文采报春苣苔PwDREB2s基因的克隆与表达分析

Cloning and expression analysis of PwDREB2s genes from Primulina wentsaii

关键词:  文采报春苣苔，DREB2转录因子，肉质叶，干旱-热激复合胁迫，表达模式

Key words:  Primulina wentsaii, DREB2 transcription factor, fleshy leaf, drought-heat shock combined stresses, expression pattern

摘要:

脱水响应元件结合蛋白2（dehydration responsive element binding protein 2, DREB2）是一种转录因子，在模式植物应答干旱、高盐和热激胁迫中起调控作用。然而在适应喀斯特干热石山环境的文采报春苣苔（Primulina wentsaii）中，这些基因的功能尚不清楚。筛选对干热双重胁迫联合响应的文采报春苣苔DREB2同源基因，可为苦苣苔抗逆种质的创制提供新的基因储备。该研究首先根据文采报春苣苔转录组的序列信息设计特异引物，以gDNA和cDNA为模板分离PwDREB2s基因，然后通过生物信息学方法，对氨基酸序列、系统进化关系及保守基序进行分析，再使用半定量RT-PCR（sqRT-PCR）分析PwDREB2s基因在低温、热激、模拟干旱（渗透）、高盐、外源ABA及氧化等单一胁迫下的表达模式，最后选择能同时应答模拟干旱与热激的成员，利用实时荧光定量PCR（qRT-PCR）检测其在单一和复合干热处理中的转录本水平。结果表明：（1）分离获得了8条缺少内含子的PwDREB2s基因，能分别编码198~386个氨基酸，拥有AP2/ERF结构域、核定位信号或转录激活域等典型特征序列；（2）PwDREB2A/2AL1/2AL2、PwDREB2D/2DL和PwDREB2F转录因子被分别归入A-2亚组的亚型1、亚型2和亚型3，PwDREB2EL1/2EL2转录因子则被归入A-6亚组；（3）SqRT-PCR分析表明PwDREB2s基因表达受多种单一胁迫的诱导，其中PwDREB2A/2AL1/2AL2/2D基因能同时应答模拟干旱与热激胁迫；（4）QRT-PCR结果揭示了在液体培养条件下，PwDREB2D基因对干热处理的应答具有组织特异性，仅在根状茎中能被强烈诱导。在土壤栽培条件下，与对照相比时，除PwDREB2D基因以外，PwDREB2A/2AL1/2AL2基因在自然干旱-热激组合胁迫中的转录反应显著增强。与单一的自然干旱和高温处理相比时，PwDREB2AL1/2AL2基因在自然干旱-热激组合胁迫中的表达水平显著较高。由此可见，PwDREB2AL1/2AL2基因在文采报春苣苔干热复合胁迫应答中可能发挥了重要的调控作用。

Abstract:

Dehydration responsive element binding protein 2 (DREB2) is a transcription factor, which plays a regulatory role to drought, high salinity and high temperature responding in model plants. However, it is still unclear for the function of DREB2 genes in Primulina wentsaii, which is adapted to limestone karst habitats with drought-high temperature environment. DREB2 homologous genes responding to dual stresses of drought and high temperature, were screened in P. wentsaii. It could provide a new gene pool for the creation of resistant germplasm in Gesneriaceae. In this study, specific primers were designed based on transcriptome sequences of P. wentsaii firstly, gDNA and cDNA were used as templates to isolate PwDREB2 genes. Then, amino acid sequences, phylogenetic relationships and consensus motifs were analyzed by bioinformatics methods. As well, the expression patterns of PwDREB2s genes were analyzed by semi-quantitative reverse transcription PCR (sqRT-PCR) with single stress, such as low temperature, heat shock, simulated drought (osmosis), high salinity, exogenous ABA and oxidation, respectively. Finally, the transcript levels of designated members, which could respond to simulated drought and heat shock, were detected with different drought-heat shock patterns by quantitative real-time PCR (qRT-PCR). The results were as follows: (1) Eight intron-lacking PwDREB2s genes were isolated, they could encode 198-386 amino acids, which contained several typical characteristic regions such as AP2/ERF domain, nuclear localization signal or transcriptional activation domain, respectively; (2) Transcription factors, such as PwDREB2A/2AL1/2AL2, PwDREB2D/2DL and PwDREB2F were classified into subtype 1, subtype 2 and subtype 3 of A-2 subgroup, respectively. While PwDREB2EL1/2EL2 were classified into A-6 subgroup; (3) Semi-quantitative RT-PCR analysis showed that the transcript levels of PwDREB2s genes were induced by a series of single abiotic stresses, among which PwDREB2A/2AL1/2AL2/2D genes could simultaneously respond to simulated drought and heat shock; (4) QRT-PCR results revealed that the PwDREB2D gene, which was responding to drought and heat shock, was tissue-specific, it could only be strongly induced in rhizomes with liquid medium. Compared with mock group, the transcriptional responses of PwDREB2A/2AL1/2AL2 genes were significantly enhanced under natural drought-heat shock combined stresses with soil matrix, except for PwDREB2D gene. However, when it was compared with the single stress of natural drought and heat shock, the expression levels of PwDREB2AL1/2AL2 genes were significantly higher in natural drought-heat shock combined stresses. In conclusion, PwDREB2AL1/2AL2 genes may play the important regulatory role in response to dual stresses of natural drought and high temperature in P. wentsaii.