烟草花青素合成相关基因及转录因子基因的克隆与功能验证研究
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摘要
花青素是多种植物体内产生的最重要的类群代谢产物之一，他不但可以吸引授粉者和种子传播者，还可以保护植物免受昆虫攻击、缓解UV-B辐射、保护叶绿体减缓过渡光照所带来的负面影响，且具有抗氧化活性及抗炎活性，近年来的研究表明，花青素的摄入还能够降低心血管疾病、糖尿病、关节炎和癌症的发病率。花青素生物合成途径是类黄酮途径的一个分支途径，对其生物合成途径基因及调控因子进行研究，不仅有助于通过产生或操控花青素来改造提升植物的抗性，还可以促进重要植物中保护健康的花青素的代谢工程，帮助人们获得具有高抗氧化性的食物。烟草是最具有农业价值的茄科农作物之一，由于其容易转化、生长周期较短，所以几十年来一直被用作植物分子生物学研究的模式植物，而且，生长快速的烟草细胞系，譬如Bright Yellow-2(BY-2)和Xanthi (用于本研究)，提供了一个研究基因表达和次级代谢的良好研究体系。但是，到目前为止，鲜有关于烟草花青素生物合成途径基因及调控因子功能特性的研究报道。本研究对影响烟草花青素生物合成的主要调控因子碱性螺旋-环-螺旋（bHLH）转录因子基因以及关键酶二氢黄酮醇-4-还原酶(DFR)基因进行了分子克隆和功能验证研究。获得了以下主要结果：
1.从烟草花器官中分离到两个bHLH转录因子基因，NtAn1a和NtAn1b，序列比对发现NtAn1a和NtAn1b与其它已知花青素/类黄酮相关bHLH转录因子序列同源性很高，进化树分析进一步将他们与矮牵牛An1、拟南芥TT8和葡萄MYC1归入同一组。
2.利用位点特异引物在cDNA和基因组DNA的PCR扩增，证实NtAn1a和NtAn1b分别起源于烟草的两个祖先毛叶烟和拟茸毛烟草，基因序列分析表明，NtAn1a和NtAn1b都含有8个内含子和9个外显子。
3.表达水平定量PCR (qPCR)分析显示，NtAn1a和NtAn1b主要在花朵中表达，且随着花朵成熟花器官中的转录本继续累积，利用基于PCR的基因组步移法，分离到一个1.1kb NtAn1a启动子片段，NtAn1a启动子驱动的GUS表达谱与NtAn1基因的表达基本一致，表明这种花器官特异表达受NtAn1启动子控制。
4.酵母反式激活（单杂交）分析和烟草原生质体反式激活分析表明， NtAn1a和NtAn1b都是转录激活因子，NtAn1a或NtAn1b的异位表达促进了烟草花朵中花青素的积累，花青素的累积与NtAn1a或NtAn1b转录本的增加呈正相关。qPCR分析表明，在表达NtAn1a和NtAn1b的转基因烟草中，类黄酮途径前期基因查耳酮合成酶（CHS）,查耳酮异构酶（CHI）和黄烷酮3-羟化酶（F3H）以及后期基因二氢黄酮醇-4-还原酶（DFR）和花青素合成酶（ANS）都上调表达。
5.酵母双杂交（Y2H）分析表明，NtAn1a或NtAn1b与R2R3-MYB转录因子NtAn2之间存在强烈的蛋白质互作。瞬时烟草原生质体分析检测表明NtAn1a、NtAn1b与NtAn2三个转录因子单独都不能激活花青素途径的两个关键基因DFR或CHS的启动子，但二重组合(NtAn1a+NtAn2或NtAn1b+NtAn2)都能激活两个启动子，这种启动子激活作用可被NtAn1a或NtAn2的显性抑制形式（将SRDX抑制子区域与转录因子融合创制）显著抑制，说明NtAn1和NtAn2蛋白共同参与烟草花朵中花青素途径的调节。
6. RT–PCR分析显示，过表达NtAn2可诱导NtAn1a和NtAn1b两个基因在叶片中的表达，而NtAn1基因的过表达对NtAn2表达无影响，这表明，NtAn1a和NtAn1b受花组织特异性MYB转录因子NtAn2的调节，但NtAn1对NtAn2没有调节作用。
7.酵母双杂交（Y2H）分析表明，玉米R2R3MYB转录因子C1与bHLH转录因子Lc存在强烈互作，但它与NtAn1a或NtAn1b都不存在互作，这解释了“C1在烟草中单独表达为何不产生花青素”这一问题。
8.从烟草花分离到两种二氢黄酮醇-4-还原酶（Dfr） cDNA，NtDfr1和NtDfr2，序列比对分析发现，这两个基因与多个分离自其它种属植物的Dfr基因具有高度的序列相似性，进化树分析将这两个基因排列在与牵牛花和马铃薯相邻的分支上。
9.转录分析显示，NtDfr1和NtDfr2均只在花中表达，而且受到严格的发育调控，它们在幼龄花中高度表达，而在成熟期花中的表达量显著下降。
10.利用位点特异引物对毛叶烟和拟茸毛烟草的花朵cDNA进行RT-PCR，PCR产物的序列分析表明，NtDfr1基因起源于拟茸毛烟草，而NtDfr2基因起源于毛叶烟。
11. NtDfr1和NtDfr2基因的过表达可引起花青素在烟草花中的积累，而RNA干涉介导的NtDfr基因的抑制会导致烟草花变为淡粉红色甚至白色，qPCR分析表明，NtDfr过表达株系花朵中，NtDfr1和NtDfr2的mRNA水平也显著提高，而NtDfr RNA干涉烟草转基因株系花朵中，NtDfr的转录水平显著低于对照，这表明，来自两种烟草祖先的这两个Dfr基因在烟草上都具有功能活性。
12.烟草原生质体瞬间检测结果显示，NtDfr是bHLH转录因子NtAn1及R2R3MYB转录因子NtAn2的直接靶基因，其表达受NtAn1和NtAn2复合体的调节。
