芥蓝二氢黄酮醇4–还原酶基因BoaDFR的克隆与功能鉴定

Cloning and Function Identification of Dihydroflavonol 4-Reductase Gene BoaDFR in Chinese Kale

以白花芥蓝‘四季粗条’为材料克隆了Boa DFR基因,Gen Bank登录号为MT472647,CDS序列长为1158 bp,编码385个氨基酸,与甘蓝型油菜、甘蓝和白菜等具有高度同源性。Boa DFR表达水平在芥蓝不同发育时期、不同器官中存在显著差异,随植株发育总体呈现先下降后上升的趋势,在幼嫩种子中表达量最高。芥蓝原生质体的亚细胞定位结果显示Boa DFR定位在细胞核上。构建了农杆菌介导的芥蓝瞬时过表达体系,将Boa DFR转入白花芥蓝‘四季粗条’和黄花芥蓝‘福州黄花’中,发现Boa DFR在两种芥蓝中均高水平表达,转基因植株叶片颜色明显变紫,花青素苷显著积累,总含量最高达461.43μg·g-1 FW,而野生型和转空载体芥蓝叶片中几乎检测不到花青素苷。通过HPLC在转基因植株叶片中共检测到8种花青素苷,均为矢车菊花青素苷,其中矢车菊–3–阿魏酰–丙二酰–槐糖苷–5–葡萄糖苷占比最高。

The Boa DFR was cloned from the cultivar‘Siji Cutiao’of white flower Chinese kale,and the Gen Bank accession number was MT472647.Boa DFR is 1158 bp-long CDS sequence,encoding 385 amino acids.Boa DFR has high homology with DFR of Brassica napus,B.oleracea and B.rapa.The expression level of Boa DFR differed significantly at different stages and organs,and generally showed a trend of decreasing first and then rising during the plant growth.The highest expression level of Boa DFR was in young seeds,which was significantly higher than other organs.The subcellular localization showed that Boa DFR was localized in the nucleus.The Agrobacterium-mediated transient overexpression system in Chinese kale was constructed,and Boa DFR was transferred into‘Siji Cutiao’and‘Fuzhou Huanghua’.It was found that Boa DFR can be expressed at high levels in both two cultivars and the leaves of transgenic plants obviously turned purple,the anthocyanin accumulated significantly and the highest content was up to 461.43μg·g-1 FW,whereas the anthocyanin almost could not be detected in both wild-type and empty vector plants.Furthermore,a total of eight anthocyanins were detected in the transgenic plant leaves by HPLC,all of which were cyanidins,and cyanidin 3-feruloylmalonylsophoroside-5-glucoside was predominant.