不结球白菜类胡萝卜素裂解双加氧酶基因BrCCD4a的克隆及表达分析  

Cloning and expression analysis of carotenoid cleavage dioxygenase gene BrCCD4a in non-heading Chinese cabbage

作　　者：尹莲 孙玉东[1,2] 陈龙正 罗德旭[1,2] 任旭琴[4] 王林闯 许文钊 于雯雯[4] 刘璐 赵建锋 YIN Lian;SUN Yu-dong;CHEN Long-zheng;LUO De-xu;REN Xu-qin;WANG Lin-chuang;XU Wen-zhao;YU Wen-wen;LIU Lu;ZHAO Jian-feng(Huaiyin Institute of Agricultural Sciences of the Xuhuai District of Jiangsu Province,Huaian 223001,China;Huaian Key Laboratory for Facility Vegetables,Huaian 223001,China;Institute of Vegetable,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;School of Life Science and Food Engineering,Huaiyin Institute of Technology,Huaian 223003,China)

机构地区：[1]江苏徐淮地区淮阴农业科学研究所,江苏淮安223001 [2]淮安市设施蔬菜重点实验室,江苏淮安223001 [3]江苏省农业科学院蔬菜研究所,江苏南京210014 [4]淮阴工学院生命科学与食品工程学院,江苏淮安223003

出　　处：《江苏农业学报》2023年第6期1386-1394,共9页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省农业科技自主创新基金项目[CX(21)2020]。

摘　　要：为探究CCD基因在不结球白菜叶片类胡萝卜素调控中的作用,本试验以不结球白菜NN2108为试验材料,克隆获得BrCCD4a基因。应用生物信息学方法对BrCCD4a蛋白氨基酸序列、理化性质及CCD4基因亲缘关系等方面进行分析,利用亚细胞定位技术明确BrCCD4a基因的空间表达。此外,以3个不结球白菜品种HB02、SX04和NN2108为材料,测定叶片中类胡萝卜素含量及BrCCD4a基因的相对表达水平。结果表明,BrCCD4a含有1个长为1 797 bp的开放阅读框(ORF),编码598个氨基酸,其编码的蛋白质相对分子质量为65 860,等电点为6.42。氨基酸序列分析结果表明,BrCCD4a属于CCD蛋白家族成员,与甘蓝CCD4氨基酸序列相似度最高。系统进化结果也显示其与甘蓝的进化关系最近。BrCCD4a蛋白主要由无规则卷曲、延伸链构成,且不存在信号肽。亚细胞定位分析发现BrCCD4a定位在叶绿体上。不同不结球白菜品种叶片中叶黄素和β-胡萝卜素含量差异较大,HB02叶片中叶黄素含量最低,为1.14 mg/g, SX04叶片中叶黄素含量最高,达2.34 mg/g。NN2108叶片中β-胡萝卜素含量最高,达2.31 mg/g,是HB02含量的1.94倍。BrCCD4a在3个品种叶片中的相对表达水平趋势与β-胡萝卜素含量趋势一致,在NN2108叶片中表达量最高,在SX04中次之,在HB02叶片中表达量最低。本研究结果为探究BrCCD4a在不结球白菜叶片类胡萝卜素降解过程中的作用提供理论基础。In order to explore the role of CCD gene on the carotenoid regulation in non-heading Chinese cabbage leaf blades,the BrCCD4a gene was cloned from experimental material NN2108.The amino acid sequence,physicochemical properites of BrCCD4a protein and genetic relationship of CCD4 were analyzed with bioinformatics methods.The spatial expression of BrCCD4a was clarified by subcellular localization technology.In addition,three varieties,HB02,SX04 and NN2108,were used to determine the carotenoid content and the relative expression level of the BrCCD4a gene.The results showed that BrCCD4a gene contained an open reading frame(ORF)with a length of 1797 bp,encoding 598 amino acids.The relative molecular mass of the protein encoded by BrCCD4a was 65860,and the isoelectric point was 6.42.BrCCD4a belonged to the CCD protein family,and had the highest similarity with the amino acid sequence of CCD4 protein in Brassica oleracea.Phylogenetic analysis also displayed that BrCCD4a had the closest evolutionary relationship with Brassica oleracea.BrCCD4a protein was mainly composed of random coils and extended chains,and there was no signal peptide.Subcellular localization analysis revealed that BrCCD4a was localized on chloroplasts.The lutein contents andβ-carotene contents in leaf blades of non-heading Chinese cabbage with different colors varied greatly.The content of lutein in the leaf blades of HB02 was the lowest(1.14 mg/g),and the content in the leaf blades of SX04 was the highest(2.34 mg/g).The content ofβ-carotene in the leaf blades of NN2108 was the highest,reaching 2.31 mg/g,which was 1.94 times higher than that of HB02.The results of relative expression analysis manifested that the trend of relative expression levels of BrCCD4a in leaf blades of the three cultivars was consistent with the trend ofβ-carotene content.The expression level of BrCCD4a was the highest in the leaf blades of NN2108,followed by SX04,and the lowest in HB02.The results of this study provide a basis for exploring the role of BrCCD4a in the degradat

关 键 词：不结球白菜 类胡萝卜素裂解双加氧酶4 类胡萝卜素 

分 类 号：Q344.13[生物学—遗传学]