Anthocyanins are one of the most important group metabolites produced in plants. Theroles of anthocyanins are their ability not only to attract pollinators and seed dispersers butalso to protect plants from insect attack, to attenuate UV-B radiation and to protectchloroplasts from the adverse effects of excess light. Recent studies suggest that theconsumption of anthocyanins is able to lower the risk of cardiovascular disease, diabetes,arthritis and cancer due to their anti-oxidant and anti-inflammatory activities. The synthesis ofanthocyanin is derived from a branch of the flavonoid biosynthetic pathway. Studies onanthocyanin biosynthesis pathway genes and regulatory factors should be possible not only toengineer improved plant resistance by generating or manipulating anthocyanins but also topromote metabolic engineering of health-protecting flavonoids in important plants, and thusto help human beings to obtain food with increased antioxidant capacity. Tobacco is amember of one of the most agriculturally important plant families (Solanaceae) and has beenused as a model in plant biotechnology for decades due to its ease of transformation andrelatively short generation time. Moreover, the fast growing tobacco cell lines such as BrightYellow-2(BY-2) and Xanthi (used in the present study) are an excellent system for studyinggene expression and secondary metabolism. However, to the best of our knowledge, thefunctional characterization of anthocyanin biosynthesis pathway genes and regulatory factorshas been reported rarely to date. Herein we report the molecular cloning and functionalanalyses of the key regulator, basic helix-loop-helix (bHLH) transcription factors (TFs), andthe key enzymes, Dihydroflavonol-4-reductase (DFRs), of anthocyanin biosynthesis pathwayin tobacco flowers. The main conclusions obtained from this project are as follows:
1. Two bHLH TFs, NtAn1a and NtAn1b, have been isolated from tobacco (Nicotianatabacum) flowers. Sequences alignment demonstrated that NtAn1a and NtAn1b share highsequence homology with other known flavonoid-related bHLH TFs. Phylogenetic analysisdivided NtAn1a and NtAn1b into the clade close to PhAn1from petunia, TT8fromArabidopsis and VvMYC1from grape.
