大豆黄酮合成酶基因的克隆与RNA干扰调控异黄酮含量的研究
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摘要
类黄酮是植物生长过程中广泛存在的一类次生代谢产物，包括异黄酮、黄酮、查耳酮、花青素等具有生物活性的物质。它们对植物生长素运输、花色调节、植物-微生物之间的互作以及对人体健康有直接而复杂的影响。研究类黄酮功能及代谢途径的调控对明确植物的防御反应机制和提高食品的功能性成分具有重要的作用。
在高等植物中，类黄酮是通过苯基丙酸类代谢途径合成的。目前，对苯基丙酸类代谢途径的研究已经进入了分子生物学领域，在大豆（Glycine max）中，途径中越来越多的编码基因被克隆并鉴定。另一方面，代谢工程，特别是RNA干扰技术的发展，为研究调控植物次生代谢产物的积累及其在植物防御中的作用开辟了新的可能性。
本研究根据其它物种中已报道的黄酮合成酶（FNS Ⅱ）基因序列及大豆基因组序列信息，进行生物信息学分析，在大豆栽培品种合丰47中通过反转录扩增，得到两个候选基因的cDNA序列，命名为GmFNSⅡ-1与GmFNSⅡ-2。根据序列分析GmFNSⅡ-1与已报道的一个大豆黄酮合成酶基因（CYP93B16）是同一基因，而GmFNSⅡ-2是一个全新的基因。对GmFNSⅡ-1与GmFNSⅡ-2的基因组序列、核苷酸序列、氨基酸序列、蛋白质的物理化学性质、蛋白质的高级结构及功能进行了生物信息学分析。
通过在大肠杆菌（Escherichia coli）及裂殖酵母（Schizosaccharomyces pombe）中融合蛋白的构建，对GmFNSⅡ-1与GmFNSⅡ-2进行酶活性分析。结果表明两个GmFNSⅡ蛋白都可以直接催化柚皮素（Naringenin）转化为大豆黄酮（Apigenin）。两个GmFNSⅡ基因的组织特异性表达情况相似，在检测的6个组织中都有表达，但总的来说，在根和未成熟种子中的表达量较低。特别的是，在受到渗透胁迫时，幼苗的根、茎及叶中，两个GmFNSⅡ基因的表达量都显著提高，表明这两个基因在大豆的胁迫信号传导过程中有重要作用。
本研究中，根据RNA干扰技术原理，以P1304+PBI121载体为基本骨架，构建了GmFNSⅡ基因的RNA干扰植物表达载体P1304+-GmFNSⅡ-RNAi；以pCAMBIA3300载体为基本骨架，构建了GmFNSⅡ基因与黄烷酮3-羟化酶基因（F3H）双价的RNA干扰植物表达载体p3300-GmFNSⅡi-F3Hi，并分别构建了两基因的单价RNA干扰植物表达载体p3300-GmFNSⅡi与p3300-F3Hi。
通过发根农杆菌（Agrobacterium rhizogenes）ATCC15834对大豆子叶节的转化，验证所构建RNA干扰植物表达载体的效率。结果发现，P1304+-GmFNSⅡ-RNAi载体转化的毛状根中，GmFNSⅡ-1与GmFNSⅡ-2基因的表达都受到的显著抑制（<10%），特别是GmFNSⅡ-2基因（<5%）。在p3300-GmFNSⅡi、p3300-F3Hi及p3300-GmFNSⅡi-F3Hi载体转化的毛状根中， GmFNSⅡ或/与F3H基因的表达受到显著抑制，表达量与对照相比降低90%以上。同时，HPLC分析结果显示，RNAi载体转化生成的毛状根中，黄酮的含量与异黄酮总含量发生了明显变化。其中双价RNAi载体对总异黄酮含量的增长具有更好的效果，转化根中总异黄酮含量是对照的两倍。研究结果为利用分子生物学方法，特别是RNAi技术提高大豆异黄酮含量提供了研究基础，并为其他相似的植物次生代谢产物的调控积累提供了依据。
本研究中，我们检测了体外条件下，不同浓度的大豆黄酮（Apigenin）与染料木素（Genistein）对导致大豆7种主要真菌病害的病原菌生长速度的影响，这7种病原菌包括： Colletotrichum truncatum， Macrophomina phaseolina， Phoma exigua，Phytophthora sojae，Pythium ultimum，Rhizoctonia solani与Sclerotinia sclerotiorum。结果发现，两种类黄酮对实验中的所有病原菌生长都有抑制作用，但抑制效果不同，如：相对其它病原菌，C. truncatum受到的抑制更小而P. exigua受到的抑制更大。并且随着两种类黄酮浓度的提高，抑制效果有明显的剂量效应，但在低浓度下，染料木素表现出更好的抑制作用。研究结果提示我们，在通过代谢工程增强大豆抗病性的研究时，染料木素可能更适宜于作为基因工程的改良的目标。
Flavonoids are one kind of secondary metabolites that are widespread throughout thegrowth and development of plants, including isoflavone, flavone, chalcone, anthocyanidin.They play diverse roles in regulation of auxin transport, modulation of flower color,plant–microbe interactions, and have direct but complex effects on human health. Theresearch of flavonoids biosynthesis pathway plays important roles in plant defenseresponse and improving functional components of food.
In all higher plants, flavonoids are produced through a branch of the phenylpropanoidpathway. At present, the research for phenylpropanoid pathway has been focus on the fieldof molecular biology, more and more coding genes in pathway has been cloned andidentified in soybean. On the other hand, the development of metabolic engineering,especially RNA interference technology, opens up new possibilities for the study ofregulation of plant secondary metabolite accumulation and their roles in plant defense.
