苦荞黄酮合成相关酶基因的克隆、芽期逆境胁迫中的应答及重组FtPAL和FtFLS的酶学活性研究
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摘要
苦荞是一种富含芦丁(槲皮素3-O-rutinoside)的药食两用小杂粮,因其具有降脂、降糖和抗氧化等保健作用而成为研究热点。现有研究集中在苦荞产品的开发、黄酮种类的鉴定和黄酮合成相关基因的克隆方面。但由于苦荞黄酮合成途径较为复杂,大多数参与黄酮(芦丁)合成相关基因在苦荞中并未得以克隆、生物学活性未得到验证、其参与逆境胁迫的研究较为薄弱。本研究通过同源克隆和RACE技术,克隆获得11个苦荞黄酮合成相关基因ORF序列；通过不同逆境胁迫,研究其黄酮合成相关基因表达与苦荞总黄酮含量的关系,分析影响苦荞总黄酮含量的关键基因；采用原核表达技术,重点分析了关键酶基因FtPAL和FtFLS编码蛋白的酶学特性。主要实验结果如下：
1.以苦荞花总RNA为材料,采用同源克隆、RT-PCR和RACE技术克隆了苦荞11个黄酮合成相关酶基因的ORF序列和1个基因的保守片段：获得苯丙氨酸解氨酶基因(FtPAL)、肉桂酸4-羟化酶基因(FtC4H)、4-香豆酰CoA连接酶基因(Ft4CL)查尔酮合酶基因(FtCHS)、查尔酮异构酶基因(FtCHI)和黄烷酮3-羟化酶基因(FtF3H)等6个黄酮合成途径上游酶基因,类黄酮3'-羟化酶基因(FtF3'H)和黄酮醇合酶基因(FtFLS)等2个黄酮醇合成支路酶基因,以及二氢黄酮醇4-还原酶基因(FtDFR)和花青素合酶基因(FtANS)等2个花青素合成支路酶基因的ORF序列；获得参与黄酮转运的谷胱甘肽转移酶基因(FtGST)的ORF序列；获得类黄酮3-葡糖苷转移酶基因(3GT)的序列片段。
2.序列分析表明,本研究所获得的11个苦荞黄酮合成相关基因分属于不同的酶蛋白家族,为典型的植物黄酮合成相关基因。而获得的3GT序列片段出现翻译提前终止的现象,可能为假基因。11个基因ORF编码的氨基酸序列与金荞和甜荞相应基因同源性均在90%以上,与其它植物同源性在38-95%之间。信号肽和亚细胞定位预测表明,所得序列均无信号肽：FtCHI定位于线粒体、FtC4H定位于内质网,其它9个酶定位于细胞质与黄酮在细胞中的合成部位一致。二级结构和三级结构预测表明,所得基因均具有相应酶蛋白典型的高级结构。
3.以萌发12d的芽期苦荞为材料,分别采用UV-B、寒冷和干旱进行胁迫8h处理。半定量RT-PCR表明,经不同胁迫处理前后FtPAL、FtCHS、FtCHI、FtF3H、 FtFLS、FtDFR和FtANS均在子叶中表达；FtPAL、FtCHS、FtCHI、FtF3H、FtDFR和FtANS均在胚轴中表达,而未检测到FtFLS的表达。统计学分析结果显示,UV-B胁迫条件下,FtF3H在子叶中、FtCHI在胚轴中的表达与对照差异不显著(P>0.05)；冷胁迫条件下,FtCHS在子叶中、FtF3H在胚轴中的表达与对照差异不显著(P>0.05)；其它基因在不同条件下胁迫8h后,其表达均处于显著水平(P<0.05)。结果表明,不同的基因表达具有组织特异性且在不同胁迫条件下表达模式有所差异,其中寒冷胁迫可诱导多数基因在子叶和胚轴中的上调表达。
4.总黄酮测定结果表明,苦荞总黄酮主要分布于子叶中,胚轴含量较低；因胁迫条件的不同,胁迫后总黄酮含量变化趋势有所差异。其中,UV-B胁迫能在短时间内(2h)引起芽期苦荞子叶和胚轴总黄酮含量的显著提高(P<0.05),且其差异随着诱导时间的延长而逐渐上升。寒冷胁迫则在长时间(8h)后引起子叶总黄酮的显著提高(P<0.05),在短时间内(2h)引起胚轴总黄酮的显著提高(P<0.05)。干旱胁迫对苦荞子叶总黄酮影响较小(P>0.05),但胚轴对该胁迫响应在处理4h后差异达到最大(P<0.05)。其中,苦荞子叶经寒冷胁迫后总黄酮含量最高可达7.40%,胚轴经干旱胁迫后总黄酮含量最高可达1.99%。
5.分析不同胁迫处理前后以及处理过程中,苦荞子叶和胚轴中总黄酮含量和基因表达量变化的相关性表明,1)苦荞子叶在UV-B和寒冷胁迫下,总黄酮含量的升高可能主要由于芦丁的合成和积累；胚轴在3种不同胁迫条件下,总黄酮的升高可能是由于花青素的合成和积累所至。2)子叶和胚轴中,FtPAL可能由单独的调节因素所控制并作为芽期苦荞黄酮合成的初级开关,参与逆境胁迫响应,并从黄酮合成途径代谢流源头上调控其的合成。子叶总黄酮含量的提高与FtFLS的表达相关,胚轴中FtDFR和FtANS可能采用协同表达的方式,调控花青素的合成和积累并引起总黄酮含量的提高。
6.Southern blot鉴定结果表明,FtPAL在苦荞基因组中为多拷贝。采用原核表达技术实现了FtPAL在大肠杆菌中的融合表达,并研究其催化特性。SDS-PAGE分析表明,重组FtPAL蛋白分子量约77KDa。酶活测定结果表明,诱导5h后FtPAL比活力达到35.7IU/g。薄层层析(TLC)结果表明,重组FtPAL具有将L-Phe转化为反式肉桂酸的催化活性。