2. PCRs by gene-specific primers using cDNA and genomic DNA clarified thatNtAn1a and NtAn1b are originated from two ancestors of tobacco, N. sylvestris and N. tomentosiformis, respectively. Analysis of the genomic sequences revealed that both NtAn1aand NtAn1b contain eight introns and nine exons.
3. Quantitative PCR (qPCR) revealed that NtAn1a and NtAn1b are predominantlyexpressed in flowers. A1.1kb NtAn1a promoter fragmen was isolated using a PCR-basedgenome walking method. The GUS expression profile driven by the NtAn1a promoter isessentially identical to that of NtAn1genes, indicating this floweral tissue-specific expressionis controlled by the NtAn1promoter
4. Yeast one-hybrid and transient protoplast assays suggested that both NtAn1a andNtAn1b are transcriptional activators. Ectopic expression of the two genes in tobacco lead toincreased anthocyanin accumulation in flowers and the elevated anthocyanin content isassociated with the increase in NtAn1a or NtAn1b transcripts. In transgenic tobaccoexpressing NtAn1a or NtAn1b, both subsets of early (Chalcone synthase, CHS; Chalconeisomerase, CHI; Flavanone3-hydroxylase, F3H) and late (Dihydroflavonol4-reductase,DFR；Anthocyanidin synthase, ANS) flavonoid pathway genes were up-regulated.
5. Yeast two-hybrid assays showed that NtAn1a and NtAn1b have strongprotein–protein interactions with R2R3-MYB TF, NtAn2. Transient tobacco protoplast assaysimplied that NtAn1a, NtAn1b and NtAn2cloud not activate the promoters of two keyanthocyanin pathway genes, dihydroflavonol reductase (DFR) and chalcone synthase (CHS)individually. However, in combination (NtAn1a+NtAn2or NtAn1b+NtAn2), they activatethe two promoters. The promoter activation is severely repressed by the dominant repressiveforms either NtAn1a or NtAn2, created by fusing the SRDX repressor domain to the TFs. thisresults pointed out that NtAn1and NtAn2act in concert to regulate the anthocyanin pathwayin tobacco flowers.
6. RT–PCR manifested that over expression of NtAn2induces expression of bothNtAn1a and NtAn1b in tobacco leaves, while over expression of NtAn1genes has no effecton NtAn2expression. These results indicate that NtAn1a and NtAn1b are regulated by NtAn2,a floral tissue specific MYB TF; however, the NtAn1proteins do not regulate NtAn2.
7. Yeast two-hybrid assays illustrated that the maize R2R3MYB TF, C1, stronglyinteracts with bHLH TF Lc, but it does not interact with either NtAn1a or NtAn1b. This resultprovides an explanation to why C1fails to induce anthocyanin when expressed alone intobacco.
8. Two dihydroflavonol4-reductase (Dfr) cDNAs, NtDfr1and NtDfr2, were isolatedfrom tobacco flowers. Sequences alignment revealed that NtDfr1and NtDfr2share highsequence homology with other known Dfr genes isolated from other species. Phylogeneticanalysis divided NtDfr1and NtDfr2into the clade close to the ones from petunia and potato.
9. Transcript analysis uncovered that both Dfr genes express only in flowers and areunder strict developmental control; highly expressed in young flowers and the expressiongoes down significantly in mature flowers.
10. RT-PCR on flower cDNA of N. sylvestris and N. tomentosiformis were performedusing allele-specific primers. Sequence analysis of the PCR products revealed that NtDfr1originated from N. tomentosiformis, whereas NtDfr2derived from N. sylvestris.