In this study, two FNS Ⅱ genes from soybean cultivar Hefeng47were clonedaccording to basic local alignment search tool (BLAST) contexts using flavone synthasesequences reported in other species. These were named GmFNSⅡ-1and GmFNSⅡ-2.Sequence alignments showed that the cDNA of GmFNSⅡ-1was identical to that ofCYP93B16, whereas GmFNSⅡ-2was clearly distinct. Both GmFNSⅡ genes wereanalysized by bioinformatics in their genome sequences, the nucleotide sequence, aminoacid sequences, physical and chemical properties of protein, and function.
Functional assays in Escherichia coli and yeast (Schizosaccharomyces pombe)suggested that these two enzymes could convert (2S)-naringenin into apigenin. The twoGmFNSⅡ genes had similar tissue-specific expression patterns, and were significantlyincreased by osmotic stress like glucose and MeJA treatment. This demonstrates that the gene plays an important role in the response to defense signals in soybean.
Here, a bivalent RNA interference (RNAi) plant-transformation vector wasconstructed to silence both the flavanone3-hydroxylase (F3H) gene and the flavonesynthase Ⅱ (GmFNSⅡ) gene. Moreover, two further unit RNAi vectors were constructedfor each of these two genes.
RNAi-mediated suppression of those genes showed a significant reduction (to <10%)in gene expression in hairy roots that arose from soybean cotyledons transformed withAgrobacterium rhizogenes (ATCC15834). HPLC showed in hairy roots，RNAi-mediatedsuppression of these genes effectively regulated flavone and isoflavone production.Notably, the bivalent RNAi vector had a significantly greater effect for increasingisoflavone production compared with the two unit RNAi vectors.
This present study highlights molecular methods that can be used to enhanceisoflavone production in soybean, and demonstrates the challenges associated with suchmetabolic engineering for the production of plant natural products.
In this study, we tested two flavonoids, apigenin and genistein, in amended culturemedia against seven soybean pathogens including Colletotrichum truncatum,Macrophomina phaseolina, Phoma exigua, Phytophthora sojae, Pythium ultimum,Rhizoctonia solani, and Sclerotinia sclerotiorum. Both compounds restricted growth of allseven pathogens, although not all equally. For example, inhibition of growth was less (P>0.05) for C. truncatum than the other pathogens tested. There also was a dosage effect asincreased concentrations of either compound further restricted colony growth. The resultssuggest that through metabolic engineering, apigenin and genistein may be useful targetsfor overexpression in planta to enhance disease resistance in soybean.