7.Southern blot鉴定结果表明,FtFLS在苦荞基因组中为多拷贝。采用原核表达技术实现了FtFLS在大肠杆菌中的融合表达,并研究其催化特性。SDS-PAGE分析表明,重组FtFLS蛋白分子量约40KDa。经His亲和层析纯化后,重组FtFLS蛋白浓度为1.93g/L。以二氢槲皮素为底物,FtFLS的总活力和比活力分别为36.55×10-3IU和18.94×10-3IU/mg；以二氢山奈酚为底物,FtFLS总活力和比活力分别为10.19×10-3IU和5.28×10-3IU/mg。可见,重组FtFLS具有催化二氢槲皮素转化为槲皮素的活性大于催化二氢山奈酚转化为山奈酚的特性。
Tartary buckwheat (Fagopyrum tataricum Garetn) is an excellent medicinal and nutrient-rich crop containing abundance of rutin, which is a kind of flavonol glycoside, and it has become one of the hottest research points for its health benefits of lipid-lowering, hypoglycemic and antioxidant activity. Nowadays, researchers have concentrated on three aspects about Tartary buckwheat:the development of flavonoids-rich products, the identification of flavonoids types, and the enzymes of flavonoids biosynthesis. However, the flavonoids biosynthetic pathway in Tartary buckwheat is more complex, and most of the flavonoids-related genes have not been cloned, biological activity has not been verified, and the stress research is relatively weak.
Due to the pivotal role of enzyme genes in flavonoids biosynthesis, our study focused on the cloning and molecular characterization of functional flavonoids-related genes from Tartary buckwheat. Thus,11ORF sequences of flavonoids-related genes were cloned from Tartary buckwheat. By analyzing the relationships between total flavonoids content and expression level of genes under different stress treatments, the key enzyme genes were found. Two key enzyme genes were expressed by prokaryotic expression, and their enzymatic characteristics were identified.
1. Using the cDNA from flowers as template,11ORF sequences and a conserved fragment of flavonoid-related genes were isolated from Tartary buckwheat, including phenylalanine ammonia-lyase (FtPAL), cinnamate-4-hydroxylase (FtC4H),4-coumarate coenzyme A ligase (Ft4CL), chalcone synthase (FtCHS), chalcone isomerase (FtCHI), flavanone3-hydroxylase (FtF3H), flavonoid3'-hydroxylase (FtF3'H), flavonol synthase (FtFLS), dihydroflavonol-4-reductase (FtDFR), anthocyanidin synthase (FtANS), and glutathione S-transferases (FtGST), and a fragment sequence was flavonoid3-glucosyl transferase(3GT).
