香鳞毛蕨DfDREB基因克隆与表达模式分析

Cloning and Expression of DfDREB Gene from Dryopteris fragrans

DREB(dehydration-responsive element-binding)转录因子是响应非生物胁迫反应的主要调节因子,为探索DREB在多年生草本药用植物香鳞毛蕨[Dryopteris fragrans(L.)Schott]抗逆过程中的功能,该研究克隆了DfDREB基因并进行生物信息学分析,采用qRT-PCR方法分析了DfDREB基因在不同激素及干旱、NaCl、高温和低温等逆境胁迫处理下的表达模式。结果表明:(1)成功获得DfDREB基因全长1203 bp,其编码401个氨基酸,相对分子质量为43.66 kD,等电点为6.13,是亲水性非分泌蛋白,该蛋白具有AP2保守结构域,属于AP2家族。(2)实时荧光定量PCR分析表明,DfDREB基因在香鳞毛蕨根、叶柄和叶中均有表达,其中在叶中表达量最高,根中表达量最低;在水杨酸(SA)、茉莉酸甲酯(MeJA)和乙烯利(ETH)处理时,DfDREB基因表达上调,并都在1 h时表达量达到峰值;在脱落酸(ABA)处理时,DfDREB基因的相对表达量仅在12 h和24 h时呈上调表达,其余时间为下调表达;在干旱、NaCl和高温处理时,DfDREB基因表达均上调;低温处理0.5 h和12 h时DfDREB基因表达显著上调,但在低温处理1~6 h和24 h时无明显变化。研究表明,DfDREB基因响应激素和非生物胁迫处理,且基因表达受胁迫诱导。该研究结果为进一步探索香鳞毛蕨抗逆分子机制奠定了基础。

DREB gene is called the main regulator of abiotic stress response.In order to explore the function of DREB in the stress resistance process of Dryopteris fragrans(L.)Schott,a perennial medicinal herb,this study cloned and analyzed the DfDREB gene with bioinformatics.The expression patterns of DfDREB gene under different hormones and stress treatments of drought,NaCl,high temperature and low temperature were studied by real-time quantitative polymerase chain reaction.The results showed that:(1)the DfDREB gene,with a total length of 1203 bp,encodes 401 amino acids.The relative molecular mass is 43.66 kD and the isoelectric point is 6.13,which is a hydrophilic non-secreted protein.This protein has an AP2 conserved domain and belongs to the AP2 family.(2)Real-time PCR analysis showed that DfDREB gene was expressed in the roots,petioles and leaves of D.fragrans,among which the highest expression was in the leaves and the lowest expression was in the roots.When SA,MeJA and ETH were treated,the expression of DfDREB was up-regulated,and the expression reached the peak at 1 h.During ABA treatment,the relative expression of DfDREB was up-regulated only at 12 and 24 h,and down-regulated during the rest of the time.Under drought,NaCl and high temperature treatments,DfDREB expression was up-regulated.The expression of DfDREB was significantly up-regulated at 0.5 and 12 h under low temperature treatment,and there was no significant change at 1 to 6 h and 24 h.It shows that DfDREB can respond to hormone and abiotic stress treatments,and gene expression is induced by stress.The results provide a scientific basis for further study on the stress resistance mechanism of D.fragrans.