在高等植物中，类黄酮是通过苯基丙酸类代谢途径合成的。目前，对苯基丙酸类代谢途径的研究已经进入了分子生物学领域，在大豆（Glycine max）中，途径中越来越多的编码基因被克隆并鉴定。另一方面，代谢工程，特别是RNA干扰技术的发展，为研究调控植物次生代谢产物的积累及其在植物防御中的作用开辟了新的可能性。
本研究根据其它物种中已报道的黄酮合成酶（FNS Ⅱ）基因序列及大豆基因组序列信息，进行生物信息学分析，在大豆栽培品种合丰47中通过反转录扩增，得到两个候选基因的cDNA序列，命名为GmFNSⅡ-1与GmFNSⅡ-2。根据序列分析GmFNSⅡ-1与已报道的一个大豆黄酮合成酶基因（CYP93B16）是同一基因，而GmFNSⅡ-2是一个全新的基因。对GmFNSⅡ-1与GmFNSⅡ-2的基因组序列、核苷酸序列、氨基酸序列、蛋白质的物理化学性质、蛋白质的高级结构及功能进行了生物信息学分析。
通过在大肠杆菌（Escherichia coli）及裂殖酵母（Schizosaccharomyces pombe）中融合蛋白的构建，对GmFNSⅡ-1与GmFNSⅡ-2进行酶活性分析。结果表明两个GmFNSⅡ蛋白都可以直接催化柚皮素（Naringenin）转化为大豆黄酮（Apigenin）。两个GmFNSⅡ基因的组织特异性表达情况相似，在检测的6个组织中都有表达，但总的来说，在根和未成熟种子中的表达量较低。特别的是，在受到渗透胁迫时，幼苗的根、茎及叶中，两个GmFNSⅡ基因的表达量都显著提高，表明这两个基因在大豆的胁迫信号传导过程中有重要作用。
本研究中，根据RNA干扰技术原理，以P1304+PBI121载体为基本骨架，构建了GmFNSⅡ基因的RNA干扰植物表达载体P1304+-GmFNSⅡ-RNAi；以pCAMBIA3300载体为基本骨架，构建了GmFNSⅡ基因与黄烷酮3-羟化酶基因（F3H）双价的RNA干扰植物表达载体p3300-GmFNSⅡi-F3Hi，并分别构建了两基因的单价RNA干扰植物表达载体p3300-GmFNSⅡi与p3300-F3Hi。
通过发根农杆菌（Agrobacterium rhizogenes）ATCC15834对大豆子叶节的转化，验证所构建RNA干扰植物表达载体的效率。结果发现，P1304+-GmFNSⅡ-RNAi载体转化的毛状根中，GmFNSⅡ-1与GmFNSⅡ-2基因的表达都受到的显著抑制（<10%），特别是GmFNSⅡ-2基因（<5%）。在p3300-GmFNSⅡi、p3300-F3Hi及p3300-GmFNSⅡi-F3Hi载体转化的毛状根中， GmFNSⅡ或/与F3H基因的表达受到显著抑制，表达量与对照相比降低90%以上。同时，HPLC分析结果显示，RNAi载体转化生成的毛状根中，黄酮的含量与异黄酮总含量发生了明显变化。其中双价RNAi载体对总异黄酮含量的增长具有更好的效果，转化根中总异黄酮含量是对照的两倍。研究结果为利用分子生物学方法，特别是RNAi技术提高大豆异黄酮含量提供了研究基础，并为其他相似的植物次生代谢产物的调控积累提供了依据。
本研究中，我们检测了体外条件下，不同浓度的大豆黄酮（Apigenin）与染料木素（Genistein）对导致大豆7种主要真菌病害的病原菌生长速度的影响，这7种病原菌包括： Colletotrichum truncatum， Macrophomina phaseolina， Phoma exigua，Phytophthora sojae，Pythium ultimum，Rhizoctonia solani与Sclerotinia sclerotiorum。结果发现，两种类黄酮对实验中的所有病原菌生长都有抑制作用，但抑制效果不同，如：相对其它病原菌，C. truncatum受到的抑制更小而P. exigua受到的抑制更大。并且随着两种类黄酮浓度的提高，抑制效果有明显的剂量效应，但在低浓度下，染料木素表现出更好的抑制作用。研究结果提示我们，在通过代谢工程增强大豆抗病性的研究时，染料木素可能更适宜于作为基因工程的改良的目标。
Flavonoids are one kind of secondary metabolites that are widespread throughout thegrowth and development of plants, including isoflavone, flavone, chalcone, anthocyanidin.They play diverse roles in regulation of auxin transport, modulation of flower color,plant–microbe interactions, and have direct but complex effects on human health. Theresearch of flavonoids biosynthesis pathway plays important roles in plant defenseresponse and improving functional components of food.
