constituents of many efficient drugs, and has prominent functions in anti-cancer, controlling tumour cell affect and transfering to lung, and anti-inflammatony antibacteria ect. At present, cultivated yield of Fagopyrum dibotrys is restricted and cultivated species are lesser than wild type in nutrition value, efficient constituents and quantity for medicine, which leads to requesting for Fagopyrum dibotrys largely, so that wild resource is destroyed greatly. In view of the conditions, it is ranked into the second degree of national protected plants. Domestically and abroad resource collection, diversity, physiological characteristic, breeding, pharmacodynamics and ecology environment and other aspects are outspread, however, in the light of functional genes research relating to active components of medicine were barely reported at home and abroad.This thesis aims to elucidate biosynthetic pathway of flavonoids through cloning and characterizing key genes,thus provides theory foundation for laying the groundwork for future producing medicinal second metabolic products on a large scale by genetic engineering.The main results in this rasearch are as follows:
1.Cloning and functional analysis of leucoanthcyanidin4-reductase(FdLAR)gene from Fagopyrum dibotrys
Based on the methods of degemerate PCR and RACE technique,the full length cDNA of leucoanthcyanidin4-reductase named as FdLAR was cloned from Fagopyrum dibotrys(GenBank accession:JN793953).The sequence analysis revealed that the full-length cDNA of FdLAR is1158 bp long fragment containing a1176bp open reading frame(ORF) encoding a polypeptide of391amino acids.Blastn and blastp analysis on NCBI showed that the amino acid sequence of the protein encoding by FdLAR gene shares a high similarity with other reported LAR proteins,especially with LAR from strawberry (Fragaria chiloensis,ADP37950.1)(75%identity).The phylogenetic analysis indicated that the genetic diatance between FdLAR and LAR from castor (Ricinus communi) is the nearest.The bioinformatics analysis showed that the FdLAR protein contains highly conserved NADPH and substrate specific binding site at N-terminal.There is no signal peptide in the deduced amino acid sequence and the FdLAR protein which is the hydrophilic protein with the typical tertiary tructure of enzyme is localized in motochondrial matrix predicted by the bioinformatics software.
The prokarytic expression vector of FdLAR and FdDFR genes were constructed and transformed into Escherichia coli Transetta(DE3),also the FdDFR and FdLAR proteins expressed and purified.The FdDFR/FdLAR-NADPH enzymatic reaction system was recongnized as a whole to determine the enzyme activity.The results showed that the recombinant protein of FdDFR/FdLAR could catalyze (+)-DHQ(Dihydroquercetin)as an initial sunbstrate to change into downstream end-product (+)-catechin which demonstrated that both of the recombinant proteins FdDFR and FdLAR had enzymatic activity.
The plant over-expression vector of FdLAR named FdLAR-2301G was consrructed and transformed into tobacco using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens GV3101(including FdLAR-2301G recombinant plasmid).15transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS(β-glucuronidase)histochemical staining and PCR and real-time quantitative PCR(qPCR) analysis.In15lines of transgenic plants, it was variable of total flavonoids contents in different lines and all of them were improved in varied degree. The content of total flavonoids in leaves of the number T2transgenic tobacco plant increased significantly, which is the highest and being up to7folds of the wild type tobacco.In subsequent molecular detection by qPCR,the high expression level of FdLAR in transgenic line number T2proved that improved flavonoids content was positively co-related with the expression of FdLAR.
2. Cloning and functional analysis of chalcone synthase (FdCHS)gene from Fagopyrum dibotrys
Based on the nuclectide sequence submitted on the NCBI(GenBank accession number:GU169470),the FdCHS gene was cloned by reverse transcription PCR(RT-PCR)from the first strand of cDNA of the Fagopyrum dibotrys. The prokarytic expression vector and plant over-expression vector were constructed respectively in order to reveal the preliminary charecterization of the FdCHS gene. the FdCHS protein was expressed in Escherichia coli Transetta(DE3) and then was purified by the His Bind Purification Kit..Took the4-Coumaroyl-CoA and Malonyl-CoA as substrates to determine the enzyme activity,and the enzyme activity determination of the recombinant protein showed that the prokaryotic expressed reconmbinant protein of FdCHS could catalyze4-Coumaroyl-CoA and Malonyl-CoA to change into downstream substances,which demonstrated that the recombinant protein had enzymatic activity. The plant over-expression vector FdCHS-2301G was constructed and transformed into tobacco plants using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens GV3101.11transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS(β-glucuronidase)histochemical staining and PCR and real-time quantitative PCR(qPCR) analysis.In11lines of transgenic plants, it was variable of total flavonoids contents in different lines and10of them were improved in varied degree. The content of total flavonoids in leaves of the number T3transgenic tobacco plant increased significantly, which is the highest and being up to3.4folds of the wild type tobacco.In subsequent molecular detection by qPCR,the high expression level of FdCHS in transgenic line number T3proved that improved flavonoids contene was positively co-related with the expression of FdCHS.However,positive transgenic line number T10did not show FdCHS function,which may be resulted from the gene silence of introduced FdCHS by co-suppression.The q-PCR also did not detect the expression the FdCHS in the number T10tobacco plant.
3The biosynthesis of flavonoids in Fagopyrum dibotrys and expression analysis of FdCHS、 FdDFR、FdLAR and transcription regulator FdMYBPl genes.
The paper analysed the relationship between the flavonoids contents in different tissues of Fagopyrum dibotrys at different stages and the three important structural genes FdCHS、FdDFR、 FdLAR and one transcription regulator FdMYBPl gene.The results showed that the the flavonoids contents display the virous patterns during different development stages.During the vegetatibe multiplication,the overall condition of the flavonoids contents was leaf>root>item and rhizome>flower>leaf>leaf during reproductive growth.The expression levels of the three structural genes in the root or rhizome approximately kept consistency with each other which was high at both ends and low in the middle during the whole development stages.Although the four genes had the high expression in item and low expression in rhizome,the flavonoids contents were high in rhizome and low in item. We sepculated that the flavonoids synthesis and accumulation occurred at different parts of Fagopyrum dibotrys plant.The item may be the main synthesis site of flavonoids synthesised and transport to the rhizome which may be the main accumulation site of flavonoids.In order to verify the assumption furthmore,we detemined the enzyme activity of the key enzyme system DFR/LAR which was closely related to the flavonoids biosynthesis in Fagopyrum dibotrys.The results showed that the enzyme activity of DFR/LAR in item are higher than other tissues during reproductive growth which suggested that there were massive enzyme reaction occurring in item. Summarization above results,the flavonoids synthesis and accumulation had the tissue specificity.
引文
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