2. Sequence analysis showed that the ORF sequences of11flavonoids-related genes encoded complete enzyme proteins, and the fragment of3GT might be a pseudogene, which appeared early termination in translation. The deduced amino acid sequence of11flavonoids-related genes showed a higher level of homology (more than90%) with the corresponded genes in Fagopyrum dibotrys and Fagopyrum esculentum, and a moderate level with other plants'genes, ranging from38-95%. Signal peptides and subcellular localization prediction showed that all the11sequences had no signal peptide existed; FtCHI located in the mitochondria, FtC4H located in the endoplasmic reticulum, and the other9genes located in the cytoplasm. Senior Structural analysis showed that the each flavonoids-related gene had similar secondary and tertiary structure with its own protein family.
3. Three different stress treatments, including UV-B, cold and drought stress, were treated with the12days old sprouts for8hours. The expression levels of FtPAL, FtCHS, FtCHI, FtF3H, FtFLS, FtDFR, and FtANS were tested by semi-quantitative RT-PCR. Results showed that FtPAL, FtCHS, FtCHI, FtF3H, FtFLS, FtDFR and FtANS could be examined in the cotyledons before and after different stress treatments. FtPAL, FtCHS, FtCHI, FtF3H, FtDFR and FtANS could be examined in the hypocotyls before and after different stress treatments. However, the expression level of FtFLS could not be detected in the hypocotyls before and after stresses. The statistical analysis results showed that, in UV-B stress condition, the expression levels of FtF3H in cotyledons and FtCHI in hypocotyls showed no significant difference with control (P>0.05). In cold stress condition, the expression levels of FtCHS in cotyledons and FtF3H in hypocotyls showed no significant difference with control (P>0.05). The expression levels of other genes in3different stress conditions showed significant differences with control (P<0.05). Thus, our results suggested that each gene expression patterns vary in different stress conditions, with a certain degree of tissue specificity, and the cold stress could up-regulate most of the genes both in cotyledons and hypocotyls.
4. Total flavonoids in Tartary buckwheat were mainly distributed in the cotyledons. The contents and change trends of total flavonoids in Tartary buckwheat showed different degrees, which were accorded to different stress treatments. Statistical analysis results show that, with in a short period of treatment's time (2h), the total flavonoids in cotyledons and hypocotyls could be induced and significantly increased by UV-B stress (P <0.05), and the significance were gradually increased along with the induction time. In cold stress, the total flavonoids in cotyledons were significantly increased after8hours' induction (P<0.05), and it was significantly increased in hypocotyls after2hours' induction (P<0.05). In drought stress condition, the total flavonoids in cotyledons could not show a certain significance after8hours' treatment (P>0.05), and it had a short response time (2h) in hypocotyls and showed a maximum after4hours'induction (P<0.05). In addition, the maximum of total flavonoids were detected in cotyledons by8hours'cold treatment and in hypocotyls by4hours'drought treatment.
5. By analyzing the relationships among total flavonoids content and expression level of genes under different stress treatments, we speculated that:1) In UV-B and cold stresses, the increase of total flavonoids might due to the rutin biosynthesis in cotyledons, and in3different stresses, the increase of total flavonoids might contribute to the accumulation of anthocyanins in hypocotyls.2) FtPAL might be controlled by some separate adjustment factors in Tartary Buckwheat and be a primary switch in flavonoids biosynthetic pathway. Its expression level of response to different stress condition might control the starting of flavonoids biosynthesis and affect the accumulation patterns of total flavonoids both in cotyledons and hypocotyls. The expression level of FtFLS might correspond with the accumulation and increase of total flavonoids in cotyledons, and the potential co-expression of FtDFR and FtANS might control the biosynthesis of anthocyanins in hypocotyls and cause the rising trend of total flavonoids in hypocotyls.
6. Southern blot analysis indicated that FtPAL was a multi-copy gene in Tartary buckwheat. A non-fusion expression vector (named pET-30b(+)-FtPAL) was successfully constructed to prokaryotic express the recombinant FtPAL protein in Escherichia coli BL21(DE3). Induced by IPTG for7hours, the SDS-PAGE result showed that the recombinant FtPAL was about77kDa. The enzymatic activity of recombinant FtPAL was tested and results showed that FtPAL reached maximum yield at5hours'cultivation and its specific activity up to35.7IU/g by using L-Phe as substrate. TLC results suggested that the recombinant FtPAL could conversion L-Phe to trans-cinnamate in vitro.
