垫状卷柏SpLEA1基因克隆及干旱胁迫下的表达分析

Cloning and expression analysis of the SpLEA1 gene of Selaginella pulvinata under drought stress

关键词:  垫状卷柏, SpLEA1, 基因克隆, 启动子克隆, 表达分析

Key words:  Selaginella pulvinata, SpLEA1, genetic cloning, promoter cloning, expression analysis

摘要:

晚期胚胎发育丰富蛋白(late embryogenesis abundant,LEA),广泛存在于生物体内,与植物抗逆性密切相关,可在干旱胁迫下保护植物细胞,减少植物损伤。垫状卷柏(Selaginella pulvinata)是一种在干旱胁迫下生存能力极强的蕨类植物,具有很强的恢复能力。为探究垫状卷柏SpLEA1基因在耐旱植物中的分子机制与表达特征,该研究以高耐旱性植物垫状卷柏为实验材料,基于转录组测序结果,采用RT-PCR技术获得SpLEA1基因cDNA序列,采用HiTail-PCR技术获得启动子序列,利用生物信息学对序列进行了分析,并采用qRT-PCR技术分析了SpLEA1基因在干旱胁迫下的表达模式。结果表明:(1)垫状卷柏SpLEA1全长为476 bp,开放阅读框(ORF)为279 bp,共编码92个氨基酸,通过在线工具预测到蛋白分子量为9 491.46 Da, 等电点为5.45,蛋白结构预测分析表明该蛋白为亲水性蛋白,含有10个磷酸化位点,二级结构以α-螺旋和无规则卷曲为主。(2)预测到SpLEA1蛋白的保守结构域为Lea-5,来源于LEA1家族。基于系统发生树和遗传距离矩阵,发现垫状卷柏SpLEA1与来自鹰嘴豆(Cicer arietinum )和红车轴草(Trifolium pratense)的Lea-5蛋白同源性较高。(3)对启动子序列进行顺式作用元件的预测分析发现SpLEA1基因启动子含有5类激素响应元件和与干旱胁迫响应有关的功能元件。(4)在自然干旱处理下SpLEA1基因表达上调,并在12 h时达到峰值,在24 h干旱后进行复水处理,表达量显著下调。综上所述,SpLEA1基因在垫状卷柏中很可能参与了干旱胁迫响应机制的相关调控。该结果为进一步研究垫状卷柏SpLEA1基因在干旱胁迫下的功能及其表达调控机制提供了参考。

Abstract:

Late embryogenesis abundant(LEA)is widely present in organisms and closely related to plant resistence, it can protect plant cells and reduce plant damage under drought stress. Selaginella pulvinata is a fern with the ability to survive drought stress, with a strong recovery ability under drought stress. To investigate the molecular mechanisms and expression characteristics of the SpLEA1 gene in drought-tolerant plants, we used the highly drought-tolerant plant S. pulvinata as experimental material and obtained the cDNA sequence of the SpLEA1 gene by RT-PCR based on the transcriptome sequencing results. The promoter sequence was obtained by the HiTail-PCR technique, and the sequence was analyzed by bioinformatics. qRT-PCR was used to analyze the expression pattern of the SpLEA1 gene under drought stress. The results were as follows:(1)The length of SpLEA1 was 476 bp, the open reading frame(ORF)was 279 bp, and it encoded 92 amino acids. The predicted molecular weight of the protein was 9 491.46 Da, and the isoelectric point was 5.45. The predicted protein structure analysis showed that the protein was hydrophilic. The protein contained ten phosphorylation sites, of which six serines, three tyrosines, and one threonine, respectively, and the predicted secondary structures showed that the protein was mainly composed of α-helix and random coil.(2)The conserved structural domain of the SpLEA1 protein was predicted to be Lea-5, derived from the LEA1 family. Based on the phylogenetic tree and genetic distanced matrix, the SpLEA1 was found to have high homology with Lea-5 protein from Cicer arietinum and Trifolium pratense.(3)Predictive analysis of cis-acting elements in promoter sequenced revealed that the SpLEA1 gene promoter contained five classes of hormone response elements and functional elements related to the drought stress response. The SpLEA1 gene was hypothesized to have multiple functions in the plant body and was closely related to drought stress response mechanisms.(4)SpLEA1 gene expression was up-regulated under natural dehydration treatment and peaked in 12 h. After rehydration treatment for 24 h, expression was significantly down-regulated. In summary, the SpLEA1 gene is likely to be involved in the regulation of drought stress response mechanisms in matted curly cypress. This results provide the reference for further studies on the function of the matted cypress SpLEA1 gene under drought stress and its expression regulation mechanism.