In all higher plants, flavonoids are produced through a branch of the phenylpropanoidpathway. At present, the research for phenylpropanoid pathway has been focus on the fieldof molecular biology, more and more coding genes in pathway has been cloned andidentified in soybean. On the other hand, the development of metabolic engineering,especially RNA interference technology, opens up new possibilities for the study ofregulation of plant secondary metabolite accumulation and their roles in plant defense.
In this study, two FNS Ⅱ genes from soybean cultivar Hefeng47were clonedaccording to basic local alignment search tool (BLAST) contexts using flavone synthasesequences reported in other species. These were named GmFNSⅡ-1and GmFNSⅡ-2.Sequence alignments showed that the cDNA of GmFNSⅡ-1was identical to that ofCYP93B16, whereas GmFNSⅡ-2was clearly distinct. Both GmFNSⅡ genes wereanalysized by bioinformatics in their genome sequences, the nucleotide sequence, aminoacid sequences, physical and chemical properties of protein, and function.
Functional assays in Escherichia coli and yeast (Schizosaccharomyces pombe)suggested that these two enzymes could convert (2S)-naringenin into apigenin. The twoGmFNSⅡ genes had similar tissue-specific expression patterns, and were significantlyincreased by osmotic stress like glucose and MeJA treatment. This demonstrates that the gene plays an important role in the response to defense signals in soybean.
Here, a bivalent RNA interference (RNAi) plant-transformation vector wasconstructed to silence both the flavanone3-hydroxylase (F3H) gene and the flavonesynthase Ⅱ (GmFNSⅡ) gene. Moreover, two further unit RNAi vectors were constructedfor each of these two genes.
RNAi-mediated suppression of those genes showed a significant reduction (to <10%)in gene expression in hairy roots that arose from soybean cotyledons transformed withAgrobacterium rhizogenes (ATCC15834). HPLC showed in hairy roots，RNAi-mediatedsuppression of these genes effectively regulated flavone and isoflavone production.Notably, the bivalent RNAi vector had a significantly greater effect for increasingisoflavone production compared with the two unit RNAi vectors.
This present study highlights molecular methods that can be used to enhanceisoflavone production in soybean, and demonstrates the challenges associated with suchmetabolic engineering for the production of plant natural products.
In this study, we tested two flavonoids, apigenin and genistein, in amended culturemedia against seven soybean pathogens including Colletotrichum truncatum,Macrophomina phaseolina, Phoma exigua, Phytophthora sojae, Pythium ultimum,Rhizoctonia solani, and Sclerotinia sclerotiorum. Both compounds restricted growth of allseven pathogens, although not all equally. For example, inhibition of growth was less (P>0.05) for C. truncatum than the other pathogens tested. There also was a dosage effect asincreased concentrations of either compound further restricted colony growth. The resultssuggest that through metabolic engineering, apigenin and genistein may be useful targetsfor overexpression in planta to enhance disease resistance in soybean.
引文
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