南京农业大学学报2025，Vol.48Issue(6)：1243-1251,9.DOI:10.7685/jnau.202406027
不结球白菜BcAP1的克隆及其启动子分析
Cloning and promoter analysis of BcAP1 in non-heading Chinese cabbage
摘要
Abstract
[Objectives]The paper aimed to investigate whether there were differences in the structure and expression of BcAP1 gene in the early and late flower varieties of non-heading Chinese cabbage.[Methods]By homologous cloning of BcAP1 gene and promoter sequence from early flowering variety'Caixin'and late flowering variety'Wuyueman'as experimental materials,amino acid sequences of the two varieties were compared and phylogenetic analysis was conducted,and the spatial expression of BcAP1 gene was studied by subcellular localization technology.Real-time quantitative fluorescent PCR(RT-qPCR)was used to analyze the expression of BcAP1 at the bud stage and flowering stage of the two varieties,and the activity of BcAP1 promoter in the two varieties was analyzed by double luciferase assay.Furthermore,the transgenic Arabidopsis thaliana was stained with β-D-glucuronidase(GUS)and the promoter insertion fragments of BcAP1 gene were predicted by PlantCARE online software.[Results]The BcAP1 CDS sequences of'Caixin'and'Wuyueman'were only different by 1 base,but the amino acid sequences translated from both ends were completely consistent.Phylogenetic analysis showed that BcAP1 had the highest homology with A.thaliana.Subcellular localization showed that the expression was located in the nucleus.The quantitative results showed that the BcAP1 gene expression of early bolting variety'Caixin'was significantly higher than that of late bolting variety'Wuyueman'during the bud stage and flowering stage.There were significant differences in the promoter sequence of BcAP1 gene between the two varieties,and the promoter sequence of'Caixin'was more than that of'Wuyueman'with a length of 896 bp.The results of double luciferase assay showed that the activity of BcAP1 promoter of'Caixin'was much higher than that of'Wuyueman'.GUS staining showed that GUS protein was more strongly expressed in A.thaliana with pCXBcAP1-BcAP1-GUS transgene.Domain analysis of BcAP1 promoter insertion fragment of'Caixin'showed that the BcAP1 promoter insertion fragment had cis-acting elements involved in biological clock regulation and gibberellin response factors.[Conclusions]The cloned BcAP1 gene was located in the nucleus and had the highest homology with A.thaliana.The BcAP1 promoter of'Caixin'had an additional insertion sequence of 896 bp in length compared to that of'Wuyueman'BcAP1 promoter,and the activity of'Caixin'BcAP1 promoter was higher than that of'Wuyueman'BcAP1 promoter.关键词
不结球白菜/BcAP1基因/克隆/启动子分析Key words
Brassica campestris ssp.chinensis/BcAP1 gene/cloning/promoter analysis分类
园艺学与植物营养学引用本文复制引用
米闵,渠天慧,徐新凤,李英,侯喜林,王建军,刘同坤..不结球白菜BcAP1的克隆及其启动子分析[J].南京农业大学学报,2025,48(6):1243-1251,9.基金项目
三亚市科技创新专项(2022KJCX80) （2022KJCX80）
国家自然科学基金项目(32372698) （32372698）
