Heterologous Expression of Platycodon grandiflorus PgF3'5'H Modifies Flower Color Pigmentation in Tobacco.
Heterologous Expression of Platycodon grandiflorus PgF3'5'H Modifies Flower Color Pigmentation in Tobacco.
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黄曲霉素-3 ′，5 ′-羟化酶（Flavonylase-3′，5 ′-hydroxylase，F3′5′H）是花色素苷合成的关键酶。本研究从桔梗中克隆了一个PgF 3 ′5′H的全长cDNA序列，该序列与风铃草F3′5′H的同源性最高。本研究将PgF 3 ′5′H亚克隆到植物过表达载体中，通过农杆菌介导法转化烟草，研究其催化功能。经潮霉素抗性筛选和PCR检测，获得了阳性转基因烟草T0代植株。PgF 3 ′5′H在所有转基因烟草中的表达量均高于对照。在花椰菜花叶病毒（CaMV）35 S启动子的驱动下，过量表达的PgF 3 ′5′H产生二氢杨梅素（DHM）和一些新的花青素（包括飞燕草素、矮牵牛素、芍药素和锦葵素衍生物），以及增加的二氢山奈酚（DHK）、花旗松素、三趾草素、矢车菊素衍生物，超高效液相色谱-串联质谱法检测转PgF 3 ′5′H基因烟草中天竺葵素和天竺葵素衍生物通过超高效液相色谱-串联质谱（UPLC-MS/MS）分析，得到从浅粉色到洋红的显著颜色变化。这些结果表明PgF 3 ′5′H产物具有F3′5′H酶活性。此外，PgF 3 ′5′H转移还影响了烟草中黄酮类色素的合成和积累。因此，PgF 3 ′5′H可作为提高花色苷积累的基因工程和蓝色花分子育种的候选基因。
Flavonoid-3′,5′-hydroxylase (F3′5′H) is the key enzyme for the biosynthesis of delphinidin-based anthocyanins, which are generally required for purple or blue flowers. Previously, we isolated a full-length cDNA of PgF3′5′H from Platycodon grandiflorus, which shared the highest homology with Campanula medium F3′5′H. In this study, PgF3′5′H was subcloned into a plant over-expression vector and transformed into tobacco via Agrobacterium tumefaciens to investigate its catalytic function. Positive transgenic tobacco T0 plants were obtained by hygromycin resistance screening and PCR detection. PgF3′5′H showed a higher expression level in all PgF3′5′H transgenic tobacco plants than in control plants. Under the drive of the cauliflower mosaic virus (CaMV) 35S promoter, the over-expressed PgF3′5′H produced dihydromyricetin (DHM) and some new anthocyanin pigments (including delphinidin, petunidin, peonidin, and malvidin derivatives), and increased dihydrokaempferol (DHK), taxifolin, tridactyl, cyanidin derivatives, and pelargonidin derivatives in PgF3′5′H transgenic tobacco plants by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) analysis, resulting in a dramatic color alteration from light pink to magenta. These results indicate that PgF3′5′H products have F3′5′H enzyme activity. In addition, PgF3′5′H transfer alters flavonoid pigment synthesis and accumulation in tobacco. Thus, PgF3′5′H may be considered a candidate gene for gene engineering to enhance anthocyanin accumulation and the molecular breeding project for blue flowers.
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影响因子：
3.1
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影响因子：
2.4
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1.6
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