11. Over-expression of NtDfr1or NtDfr2in tobacco results in pigment accumulation inflowers, while RNAi-mediated suppression of NtDfr in tobacco resulted in white to pale pinkflowers. The levels of NtDfr1or NtDfr2mRNA in flowers from transgenic tobacco lines weresignificantly higher than control. However, the NtDfr trascript levels in transgenic line weresignificantly reduced compared to the control flowers. This suggests that the Dfr genesderived from both parents are functional in tobacco.
12. Tobacco protoplast transient assay demonstrated that NtDfr is a direct target of thebHLH TF, NtAn1and R2R3-MYB, NtAn2. NtDfr expression is regulated by a complex ofNtAn1and NtAn2.
1.从烟草花器官中分离到两个bHLH转录因子基因，NtAn1a和NtAn1b，序列比对发现NtAn1a和NtAn1b与其它已知花青素/类黄酮相关bHLH转录因子序列同源性很高，进化树分析进一步将他们与矮牵牛An1、拟南芥TT8和葡萄MYC1归入同一组。
2.利用位点特异引物在cDNA和基因组DNA的PCR扩增，证实NtAn1a和NtAn1b分别起源于烟草的两个祖先毛叶烟和拟茸毛烟草，基因序列分析表明，NtAn1a和NtAn1b都含有8个内含子和9个外显子。
3.表达水平定量PCR (qPCR)分析显示，NtAn1a和NtAn1b主要在花朵中表达，且随着花朵成熟花器官中的转录本继续累积，利用基于PCR的基因组步移法，分离到一个1.1kb NtAn1a启动子片段，NtAn1a启动子驱动的GUS表达谱与NtAn1基因的表达基本一致，表明这种花器官特异表达受NtAn1启动子控制。
4.酵母反式激活（单杂交）分析和烟草原生质体反式激活分析表明， NtAn1a和NtAn1b都是转录激活因子，NtAn1a或NtAn1b的异位表达促进了烟草花朵中花青素的积累，花青素的累积与NtAn1a或NtAn1b转录本的增加呈正相关。qPCR分析表明，在表达NtAn1a和NtAn1b的转基因烟草中，类黄酮途径前期基因查耳酮合成酶（CHS）,查耳酮异构酶（CHI）和黄烷酮3-羟化酶（F3H）以及后期基因二氢黄酮醇-4-还原酶（DFR）和花青素合成酶（ANS）都上调表达。
5.酵母双杂交（Y2H）分析表明，NtAn1a或NtAn1b与R2R3-MYB转录因子NtAn2之间存在强烈的蛋白质互作。瞬时烟草原生质体分析检测表明NtAn1a、NtAn1b与NtAn2三个转录因子单独都不能激活花青素途径的两个关键基因DFR或CHS的启动子，但二重组合(NtAn1a+NtAn2或NtAn1b+NtAn2)都能激活两个启动子，这种启动子激活作用可被NtAn1a或NtAn2的显性抑制形式（将SRDX抑制子区域与转录因子融合创制）显著抑制，说明NtAn1和NtAn2蛋白共同参与烟草花朵中花青素途径的调节。
6. RT–PCR分析显示，过表达NtAn2可诱导NtAn1a和NtAn1b两个基因在叶片中的表达，而NtAn1基因的过表达对NtAn2表达无影响，这表明，NtAn1a和NtAn1b受花组织特异性MYB转录因子NtAn2的调节，但NtAn1对NtAn2没有调节作用。
7.酵母双杂交（Y2H）分析表明，玉米R2R3MYB转录因子C1与bHLH转录因子Lc存在强烈互作，但它与NtAn1a或NtAn1b都不存在互作，这解释了“C1在烟草中单独表达为何不产生花青素”这一问题。
8.从烟草花分离到两种二氢黄酮醇-4-还原酶（Dfr） cDNA，NtDfr1和NtDfr2，序列比对分析发现，这两个基因与多个分离自其它种属植物的Dfr基因具有高度的序列相似性，进化树分析将这两个基因排列在与牵牛花和马铃薯相邻的分支上。
9.转录分析显示，NtDfr1和NtDfr2均只在花中表达，而且受到严格的发育调控，它们在幼龄花中高度表达，而在成熟期花中的表达量显著下降。
10.利用位点特异引物对毛叶烟和拟茸毛烟草的花朵cDNA进行RT-PCR，PCR产物的序列分析表明，NtDfr1基因起源于拟茸毛烟草，而NtDfr2基因起源于毛叶烟。
11. NtDfr1和NtDfr2基因的过表达可引起花青素在烟草花中的积累，而RNA干涉介导的NtDfr基因的抑制会导致烟草花变为淡粉红色甚至白色，qPCR分析表明，NtDfr过表达株系花朵中，NtDfr1和NtDfr2的mRNA水平也显著提高，而NtDfr RNA干涉烟草转基因株系花朵中，NtDfr的转录水平显著低于对照，这表明，来自两种烟草祖先的这两个Dfr基因在烟草上都具有功能活性。
12.烟草原生质体瞬间检测结果显示，NtDfr是bHLH转录因子NtAn1及R2R3MYB转录因子NtAn2的直接靶基因，其表达受NtAn1和NtAn2复合体的调节。
