不结球白菜BcCNL基因的克隆及功能分析

Cloning and functional analysis of BcCNL gene in non-heading Chinese cabbage

[目的]CC-NBS-LRR(卷曲螺旋-核苷酸结合位点-富含亮氨酸重复,CNL)是高度保守的植物抗病蛋白家族。本文旨在克隆和研究CNL基因在不结球白菜抗霜霉病中的作用。[方法]以‘苏州青’c DNA为模板克隆获得BcCNL基因全长,并进行生物信息学分析、实时荧光定量PCR(RT-qPCR)检测、亚细胞定位及病毒诱导基因沉默(VIGS)验证。[结果]BcCNL基因含有2 790 bp开放阅读框,编码929个氨基酸,蛋白质相对分子质量为226.7,等电点为2.7。系统进化树结果显示,不结球白菜BcCNL蛋白与野甘蓝的进化关系最近,含有RX-CC、NB-ARC和LRR-3结构域;亚细胞定位显示其定位于细胞膜上。霜霉菌侵染后,BcCNL基因在抗病品种‘苏州青’中的表达量高于感病品种‘矮脚黄’; BcCNL基因的沉默降低了‘苏州青’对霜霉病的抗性。[结论]BcCNL蛋白在进化过程中保守性较高,定位于细胞膜上,BcCNL基因的沉默降低了‘苏州青’对霜霉病的抗性。

[Objectives] The CC-NBS-LRR(coiled-coil-nucleotide binding-site-leucine-rich repeat,CNL)is a highly conserved plant disease resistance protein family.In this paper,we aimed to clone and study the role of CNL gene against downy mildew in non-heading Chinese cabbage(Brassica campestris ssp.chinensis).[Methods] The full length cDNA of BcCNL was cloned by variety‘Suzhouqing’,analyzed by bioinformatics analysis,RT-qPCR detection,subcellular localization and virus-induced gene silencing(VIGS)validation.[Results] The BcCNL gene contained a 2790 bp open reading frame,encoding 929 amino acids,with a molecular weight of 226.7 and an isoelectric point of 2.7.The evolutionary relationship between BcCNL and CNL protein of Brassica oleracea var.oleracea was the most recent,and BcCNL contained RX-CC,NB-ARC and LRR-3 domains.The subcellular localization of BcCNL protein was located in the cell membrane.The expression of BcCNL gene in resistance variety‘Suzhouqing’was higher than that of susceptible variety‘Aijiaohuang’after infected with downy mildew.Silencing of BcCNL gene reduced the‘Suzhouqing’against downy mildew.[Conclusions] The BcCNL protein was highly conserved during evolution and was located in the cell membrane.The silencing of the BcCNL gene reduced the‘Suzhouqing’against downy mildew.