7. Southern blot analysis indicated that FtFLS was a multi-copy gene in Tartary buckwheat. A fusion expression vector (named pET-30b(+)-FtFLS) was successfully constructed to prokaryotic express the recombinant FtFLS protein in Escherichia coli BL21(DE3). After10hours of induction, the SDS-PAGE result showed that the recombinant FtFLS was about40kDa. His affinity chromatography results showed that the concentration of purified FtFLS was1.93g/L. Enzymatic activity of FtFLS showed that the total and specific activities of dihydroquercetin were36.55×10-3IU and18.94×10-3IU/mg, respectively, and the total and specific activities of dihydrokaempferol were10.19×10-3IU and5.28×10-3IU/mg. Thus, our study suggested that the recombinant FtFLS had a higher catalytic efficiency of dihydroquercetin than dihydrokaempferol in vitro.
1.以苦荞花总RNA为材料,采用同源克隆、RT-PCR和RACE技术克隆了苦荞11个黄酮合成相关酶基因的ORF序列和1个基因的保守片段：获得苯丙氨酸解氨酶基因(FtPAL)、肉桂酸4-羟化酶基因(FtC4H)、4-香豆酰CoA连接酶基因(Ft4CL)查尔酮合酶基因(FtCHS)、查尔酮异构酶基因(FtCHI)和黄烷酮3-羟化酶基因(FtF3H)等6个黄酮合成途径上游酶基因,类黄酮3'-羟化酶基因(FtF3'H)和黄酮醇合酶基因(FtFLS)等2个黄酮醇合成支路酶基因,以及二氢黄酮醇4-还原酶基因(FtDFR)和花青素合酶基因(FtANS)等2个花青素合成支路酶基因的ORF序列；获得参与黄酮转运的谷胱甘肽转移酶基因(FtGST)的ORF序列；获得类黄酮3-葡糖苷转移酶基因(3GT)的序列片段。
2.序列分析表明,本研究所获得的11个苦荞黄酮合成相关基因分属于不同的酶蛋白家族,为典型的植物黄酮合成相关基因。而获得的3GT序列片段出现翻译提前终止的现象,可能为假基因。11个基因ORF编码的氨基酸序列与金荞和甜荞相应基因同源性均在90%以上,与其它植物同源性在38-95%之间。信号肽和亚细胞定位预测表明,所得序列均无信号肽：FtCHI定位于线粒体、FtC4H定位于内质网,其它9个酶定位于细胞质与黄酮在细胞中的合成部位一致。二级结构和三级结构预测表明,所得基因均具有相应酶蛋白典型的高级结构。
3.以萌发12d的芽期苦荞为材料,分别采用UV-B、寒冷和干旱进行胁迫8h处理。半定量RT-PCR表明,经不同胁迫处理前后FtPAL、FtCHS、FtCHI、FtF3H、 FtFLS、FtDFR和FtANS均在子叶中表达；FtPAL、FtCHS、FtCHI、FtF3H、FtDFR和FtANS均在胚轴中表达,而未检测到FtFLS的表达。统计学分析结果显示,UV-B胁迫条件下,FtF3H在子叶中、FtCHI在胚轴中的表达与对照差异不显著(P>0.05)；冷胁迫条件下,FtCHS在子叶中、FtF3H在胚轴中的表达与对照差异不显著(P>0.05)；其它基因在不同条件下胁迫8h后,其表达均处于显著水平(P<0.05)。结果表明,不同的基因表达具有组织特异性且在不同胁迫条件下表达模式有所差异,其中寒冷胁迫可诱导多数基因在子叶和胚轴中的上调表达。
4.总黄酮测定结果表明,苦荞总黄酮主要分布于子叶中,胚轴含量较低；因胁迫条件的不同,胁迫后总黄酮含量变化趋势有所差异。其中,UV-B胁迫能在短时间内(2h)引起芽期苦荞子叶和胚轴总黄酮含量的显著提高(P<0.05),且其差异随着诱导时间的延长而逐渐上升。寒冷胁迫则在长时间(8h)后引起子叶总黄酮的显著提高(P<0.05),在短时间内(2h)引起胚轴总黄酮的显著提高(P<0.05)。干旱胁迫对苦荞子叶总黄酮影响较小(P>0.05),但胚轴对该胁迫响应在处理4h后差异达到最大(P<0.05)。其中,苦荞子叶经寒冷胁迫后总黄酮含量最高可达7.40%,胚轴经干旱胁迫后总黄酮含量最高可达1.99%。
5.分析不同胁迫处理前后以及处理过程中,苦荞子叶和胚轴中总黄酮含量和基因表达量变化的相关性表明,1)苦荞子叶在UV-B和寒冷胁迫下,总黄酮含量的升高可能主要由于芦丁的合成和积累；胚轴在3种不同胁迫条件下,总黄酮的升高可能是由于花青素的合成和积累所至。2)子叶和胚轴中,FtPAL可能由单独的调节因素所控制并作为芽期苦荞黄酮合成的初级开关,参与逆境胁迫响应,并从黄酮合成途径代谢流源头上调控其的合成。子叶总黄酮含量的提高与FtFLS的表达相关,胚轴中FtDFR和FtANS可能采用协同表达的方式,调控花青素的合成和积累并引起总黄酮含量的提高。
6.Southern blot鉴定结果表明,FtPAL在苦荞基因组中为多拷贝。采用原核表达技术实现了FtPAL在大肠杆菌中的融合表达,并研究其催化特性。SDS-PAGE分析表明,重组FtPAL蛋白分子量约77KDa。酶活测定结果表明,诱导5h后FtPAL比活力达到35.7IU/g。薄层层析(TLC)结果表明,重组FtPAL具有将L-Phe转化为反式肉桂酸的催化活性。
7.Southern blot鉴定结果表明,FtFLS在苦荞基因组中为多拷贝。采用原核表达技术实现了FtFLS在大肠杆菌中的融合表达,并研究其催化特性。SDS-PAGE分析表明,重组FtFLS蛋白分子量约40KDa。经His亲和层析纯化后,重组FtFLS蛋白浓度为1.93g/L。以二氢槲皮素为底物,FtFLS的总活力和比活力分别为36.55×10-3IU和18.94×10-3IU/mg；以二氢山奈酚为底物,FtFLS总活力和比活力分别为10.19×10-3IU和5.28×10-3IU/mg。可见,重组FtFLS具有催化二氢槲皮素转化为槲皮素的活性大于催化二氢山奈酚转化为山奈酚的特性。
