不结球白菜耐热相关基因BcNAC036的克隆、亚细胞定位及功能分析

Cloning,subcellular localization and functional analysis of the heat resistance-associated gene BcNAC036 in non-heading Chinese cabbage

[目的]本文旨在研究不结球白菜基因BcNAC 036的亚细胞定位及高温胁迫下的表达分析,并探索其功能及调控机制。[方法]以不结球白菜‘苏州青’为试验材料,提取叶片总RNA并且反转录成cDNA,同源克隆BcNAC 036的全长基因序列,运用在线软件工具对其进行生物信息学分析。采用同源重组的方法构建过表达载体pCAMBIA1302-BcNAC 036-GFP,农杆菌介导法将构建的过表达载体注射入本氏烟草叶片背面,通过激光共聚焦显微镜观察其亚细胞表达部位。采用RT-qPCR技术检测高温处理下不结球白菜耐高温品种‘苏州青’和热敏感品种‘矮脚黄’以及转基因拟南芥体内BcNAC 036基因的表达量。对转基因拟南芥进行高温处理并统计其成活率。[结果]同源克隆的方法获得BcNAC 036基因,其开放阅读框(ORF)为840 bp,编码279个氨基酸,相对分子质量为32154.83,等电点是8.96。氨基酸序列比对以及同源进化树分析结果显示,BcNAC036蛋白与芜菁、甘蓝型油菜NAC036蛋白同源关系较近。亚细胞定位结果显示,BcNAC036的亚细胞定位表达在细胞核。RT-qPCR表明,BcNAC 036在耐高温和热敏感品种中表达差异较大且表达量均呈明显下降趋势。转基因拟南芥与野生型相比,其成活率较低。[结论]BcNAC036蛋白在细胞核表达,BcNAC 036基因与不结球白菜耐高温性呈负相关。

[Objectives]This paper aimed to study the subcellular localization and expression analysis of BcNAC 036 gene in non-heading Chinese cabbage under high temperature stress,and to explore its function and its regulatory mechanism.[Methods]Total RNA of the leaves was extracted and reverse transcribed into cDNA using non-heading Chinese cabbage‘Suzhouqing’as the test material.The full-length gene sequence of BcNAC 036 was homologously cloned and the bioinformatics analysis was conducted using online software tools.The overexpression vector pCAMBIA1302-BcNAC 036-GFP was constructed by homologous recombination method,and Agrobacterium-mediated injection of the overexpression vector into the abaxial surface of the leaves of Nicotinan benthaminan was performed,and the subcellular expression sites were observed with laser confocal microscopy.RT-qPCR was used to detect the expression level of the heat-resistant variety‘Suzhouqing’and the heat-sensitive variety‘Aijiaohuang’of non-heading Chinese cabbage and the transgenic Arabidopsis thaliana gene BcNAC 036 under high temperature treatment.The survival rate of transgenic A.thaliana was counted by high temperature treatment.[Results]The BcNAC 036 gene was obtained by homologous cloning,with an open reading frame(ORF)of 840 bp,encoding 279 amino acids,a relative molecular mass of 32154.83 and an isoelectric point of 8.96.The amino acid sequence comparison and phylogenetic tree analysis showed that the protein BcNAC036 was closely related to NAC036 protein in Brassica rapa and B.napus.The subcellular localization results showed that subcellular localization of BcNAC036 was expressed in the nucleus.RT-qPCR showed that BcNAC 036 gene was differentially expressed in heat-resistant and heat-sensitive varieties with a significant decrease in expression.The viability of the transgenic A.thaliana had lower survival rate compared to wild-type.[Conclusions]BcNAC036 protein was expressed in the nucleus and BcNAC 036 gene was negatively correlated with the heat resistance of non-heading Chinese cabbage.