摘要
花青素合酶(ANS)是植物花青素合成过程中的关键酶,可以将无色的花色苷催化成红色、橙色、蓝色等不同颜色的花青素。为了探究ANS基因调控菊苣叶色的分子机制,首先对该基因进行生物信息学分析,随后选取红色结球型菊苣(印地欧)和绿色菊苣(普那)为材料,克隆得到菊苣ANS基因(CiANS),并对该基因在这2个材料中的氨基酸序列及蛋白结构进行了差异分析,最后对ANS基因的组织特异表达和蛋白的亚细胞定位进行了检测,并对ANS蛋白进行了原核表达。结果显示,CiANS与莴苣及栽培菊苣的ANS亲缘关系最近,Motif分析发现,CiANS蛋白基序在不同植物中均较为保守。2种菊苣中ANS的克隆结果表明,ANS基因全长均为1068 bp,编码355个氨基酸,具有9个差异氨基酸,但在预测的三级结构上并未表现出明显的差异。qRT-PCR结果显示,CiANS基因在不同组织中均有表达,但在叶片中表达量最高,并且红色的结球菊苣中CiANS基因的表达水平显著高于绿色的普那菊苣。亚细胞定位结果显示,CiANS蛋白定位于细胞质和细胞核中。CiANS蛋白在原核载体上诱导表达后大小共为45 ku,与预期蛋白大小一致。综上所述,菊苣叶色差异的形成很可能与ANS基因的表达量差异有关,研究结果为解析菊苣叶色分子调控网络及花青素代谢遗传改良提供了理论依据。
Anthocyanin synthase(ANS)is a key enzyme in the anthocyanin biosynthesis pathway in plants,catalyzing the conversion of colorless leucoanthocyanidins into colorful anthocyanins such as red,orange,and blue.To investigate the molecular mechanism of ANS gene in leaf coloration regulation in chicory(Cichorium intybus),bioinformatics analysis of ANS was first conducted.Then,the red heading C.intybus(Indiou)and green forage variety(Puna)were selected as the materials to clone the C.intybus ANS(CiANS)gene.Differences in amino acid sequences and protein structures of CiANS between the two materials were analyzed.Finally,tissue-specific expression of the ANS gene and subcellular localization of CiANS were characterized,and prokaryotic expression of the CiANS was performed.The results showed that CiANS shared the closest phylogenetic relationship with ANS from lettuce(Lactuca sativa)and endive(C.endivia).Motif analysis revealed that the protein motifs of CiANS were relatively conserved across different plant species.Cloning results indicated that the full-length ANS gene in both chicory varieties was 1068 bp,encoding 355 amino acids with 9 divergent residues,though no significant differences were observed in predicted tertiary structures.qRT-PCR results demonstrated that CiANS was expressed in all tissues,with the highest expression level in leaves,and its expression in red heading Indiou was significantly higher than in green Puna.Subcellular localization revealed that CiANS protein was localized in both the cytoplasm and nucleus.After prokaryotic expression,the induced CiANS protein exhibited a molecular weight of 45 ku,consistent with the predicted size.In conclusion,the observed leaf color variation in C.intybus is likely associated with differential expression levels of the CiANS gene.This study provides theoretical insights for elucidating the molecular regulatory network of Cichorium intybus leaf coloration and genetic improvement of anthocyanin metabolism.
作者
冯清香
朱哈修
杜洋
徐艳茹
王晨晨
刘雪
李大勇
周军
张彬
FENG Qingxiang;ZHU Haxiu;DU Yang;XU Yanru;WANG Chenchen;LIU Xue;LI Dayong;ZHOU Jun;ZHANG Bin(Beijing Vegetable Research Center,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,China;College of Life Sciences,Shandong Normal University,Jinan 250300,China;College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650000,China;School of Grassland Science,Beijing Forestry University,Beijing 100083,China)
出处
《华北农学报》
北大核心
2025年第3期51-59,共9页
Acta Agriculturae Boreali-Sinica
基金
北京市农林科学院创新能力建设专项科技攻关项目(KJCX20251008)
现代农业产业技术体系北京市设施蔬菜创新团队建设项目(BAIC01-2024-04)
中央本级重大增减支项目(2060302)。
关键词
菊苣
花青素合酶
表达分析
亚细胞定位
原核表达
Cichorium intybus
Anthocyanin synthase
Expression analysis
Subcellular localization
Prokaryotic expression