Tartary buckwheat (Fagopyrum tataricum Garetn) is an excellent medicinal and nutrient-rich crop containing abundance of rutin, which is a kind of flavonol glycoside, and it has become one of the hottest research points for its health benefits of lipid-lowering, hypoglycemic and antioxidant activity. Nowadays, researchers have concentrated on three aspects about Tartary buckwheat:the development of flavonoids-rich products, the identification of flavonoids types, and the enzymes of flavonoids biosynthesis. However, the flavonoids biosynthetic pathway in Tartary buckwheat is more complex, and most of the flavonoids-related genes have not been cloned, biological activity has not been verified, and the stress research is relatively weak.
Due to the pivotal role of enzyme genes in flavonoids biosynthesis, our study focused on the cloning and molecular characterization of functional flavonoids-related genes from Tartary buckwheat. Thus,11ORF sequences of flavonoids-related genes were cloned from Tartary buckwheat. By analyzing the relationships between total flavonoids content and expression level of genes under different stress treatments, the key enzyme genes were found. Two key enzyme genes were expressed by prokaryotic expression, and their enzymatic characteristics were identified.
1. Using the cDNA from flowers as template,11ORF sequences and a conserved fragment of flavonoid-related genes were isolated from Tartary buckwheat, including phenylalanine ammonia-lyase (FtPAL), cinnamate-4-hydroxylase (FtC4H),4-coumarate coenzyme A ligase (Ft4CL), chalcone synthase (FtCHS), chalcone isomerase (FtCHI), flavanone3-hydroxylase (FtF3H), flavonoid3'-hydroxylase (FtF3'H), flavonol synthase (FtFLS), dihydroflavonol-4-reductase (FtDFR), anthocyanidin synthase (FtANS), and glutathione S-transferases (FtGST), and a fragment sequence was flavonoid3-glucosyl transferase(3GT).
2. Sequence analysis showed that the ORF sequences of11flavonoids-related genes encoded complete enzyme proteins, and the fragment of3GT might be a pseudogene, which appeared early termination in translation. The deduced amino acid sequence of11flavonoids-related genes showed a higher level of homology (more than90%) with the corresponded genes in Fagopyrum dibotrys and Fagopyrum esculentum, and a moderate level with other plants'genes, ranging from38-95%. Signal peptides and subcellular localization prediction showed that all the11sequences had no signal peptide existed; FtCHI located in the mitochondria, FtC4H located in the endoplasmic reticulum, and the other9genes located in the cytoplasm. Senior Structural analysis showed that the each flavonoids-related gene had similar secondary and tertiary structure with its own protein family.
