摘要
【目的】热激转录因子(HSF)在植物对非生物胁迫应答中起重要的调节作用。本研究拟从白桦中克隆获得HSFA4基因(命名为BpHSFA4),研究该基因的抗逆功能,为该基因用于林木基因工程育种奠定理论基础。【方法】在白桦转录组中筛选获得1条HSFA4基因,通过RT-PCR克隆获得BpHSFA4基因序列。利用生物信息学工具进行序列分析。利用实时荧光定量RT-PCR(qRT-PCR)分析不同逆境胁迫下BpHSFA4基因在白桦叶、茎和根中的表达模式。构建植物过表达载体pROKⅡ-BpHSFA4和抑制表达载体pFGC5941-BpHSFA4,进行白桦瞬时转化,同时以pROKⅡ瞬时侵染白桦作为对照。分析NaCl胁迫下不同瞬时转化白桦株系的MDA、H2O2、SOD、POD、相对电导率及二氨基联苯胺(DAB)、氯化硝基四氮唑蓝(NBT)及伊文思蓝(Evans blue)染色情况,以综合评定BpHSFA4基因的耐盐功能。【结果】BpHSFA4基因cDNA全长为1170 bp,编码389个氨基酸,编码蛋白的相对分子量为44.15 kDa,理论等电点为5.14,具有典型HSF结构域。非生物胁迫(NaCl、PEG6000、镉、高温和低温)条件下和激素(ABA、GA3和JA)处理下,BpHSFA4基因的表达均发生了不同程度的改变,且每种处理后至少有1个时间点发生了明显上调或者下调,其中盐胁迫下表达变化尤为明显。3种瞬时表达白桦株系中的BpHSFA4基因的表达分析结果显示成功获得了瞬时过表达转基因株系(OE)和抑制表达转基因株系(SE)。进一步对不同转基因白桦株系的生理指标和生理染色进行分析,结果显示:NaCl胁迫条件下,过表达BpHSFA4植株H2O2含量、MDA含量和相对电导率明显低于对照植株,抑制表达BpHSFA4植株的H2O2含量、MDA含量和相对电导率则明显高于对照植株;过表达BpHSFA4植株的SOD活性和POD活性明显高于对照植株,而抑制表达BpHSFA4植株的SOD活性和POD活性明显低于对照株系;NBT、DAB染色结果与H2O2含量测定结果一致,显示OE植株的着色明�
【Objective】Heat shock transcription factor(HSF)plays an important regulatory role in plant response to abiotic stress.The aim of this study was to identify the stress resistance function of BpHSFA4 in birch(Betula platyphylla)in order to lay a theoretical foundation for using the gene in tree breeding through genetic engineering.【Method】The BpHSFA4 cDNA sequence was obtained by searching with birch transcriptome libraries.Further,the BpHSFA4 gene was cloned by RT-PCR.And the BpHSFA4 cDNA sequence was analyzed using bioinformatics tools.Quantitative real-time RT-PCR was used to monitor the expression patterns of BpHSFA4 genes in the leaves,stems and roots of birch under different stress treatments.The vector of overexpression pROKⅡ-BpHSFA4 and silencing expression vector of pFGC5941-BpHSFA4 were constructed and transient transformed into birch.The empty pROKⅡwas also transient infected into birch as a control.The MDA content,H2O2,SOD activity,POD activity,electrolyte leakage and diaminobenzidine(DAB),nitrotetrazolium blue chloride(NBT)and Evans blue staining under NaCl stress were measured and compared between transgenic(OE,SE)and control(CK)plants.【Result】The results showed that the cDNA length of BpHSFA4 was 1170 bp.The protein encoded by this BpHSFA4 gene was 389 amino acids.The relative molecular weight of BpHSFA4 protein was 44.15 kDa,with isoelectric point 5.14.The typical HSF domain was found in the BpHSFA4 sequence.The results of qRT-PCR showed that the expressions of BpHSFA4 gene were different under abiotic stresses and hormone treatments compared to the control.Furthermore,the expressions were significantly different at least at one stress point time for each treatment.In addition,the expression patterns of BpHSFA4 gene were more significantly changed under NaCl stress.To determine whether BpHSFA4 displayed a successful overexpression(OE)or a suppressed expression(SE),the transcript levels of BpHSFA4 in the three types of transformed birch plants were measured by qRT-PCR.The results show
作者
刘中原
刘峥
徐颖
刘珊珊
田志兰
解庆军
高彩球
Liu Zhongyuan;Liu Zheng;Xu Ying;Liu Shanshan;Tian Zhilan;Xie Qingjun;Gao Caiqiu(State Key Laboratory of Tree Genetics and Breeding,Northeast Forestry University, Harbin 150040)
出处
《林业科学》
EI
CAS
CSCD
北大核心
2020年第5期69-79,共11页
Scientia Silvae Sinicae
基金
中央高校基本科研业务费专项资金资助项目(2572018AA12)
大学生创新实验训练项目(201810225473)
国家自然科学基金面上项目(31670679)。
关键词
白桦
HSFA4转录因子
基因表达
耐盐
Betula platyphylla
HSFA4 transcription factor
gene expression
salt tolerance
