蝴蝶兰PhNAC1基因序列分析及对低温胁迫的响应

Sequence analysis of PhNAC1 gene from Phalaenopsis and its response to cold stress

关键词:  蝴蝶兰, NAC转录因子, 表达特性, 低温胁迫, 序列分析

Key words:   Phalaenopsis, NAC transcription factor, expression pattern, cold stress, sequence analysis

摘要:

为探讨NAC转录因子在蝴蝶兰低温胁迫响应中的分子调控机理,该研究以蝴蝶兰的叶片为材料,运用RT-PCR及RACE技术克隆得到一条蝴蝶兰的NAC转录因子基因完整的cDNA序列,命名为PhNAC1(GenBank登录号MF797909),并分析了其在两种低温条件下的表达模式。结果表明:PhNAC1基因cDNA序列全长1 442 bp,ORF全长942 bp,编码313个氨基酸。预测其蛋白分子量为35.22 kDa,等电点为6.95,属于稳定亲水性蛋白。二级结构预测表明,无规则卷曲和延伸链为该蛋白的主要结构元件,与三级结构预测结果基本相符。PhNAC1编码的氨基酸序列与其他已登录的兰科植物NAC蛋白进行同源序列比对,表明与小兰屿蝴蝶兰(XP_0205763790)亲缘关系较近,序列一致性达97%,其次为铁皮石斛(XP_020695081),一致性为84%。实时荧光定量PCR分析表明,PhNAC1基因在营养器官和生殖器官中均有表达,在蕊柱中的表达量最高。在11 ℃/6 ℃低温条件下,PhNAC1基因的转录表达水平在前5天随着处理时间逐渐升高,到第7天开始下降; 在4 ℃低温条件下,PhNAC1基因的表达水平在处理0.5 h时表达量有所下降,1 h后表达量上升至对照水平,之后无明显变化,在处理24至48 h又逐渐升高,推测PhNAC1基因参与蝴蝶兰低温胁迫响应。

Abstract:

NAC transcription factors are involved in many processes of plant development, which play an important role in stress response. The NAC transcription factor named PhNAC1(GenBank accession No. MF797909)was cloned from the leaves of Phalaenopsis using RT-PCR and RACE method. The full-length of PhNAC1 was 1 442 bp, which contained a 942 bp ORF that encoding a protein with 313 amino acids residues. The molecular weight of the putative protein was 35.22 kDa and the theoretical pI was 6.95, a hydrophilic and unstable protein. Prediction of secondary structure showed that the random coil and extended strand were the main structural elements of the protein, which conformed to the prediction of tertiary structure. Amino acid sequence alignment and phylogenetic tree analysis between the protein of PhNAC1 and NACs from other Orchids showed that PhNAC1 was close to Phalaenopsis equestris(XP_020576379)with the sequence identity of 97%, followed by the Dendrobium catenatum(XP_020695081)with 84%. The qRT-PCR analysis indicated that the PhNAC1 gene was expressed in both of the vegetative and reproductive organs, and the expression level was the highest in the column. Under the cold stress of 11 ℃/6 ℃, the expression level of PhNAC1 gene was increased significantly with treatment time in leaves during the first five days, and then decreased at the 7th day. Under the cold stress of 4 ℃, the expression level was decreased slightly at 0.5 h, then recovered and remained to the initial level at 1 h after treatment, whereas the expression level was increased obviously after treating for 24、48 h, indicating that PhNAC1 may be involved in cold stress response of Phalaenopsis. This research will be useful for study of molecular mechanism of NAC transcription factor in cold stress response in Phalaenopsis species.