ZHANG Ya,HUANG Tianhong,ZHANG Xilin,et al.Cloning and expression analysis of BcSERK1 from non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2019,42(6):1014-1021.[doi:10.7685/jnau.201901034]





Cloning and expression analysis of BcSERK1 from non-heading Chinese cabbage
张娅 黄天虹 张西林 刘同坤 侯喜林 李英
南京农业大学作物遗传与种质创新国家重点实验室/农业农村部华东地区园艺作物生物学与种质创新重点实验室/园艺学院, 江苏 南京 210095
ZHANG Ya HUANG Tianhong ZHANG Xilin LIU Tongkun HOU Xilin LI Ying
State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture and Rural Affairs/College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
non-heading Chinese cabbageBcSERK1clonesequence analysissubcellular localizationembryogenesis
[目的] 本文旨在研究不结球白菜BcSERK1基因及其在胚胎发生中的作用。[方法] 采用同源克隆的方法从不结球白菜‘二桩白’中克隆到BcSERK1基因全长序列,利用生物信息学分析其结构特征,并与其他物种进行氨基酸序列比对和进化分析,同时利用基因枪介导的方法进行亚细胞定位;利用小孢子培养技术研究不结球白菜‘二桩白’和‘四九菜心’小孢子培养出胚情况;采用实时荧光定量PCR (RT-qPCR)分析BcSERK1基因在2个品种不同组织和在小孢子胚胎发生早期的表达特征。[结果] BcSERK1含有1个1 878 bp开放阅读框,编码625个氨基酸。与其他物种的氨基酸序列比对和系统进化分析结果显示:BcSERK1在进化过程中保守程度较高,与甘蓝型油菜亲缘关系最近。亚细胞定位结果显示:BcSERK1蛋白定位于细胞膜上。游离小孢子培养结果显示:‘二桩白’出胚率较高,每蕾6.67个胚,而‘四九菜心’不出胚。RT-qPCR结果表明:BcSERK1在2个品种不同组织中均有表达,除根外‘四九菜心’的组织中其表达量均低于‘二桩白’;在小孢子胚胎发生早期,BcSERK1基因在‘二桩白’不同培养时间的表达量均高于‘四九菜心’。[结论] BcSERK1蛋白在进化过程中保守性较高,定位于细胞膜上;SERK1基因在2个不结球白菜品种中的差异表达表明该基因对胚胎发生具有重要作用。
[Objectives] The aim of this study was to clone and study the effect of BcSERK1 gene in embryogenesis.[Methods] The full-length sequence of BcSERK1 gene was cloned from the non-heading Chinese cabbage ‘Erzhuangbai’ by homologous cloning. The structural characteristics were analyzed by bioinformatics,and amino acid sequence alignment and evolution analysis were performed with other species,and subcellular localization was performed by particle bombardment. Isolated microspore culture technique was used to study the microspore embryogenesis frequency of the non-heading Chinese cabbage ‘Erzhuangbai’ and ‘Sijiucaixin’. Real-time quantitative PCR(RT-qPCR)was used to analyze the expression characteristics of BcSERK1 gene in different tissues and during the early stage of microspore embryogenesis of two cultivars.[Results] BcSERK1 contained a 1 878 bp open reading frame encoding 625 amino acids. The amino acid sequence alignment and phylogenetic analysis with other species showed that BcSERK1 was highly conserved in the evolutionary process,and it was closely related to Brassica napus. Subcellular localization results showed that BcSERK1 protein was localized on the cell membrane. The results of isolated microspore culture showed that the microspore embryogenesis frequency of ‘Erzhuangbai’ was higher,reaching 6.67 embryos per bud,while the ‘Sijiucaixin’ did not produce embryos. RT-qPCR results indicated BcSERK1 expressed in different tissues of two cultivars,but the expression level of each tissue except for root in ‘Sijiucaixin’ was lower than that in ‘Erzhuangbai’,and in the early stage of microspore embryogenesis,the expression level of BcSERK1 gene in ‘Erzhuangbai’ was higher than that in ‘Sijiucaixin’.[Conclusions] BcSERK1 protein was highly conserved during evolution and localizes on the cell membrane. The differential expression of BcSERK1 gene in non-heading Chinese cabbage showed that this gene played an important role in embryogenesis.


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更新日期/Last Update: 1900-01-01