3. Three different stress treatments, including UV-B, cold and drought stress, were treated with the12days old sprouts for8hours. The expression levels of FtPAL, FtCHS, FtCHI, FtF3H, FtFLS, FtDFR, and FtANS were tested by semi-quantitative RT-PCR. Results showed that FtPAL, FtCHS, FtCHI, FtF3H, FtFLS, FtDFR and FtANS could be examined in the cotyledons before and after different stress treatments. FtPAL, FtCHS, FtCHI, FtF3H, FtDFR and FtANS could be examined in the hypocotyls before and after different stress treatments. However, the expression level of FtFLS could not be detected in the hypocotyls before and after stresses. The statistical analysis results showed that, in UV-B stress condition, the expression levels of FtF3H in cotyledons and FtCHI in hypocotyls showed no significant difference with control (P>0.05). In cold stress condition, the expression levels of FtCHS in cotyledons and FtF3H in hypocotyls showed no significant difference with control (P>0.05). The expression levels of other genes in3different stress conditions showed significant differences with control (P<0.05). Thus, our results suggested that each gene expression patterns vary in different stress conditions, with a certain degree of tissue specificity, and the cold stress could up-regulate most of the genes both in cotyledons and hypocotyls.
4. Total flavonoids in Tartary buckwheat were mainly distributed in the cotyledons. The contents and change trends of total flavonoids in Tartary buckwheat showed different degrees, which were accorded to different stress treatments. Statistical analysis results show that, with in a short period of treatment's time (2h), the total flavonoids in cotyledons and hypocotyls could be induced and significantly increased by UV-B stress (P <0.05), and the significance were gradually increased along with the induction time. In cold stress, the total flavonoids in cotyledons were significantly increased after8hours' induction (P<0.05), and it was significantly increased in hypocotyls after2hours' induction (P<0.05). In drought stress condition, the total flavonoids in cotyledons could not show a certain significance after8hours' treatment (P>0.05), and it had a short response time (2h) in hypocotyls and showed a maximum after4hours'induction (P<0.05). In addition, the maximum of total flavonoids were detected in cotyledons by8hours'cold treatment and in hypocotyls by4hours'drought treatment.
5. By analyzing the relationships among total flavonoids content and expression level of genes under different stress treatments, we speculated that:1) In UV-B and cold stresses, the increase of total flavonoids might due to the rutin biosynthesis in cotyledons, and in3different stresses, the increase of total flavonoids might contribute to the accumulation of anthocyanins in hypocotyls.2) FtPAL might be controlled by some separate adjustment factors in Tartary Buckwheat and be a primary switch in flavonoids biosynthetic pathway. Its expression level of response to different stress condition might control the starting of flavonoids biosynthesis and affect the accumulation patterns of total flavonoids both in cotyledons and hypocotyls. The expression level of FtFLS might correspond with the accumulation and increase of total flavonoids in cotyledons, and the potential co-expression of FtDFR and FtANS might control the biosynthesis of anthocyanins in hypocotyls and cause the rising trend of total flavonoids in hypocotyls.
6. Southern blot analysis indicated that FtPAL was a multi-copy gene in Tartary buckwheat. A non-fusion expression vector (named pET-30b(+)-FtPAL) was successfully constructed to prokaryotic express the recombinant FtPAL protein in Escherichia coli BL21(DE3). Induced by IPTG for7hours, the SDS-PAGE result showed that the recombinant FtPAL was about77kDa. The enzymatic activity of recombinant FtPAL was tested and results showed that FtPAL reached maximum yield at5hours'cultivation and its specific activity up to35.7IU/g by using L-Phe as substrate. TLC results suggested that the recombinant FtPAL could conversion L-Phe to trans-cinnamate in vitro.
7. Southern blot analysis indicated that FtFLS was a multi-copy gene in Tartary buckwheat. A fusion expression vector (named pET-30b(+)-FtFLS) was successfully constructed to prokaryotic express the recombinant FtFLS protein in Escherichia coli BL21(DE3). After10hours of induction, the SDS-PAGE result showed that the recombinant FtFLS was about40kDa. His affinity chromatography results showed that the concentration of purified FtFLS was1.93g/L. Enzymatic activity of FtFLS showed that the total and specific activities of dihydroquercetin were36.55×10-3IU and18.94×10-3IU/mg, respectively, and the total and specific activities of dihydrokaempferol were10.19×10-3IU and5.28×10-3IU/mg. Thus, our study suggested that the recombinant FtFLS had a higher catalytic efficiency of dihydroquercetin than dihydrokaempferol in vitro.
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