铁皮石斛DcbHLH14基因克隆及表达分析

Cloning and Expression of DcbHLH14 from Dendrobium catenatum Lindl.

【目的】克隆铁皮石斛转录因子DcbHLH14基因并分析其在非生物胁迫响应中的表达情况,为研究DcbHLH14基因的功能提供理论参考。【方法】通过同源克隆从铁皮石斛叶组织中得到DcbHLH14基因,对其编码的蛋白序列特征和组织表达特性进行分析,并采用qRT-PCR对DcbHLH14基因在低温、干旱和ABA处理过程中的表达量进行分析。【结果】DcbHLH14基因开放阅读框(Open reading frame,ORF)为1269 bp,与参考序列存在7个碱基差异,仅含有1个外显子且无内含子,编码422个氨基酸。DcbHLH14蛋白的分子式为C_(2011)H_(3192)N_(586)O_(613)S_(13),理论分子量为45.8 kD,理论等电点(pI)为5.98,含有bHLH家族保守结构域bHLH-MYC-N和HLH,属于bHLH家族,与鼓槌石斛(Dendrobium chrysotoxum)和春兰(Cymbidium goeringii)bHLH蛋白的氨基酸序列同源性较高,分别为97.16%和86.90%。转录组分析结果显示,DcbHLH14基因在云南产地野生铁皮石斛花蕾中的表达量最高,在叶中的表达量最低。进一步qRT-PCR分析结果表明,该基因在广东丹霞铁皮石斛叶中的表达量最高,而在茎中的表达量最低。DcbHLH14基因启动子富含多种与水分胁迫、低温、脱水以及ABA响应等相关的顺式作用元件。DcbHLH14基因明显受到低温、干旱和ABA诱导,低温和ABA处理6 h后DcbHLH14表达量被显著提高并达到峰值,分别是处理前的12.6倍和3.7倍;干旱处理9 h后,DcbHLH14表达量最高,是处理前的6.5倍,达到极显著差异水平。【结论】DcbHLH14基因可能在转录水平上通过依赖于ABA信号通路途径响应低温和干旱胁迫,从而调控下游功能基因表达,提高铁皮石斛抗逆性。

【Objective】Functions of DcbHLH14,a basic helix-loop-helix transcription factor of Dendrobium catenatum Lindl.,in response to abiotic stresses were studied.【Method】DcbHLH14 was cloned from D.catenatum leaves using homologous cloning method for a bioinformatic analysis on the gene and expression in tissues.Gene expressions under low temperature,drought,and abscisic acid(ABA)stresses were determined.【Result】The ORF of DcbHLH14 was 1269 bp and encoded 422 amino acids.It contained one exon,no intron,and 7 bases that were different from the reference sequences.The theoretical molecular weight was 45.8 kD,isoelectric point pH 5.98,and molecular formula C_(2011)H_(3192)N_(586)O_(613)S_(13).Its conserved domains contained bHLH-MYC-N and HLH proteins that had high similarities with the bHLH proteins in D.chrysotoxum at 97.16%and in Cymbidium goeringii at 86.90%.The transcriptome analysis revealed high expressions in the flower buds and columns but low in the leaves of the wild D.catenatum from Yunnan,whereas the qRT-PCR analysis showed high expressions in the leaves and low in the stems of the sample from Danxia,Guangdong.The promoters of DcbHLH14 contained numerous cis-acting elements associated with the responses to water-depletion,low temperature,dehydration,and ABA stresses,which significantly affected expression of the gene.For instance,DcbHLH14 was upregulated to peak in 6 h after a low temperature or ABA treatment reaching 12.6 or 3.7 times,respectively,as well as by a 9 h drought stress to become as high as 6.5 times of control.【Conclusion】It was postulated that DcbHLH14 responded to low temperature or drought stress through the ABA signaling pathway at transcription level.Hence,the tolerance of D.catenatum to the abiotic stresses could be manipulated by regulating the expression of the downstream functional gene.