不结球白菜BrABF3基因的克隆与功能分析

Cloning and function analysis of BrABF3 gene in non-heading Chinese cabbage

[目的]本文旨在研究BrABF3基因在不结球白菜开花时间调控中的功能。[方法]以不结球白菜‘苏州青’为材料,通过同源克隆获得BrABF3基因全长序列,再与其他物种中的同源序列进行进化树分析;利用注射法将农杆菌导入烟草叶片研究BrABF3基因的亚细胞定位;利用PlantCARE在线软件分析启动子motif元件;采用荧光定量PCR(RT-qPCR)技术分析BrABF3基因在早花品种‘苏州青’和晚花‘五月慢’品种不同生长期的表达情况。[结果]BrABF3基因含有1242 bp开放阅读框,编码413个氨基酸,其蛋白定位于细胞核。在进化过程中BrABF3的氨基酸序列与甘蓝型油菜亲缘关系最近。BrABF3启动子序列中含有大量的motif元件,如光响应元件、激素响应元件和胁迫响应元件等。RT-qPCR结果表明,开花前BrABF3基因在晚花品种‘五月慢’中的表达显著高于早花品种‘苏州青’。[结论]本试验克隆并获得BrABF3基因,其蛋白定位于细胞核,而且在晚花品种中高表达,推测该基因可能参与调控不结球白菜的开花时间。

[Objectives]The paper aimed to study the function of BrABF3 gene in regulating flowering time of non-heading Chinese cabbage.[Methods]Using non-heading Chinese cabbage‘Suzhouqing’as the material,the full-length BrABF3 was cloned by homology cloning strategy.Based on alignments of amino acid sequences of ABF3 gene,the phylogenetic tree was constructed.Then,subcellular localization of BrABF3 was studied by injecting the agrobacterium into tobacco leaves.In addition,the motif element of BrABF3 promoter was investigated using the online software PlantCARE.Moreover,the expression patterns of BrABF3 gene in different growth stages of early-flowering cultivar‘Suzhouqing’and late-flowering cultivar‘Wuyueman’were examined by RT-qPCR.[Results]The open reading frame length of BrABF3 gene was 1242 bp,encoding 413 amino acids.Subcellular localization results showed that BrABF3 protein expressed in the nucleus of tobacco leaves.During the plants evolution,the amino acid sequence of BrABF3 had a close relationship with that of Brassica napus.In addition,the promoter of BrABF3 contained multiple motif elements including light-responsive elements,hormone-responsive elements and stress-responsive elements,etc..Before flowering,the expression level of BrABF3 in late-flowering cultivar‘Wuyueman’was significantly higher than that in early-flowering cultivar‘Suzhouqing’.[Conclusions]In this study,BrABF3 gene was cloned from non-heading Chinese cabbage.Function analysis showed that BrABF3 protein expressed in the nucleus of tobacco.Compared with early-flowering cultivar,BrABF3 was highly expressed in late-flowering cultivar of non-heading Chinese cabbage.From these results,we speculated that BrABF3 gene may be involved in regulating flowering time of non-heading Chinese cabbage.