Anthocyanins are one of the most important group metabolites produced in plants. Theroles of anthocyanins are their ability not only to attract pollinators and seed dispersers butalso to protect plants from insect attack, to attenuate UV-B radiation and to protectchloroplasts from the adverse effects of excess light. Recent studies suggest that theconsumption of anthocyanins is able to lower the risk of cardiovascular disease, diabetes,arthritis and cancer due to their anti-oxidant and anti-inflammatory activities. The synthesis ofanthocyanin is derived from a branch of the flavonoid biosynthetic pathway. Studies onanthocyanin biosynthesis pathway genes and regulatory factors should be possible not only toengineer improved plant resistance by generating or manipulating anthocyanins but also topromote metabolic engineering of health-protecting flavonoids in important plants, and thusto help human beings to obtain food with increased antioxidant capacity. Tobacco is amember of one of the most agriculturally important plant families (Solanaceae) and has beenused as a model in plant biotechnology for decades due to its ease of transformation andrelatively short generation time. Moreover, the fast growing tobacco cell lines such as BrightYellow-2(BY-2) and Xanthi (used in the present study) are an excellent system for studyinggene expression and secondary metabolism. However, to the best of our knowledge, thefunctional characterization of anthocyanin biosynthesis pathway genes and regulatory factorshas been reported rarely to date. Herein we report the molecular cloning and functionalanalyses of the key regulator, basic helix-loop-helix (bHLH) transcription factors (TFs), andthe key enzymes, Dihydroflavonol-4-reductase (DFRs), of anthocyanin biosynthesis pathwayin tobacco flowers. The main conclusions obtained from this project are as follows:
1. Two bHLH TFs, NtAn1a and NtAn1b, have been isolated from tobacco (Nicotianatabacum) flowers. Sequences alignment demonstrated that NtAn1a and NtAn1b share highsequence homology with other known flavonoid-related bHLH TFs. Phylogenetic analysisdivided NtAn1a and NtAn1b into the clade close to PhAn1from petunia, TT8fromArabidopsis and VvMYC1from grape.
2. PCRs by gene-specific primers using cDNA and genomic DNA clarified thatNtAn1a and NtAn1b are originated from two ancestors of tobacco, N. sylvestris and N. tomentosiformis, respectively. Analysis of the genomic sequences revealed that both NtAn1aand NtAn1b contain eight introns and nine exons.
