甘蓝型油菜BnPHL12基因克隆及功能分析

Molecular cloning and functional analysis of BnPHL12 homologous gene in Brassica napus L.

【目的】在甘蓝型油菜中克隆拟南芥MYB-CC家族转录因子AtPHL12的同源基因BnPHL12并进行生物信息学、组织表达模式、亚细胞定位及转录活性研究,为BnPHL12的基因功能研究奠定基础。【方法】利用序列比对方法(BLAST)在甘蓝型油菜泛基因组数据库BnIR中鉴定AtPHL12的同源基因BnPHL12,在“Westar”品系中克隆AtPHL12的同源基因,进行生物信息学及进化分析。利用qRT-PCR及GUS染色方法检测AtPHL12同源基因在不同组织中的表达水平,利用原生质体转化的方法检测BnPHL12的亚细胞定位及转录活性。利用农杆菌侵染介导的转化方法,创建BnA01PHL12过量表达转化植株,并进行BnA01PHL12功能分析。【结果】在“Weatar”材料中克隆到4个AtPHL12的同源基因,命名为BnA01PHL12(705 bp),BnC01PHL12(705 bp),BnA05PHL12(705 bp)和BnC05PHL12(696 bp),分别编码234、234、234、231个氨基酸,与AtPHL12的序列相似性大于70%,均含有保守的MYB DNA结合结构域和Coiled-Coiled结构域。BnA01PHL12与BnC01PHL12分别来源于白菜的BraA01t04110Z、甘蓝的BolC01g050170.2J。BnPHL12的同源基因在根、茎、叶、花蕾中均有表达,在根和叶的表达水平更高;BnA01PHL12与BnC01PHL12在花药发育的早期和晚期均有较高的表达水平。BnA01PHL12定位在细胞核中并具有转录激活活性。在甘蓝型油菜和拟南芥中,利用绒毡层特异表达基因BnA9启动子驱动BnA01PHL12表达,导致转基因材料雄性不育。【结论】在甘蓝型油菜中,AtPHL12的4个同源基因具有保守的MYB和CC结构域,在营养器官和生殖器官中均有表达。通过在绒毡层细胞中过量表达BnA01PHL12,创制了新的雄性不育材料,为研究花药发育提供良好基础。

【Objective】 Homologous genes of MYB-CC family transcription factor AtPHL12 were cloned in Brassica napus, and their bioinformatics, tissue expression pattern, subcellular localization and transcriptional activity were analyzed, which could facilitate the functional analysis of BnPHL12.【Method】Homologous genes of AtPHL12 were identified in the B. napus pan-genome database(BnIR) by blasting, and they were cloned in ‘Westar’ line for bioinformatics and evolutionary analysis. The relative expression levels of AtPHL12 homologous genes in different tissues were detected by qRT-PCR and GUS staining, and the subcellular localization and transcriptional activity of BnPHL12 were detected by protoplast transformation. Using the transformation method mediated by Agrobacterium infection, the overexpression transformed plants of BnA01PHL12 were created, and the function was analyzed.【Result】Four homologous genes of AtPHL12 were cloned in ‘Westar’ line, named as BnA01PHL12(705 bp), BnC01 PHL12(705 bp), BnA05PHL12(705 bp) and BnC05 PHL12(696 bp), which encoded 234, 234, 234 and 231 amino acids, respectively. The homologous genes of BnPHL12 and AtPHL12 had more than 70% sequence similarity and both contained the conserved MYB DNA-binding domain and Coiled-Coiled domain. BnA01PHL12 and BnC01PHL12 was evolved from BraA01 t04110 Z of B. rapa and BolC01 g050170.2J of B. oleracea, respectively. The homologous genes of BnPHL12 were expressed in roots, stems, leaves and flower buds, and the expression levels were higher in roots and leaves. Interestingly, BnA01PHL12 and BnC01 PHL12 had higher expression levels in the early and late stages of anther development. BnA01PHL12 localized in the nucleus and had transcriptional activation activity. The overexpression of BnA01PHL12 was driven by the promoter of the tapetum-specific expression gene BnA9 in B. napus and Arabidopsis thaliana, resulting in male sterility of the transgenic materials.【Conclusion】Four homologous genes of AtPHL12 were cloned in B. napus, which had conserved MYB and CC domains, and expressed in both vegetative and reproductive organs. A new male sterile line was created by overexpressing BnA01PHL12 in tapetum cells, which could provide a good foundation for the further research on anther development.