观赏羽衣甘蓝BoDFR基因的克隆及表达分析  

Cloning and Expression Analysis of DFR in Ornamental Kale(Brassica oleracea L.var.acephala)

作　　者：王玉书[1,2] 杨旭妍 付震 夏爽 段钰萌 罗大森 曹蕾 WANG Yushu;YANG Xuyan;FU Zhen;XIA Shuang;DUAN Yumeng;LUO Dasen;CAO Lei(College of Life Sciences, Agriculture and Forestry, Qiqihaer University, Qiqihaer,Heilongjiang 161006, China;Heilongjiang Provincial Key Laboratory of Resistance Gene Engineering and Preservation of Biodiversity in Cold Areas, Qiqihaer,Heilongjiang 161006, China;Inspection and Testing Certification Center of Anshan City,Anshan,Liaoning 114000, China)

机构地区：[1]齐齐哈尔大学生命科学与农林学院,黑龙江齐齐哈尔161006 [2]黑龙江省抗性基因工程与寒地生物多样性保护重点实验室,黑龙江齐齐哈尔161006 [3]鞍山市检验检测认证中心,辽宁鞍山114000

出　　处：《西北植物学报》2020年第9期1483-1489,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31401908);黑龙江省自然科学基金(C2016056);黑龙江省普通高等学校青年创新人才培养计划(UNPYSCT-2017155);黑龙江省省属高等学校基本科研业务费(135209313);植物性食品加工技术特色学科专项(YSTSXK201888);齐齐哈尔大学大学生创新创业项目(201910232346,201910232118,202010232104,202010232197,202010232801)。

摘　　要：为揭示羽衣甘蓝二氢黄酮醇4-还原酶(DFR)基因调控花青素合成的功能,该研究对不同叶色羽衣甘蓝的叶片花青素含量进行测定,根据结球甘蓝DFR序列信息,利用RT-PCR技术克隆羽衣甘蓝BoDFR基因并进行实时荧光定量表达分析。结果表明:BoDFR的cDNA全长为1158 bp,编码385个氨基酸,其蛋白相对分子质量为42925.06 Da,预测亚细胞定位为细胞质;蛋白质二级结构分析表明α-螺旋和无规则卷曲为DFR蛋白的主要二级结构元件。序列比对显示DFR蛋白具有NADPH结合位点和底物结合位点,属于NADB-Rossmann超基因家族。系统进化分析表明,BoDFR与结球甘蓝(Brassica oleracea var.capitata)DFR亲缘关系最近。花青素含量测定显示,紫叶羽衣甘蓝叶片中花青素含量最高,粉叶羽衣甘蓝含量较高,而白叶羽衣甘蓝叶片中检测不到花青素。实时荧光定量PCR分析表明,BoDFR基因表达量与花青素含量高低一致,其中紫叶羽衣甘蓝叶片中BoDFR基因的表达量最高,而白叶羽衣甘蓝心叶中仅微量表达。In order to reveal the function of dihydroflavonol 4-reductase(DFR)gene in the regulation of anthocyanin synthesis of kale,we determined the anthocyanin content of kale leaves with different colors.Meanwhile,according to the DFR sequence information of cabbage,we cloned the BoDFR gene of kale by RT-PCR and analyzed by real-time fluorescence quantitative expression.The results showed that:the cDNA sequence of BoDFR was 1158 bp in length and corresponding to 385 amino acid residues,and the relative molecular weight of coding protein was 42925.06 Da.The subcellular localization most likely be cytoplasm.Results of secondary structure analysis exhibited thatα-helix and random coil were primary secondary structural components of the DFR protein.Sequence alignment showed that DFR protein has a NADPH binding domain and substrate specific binding domain,belonging to the NADB-Rossmann supergene family.Phylogenetic analysis showed that BoDFR had the closest relationship with DFR of cabbage(Brassica oleracea var.capitata).Results of anthocyanins measurement showed that the highest contents of anthocyanins were detected in purple leaves,the higher content of pink leaves,while no anthocyanins were detected in white leaves.Real-time fluorescence quantitative PCR analysis showed that the expression of BoDFR was positively correlated with the change of anthocyanin content.The highest expression was found in purple-leaf kale,but very weak expression was detected in white-leaf kale.

关 键 词：羽衣甘蓝 花青素 二氢黄酮醇4-还原酶(DFR)基因 基因克隆 基因表达 

分 类 号：Q786[生物学—分子生物学] Q789