3. Quantitative PCR (qPCR) revealed that NtAn1a and NtAn1b are predominantlyexpressed in flowers. A1.1kb NtAn1a promoter fragmen was isolated using a PCR-basedgenome walking method. The GUS expression profile driven by the NtAn1a promoter isessentially identical to that of NtAn1genes, indicating this floweral tissue-specific expressionis controlled by the NtAn1promoter
4. Yeast one-hybrid and transient protoplast assays suggested that both NtAn1a andNtAn1b are transcriptional activators. Ectopic expression of the two genes in tobacco lead toincreased anthocyanin accumulation in flowers and the elevated anthocyanin content isassociated with the increase in NtAn1a or NtAn1b transcripts. In transgenic tobaccoexpressing NtAn1a or NtAn1b, both subsets of early (Chalcone synthase, CHS; Chalconeisomerase, CHI; Flavanone3-hydroxylase, F3H) and late (Dihydroflavonol4-reductase,DFR；Anthocyanidin synthase, ANS) flavonoid pathway genes were up-regulated.
5. Yeast two-hybrid assays showed that NtAn1a and NtAn1b have strongprotein–protein interactions with R2R3-MYB TF, NtAn2. Transient tobacco protoplast assaysimplied that NtAn1a, NtAn1b and NtAn2cloud not activate the promoters of two keyanthocyanin pathway genes, dihydroflavonol reductase (DFR) and chalcone synthase (CHS)individually. However, in combination (NtAn1a+NtAn2or NtAn1b+NtAn2), they activatethe two promoters. The promoter activation is severely repressed by the dominant repressiveforms either NtAn1a or NtAn2, created by fusing the SRDX repressor domain to the TFs. thisresults pointed out that NtAn1and NtAn2act in concert to regulate the anthocyanin pathwayin tobacco flowers.
6. RT–PCR manifested that over expression of NtAn2induces expression of bothNtAn1a and NtAn1b in tobacco leaves, while over expression of NtAn1genes has no effecton NtAn2expression. These results indicate that NtAn1a and NtAn1b are regulated by NtAn2,a floral tissue specific MYB TF; however, the NtAn1proteins do not regulate NtAn2.
7. Yeast two-hybrid assays illustrated that the maize R2R3MYB TF, C1, stronglyinteracts with bHLH TF Lc, but it does not interact with either NtAn1a or NtAn1b. This resultprovides an explanation to why C1fails to induce anthocyanin when expressed alone intobacco.
8. Two dihydroflavonol4-reductase (Dfr) cDNAs, NtDfr1and NtDfr2, were isolatedfrom tobacco flowers. Sequences alignment revealed that NtDfr1and NtDfr2share highsequence homology with other known Dfr genes isolated from other species. Phylogeneticanalysis divided NtDfr1and NtDfr2into the clade close to the ones from petunia and potato.
9. Transcript analysis uncovered that both Dfr genes express only in flowers and areunder strict developmental control; highly expressed in young flowers and the expressiongoes down significantly in mature flowers.
10. RT-PCR on flower cDNA of N. sylvestris and N. tomentosiformis were performedusing allele-specific primers. Sequence analysis of the PCR products revealed that NtDfr1originated from N. tomentosiformis, whereas NtDfr2derived from N. sylvestris.
11. Over-expression of NtDfr1or NtDfr2in tobacco results in pigment accumulation inflowers, while RNAi-mediated suppression of NtDfr in tobacco resulted in white to pale pinkflowers. The levels of NtDfr1or NtDfr2mRNA in flowers from transgenic tobacco lines weresignificantly higher than control. However, the NtDfr trascript levels in transgenic line weresignificantly reduced compared to the control flowers. This suggests that the Dfr genesderived from both parents are functional in tobacco.
12. Tobacco protoplast transient assay demonstrated that NtDfr is a direct target of thebHLH TF, NtAn1and R2R3-MYB, NtAn2. NtDfr expression is regulated by a complex ofNtAn1and NtAn2.
引文
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