砂藓SMPD基因的克隆及对干旱和高温胁迫响应分析

Cloning of SMPD in Racomitrium canescens and analysis of its responses to drought and high-temperature stress

关键词:  砂藓, 鞘磷脂磷酸二酯酶基因, 表达分析, 干旱胁迫, 高温胁迫

Key words:  Racomitrium canescens,sphingomyelin phosphodiesterase gene,expression analysis,drought stress,high-temperature stress

摘要:

鞘磷脂磷酸二酯酶(sphingomyelin phosphodiesterase,SMPD)能够水解鞘磷脂,形成磷脂酰胆碱和神经酰胺,在鞘脂代谢中起关键作用。为研究强耐脱水性植物砂藓(Racomitrium canescens)中1个SMPD基因(RcSMPD)的耐旱和抗高温相关功能,该文利用实时荧光定量PCR检测RcSMPD在砂藓脱水胁迫及高温处理下的表达量,对其编码序列进行克隆,对其编码蛋白质进行生物信息学预测,并构建其过表达转基因拟南芥(Arabidopsis thaliana)株系以检测RcSMPD的耐旱及抗高温能力。结果表明:(1)RcSMPD能够响应砂藓的脱水胁迫及高温处理。(2)RcSMPD总氨基酸残基数为326,为亲水性蛋白质,可能为酸性或中性SMPD并定位于叶绿体或液泡。(3)RcSMPD过表达转基因拟南芥与野生型植株相比形态发生改变,但在模拟干旱胁迫和高温胁迫处理下的抗逆能力无显著性差异。综上认为,RcSMPD能够参与砂藓对脱水和高温胁迫的应答,过表达RcSMPD能够改变拟南芥形态,但未对其耐旱性和抗高温性产生显著影响。

Abstract:

Sphingomyelin phosphodiesterase(SMPD)plays a crucial role in the sphingolipid metabolism process by hydrolyzing sphingomyelin into phosphatidylcholines and ceramides. To investigate the functions related to drought tolerance and high-temperature tolerance of an SMPD gene(RcSMPD)in the strongly desiccation-tolerant plant Racomitrium canescens, quantitative real-time PCR(qPCR)was employed to detect the expression of RcSMPD under dehydration stress and high-temperature treatments. The coding sequence of RcSMPD was cloned, and bioinformatics analysis was carried out to predict the protein encoded by RcSMPD. Furthermore, transgenic Arabidopsis thaliana lines overexpressing RcSMPD were constructed to evaluate the drought tolerance and high-temperature tolerance of RcSMPD. The results were as follows:(1)RcSMPD was found to respond to the dehydration stress and high-temperature treatments of Racomitrium canescens to different degrees.(2)RcSMPD had a total of 326 amino acid residues and was a hydrophilic protein. RcSMPD could be an acidic or neutral SMPD and was predicted to be located in chloroplasts or vacuoles.(3)Compared with wild-type plants, the morphology of the RcSMPD overexpression transgenic Arabidopsis thaliana showed differences, yet there was no significant difference in stress resistance under simulated drought stress and high-temperature stress treatments among the A. thaliana lines. In conclusion, RcSMPD can participate in the response to Racomitrium canescens to dehydration and high-temperature stress. Overexpression of RcSMPD can change the morphology of Arabidopsis thaliana, but has no significant impact on its drought tolerance and high-temperature resistance.