小桐子JcMAPK3基因的克隆及低温表达分析

Cloning and Expression Analysis of JcMAPK3 Gene from Jatropha curcas Under Cold Stress

【目的】低温严重影响着能源植物小桐子的生长发育及桐油产量,对小桐子抗冷性相关基因的挖掘和抗冷性机理的研究显得尤为重要,探究小桐子JcMAPK3基因在低温下的表达规律,为深入研究JcMAPK3基因在小桐子抗冷性形成中的功能提供理论基础。【方法】基于小桐子低温锻炼条件下的转录组数据,通过RT-PCR技术克隆小桐子JcMAPK3基因,进行生物信息学分析,采用实时荧光定量qRT-PCR研究JcMAPK3在小桐子不同器官中的表达差异,以及在12℃低温锻炼和1℃低温胁迫下的表达模式。【结果】克隆到的JcMAPK3基因开放阅读框为1119 bp,编码372个氨基酸,含有STKcTEYMAPK激酶催化结构域,蛋白相对分子量为42.82 kD,理论等电点为5.6,分子式为C1920H2985N521O551S20。qRT-PCR分析显示,JcMAPK3基因在根中表达量最高,叶片中表达量最低。在1℃低温胁迫下72 h表达量最高,为对照(0 h)的13.5倍;在12℃低温锻炼下,JcMAPK3基因表达量在24 h最高,为对照(0 h)的5.58倍。【结论】小桐子JcMAPK3在12℃低温锻炼和1℃低温胁迫下受到低温诱导表达显著,表明该基因参与小桐子对低温的响应与适应过程。

【Objective】Low temperature seriously affects the growth and development of energy plant Jatropha curcas and the yield of oil.It is particularly important to explore the genes related to chilling resistance and study the mechanism of chilling resistance in J.curcas.In order to provide a theory for further study on the function of JcMAPK3 gene in the chilling resistance formation of J.curcas,the expression pattern of JcMAPK3 gene in J.curcas at low temperature was explored.【Method】Based on transcriptome data of J.curcas under chill-hardening,JcMAPK3 gene was cloned by RT-PCR,and its bioinformatics analysis was carried out.The expression differences of JcMAPK3 in different organs of J.curcas were studied by real-time fluorescence quantitative(qRT-PCR)technology,and the expression patterns of JcMAPK3 under chill-hardening at 12℃and chilling stress at 1℃were also studied.【Result】The open reading frame of JcMAPK3 gene was 1119 bp,encoding 372 amino acids,containing the catalytic domain of STKcTEYMAPK kinase.The relative molecular weight of the protein was 42.82 kD,the JcMAPK3 theoretical isoelectric point was 5.6,and the molecular formula was C1920H2985N521O551S20.The qRT-PCR analysis showed that JcMAPK3 gene had the highest expression in roots and the lowest expression in leaves.The highest expression of JcMAPK3 gene was found at 72 hours under 1℃chilling stress,which was 13.5 folds higher than that of control(0 hours).The highest expression of JcMAPK3 gene was found at 24 hours under 12℃chill-hardening,which was 5.58 folds higher than that of control(0 hours).【Conclusion】JcMAPK3 of J.curcas was significantly induced to express under low temperature stress at 12 and 1℃,which indicated that the gene was involved in the response and adaptation process of J.curcas to low temperature.