摘要
脱水反应元件结合蛋白(DREBs)在植物非生物逆境胁迫中可调节下游一系列抗逆基因的表达。为探究巴哈雀稗DREB2基因在逆境胁迫下的功能,本研究采用RNA-Seq结合RT-PCR技术从巴哈雀稗中获得一个DREB2基因CDS区全序列,命名为PnDREB2(GenBank登录号:MH150946)。核苷酸序列分析表明,该基因开放阅读框全长774 bp,编码257个氨基酸,相对分子质量为28.09 kD,理论等电点为5.25;该植物蛋白中具有DREB类基因家族典型的保守域AP2结构域,属于DREB2类转录因子A类中A2亚类的亚型1。进化和聚类分析结果表明,巴哈雀稗PnDREB2基因与高粱、割手密、玉米、谷子及牛鞭草亲缘关系较近,氨基酸序列同源性分别为91.10%、89.50%、88.20%、87.60%和87.60%。RT-qPCR和生物信息学分析表明,PnDREB2基因在茎中表达量最高,幼穗次之,叶最低;表达产物定位于细胞质中,具有26个磷酸化位点,并具有结构和功能的特异性。PnDREB2表达量受干旱(PEG-6000,20%)和高温(40℃)的强烈诱导,同时也受到高盐(NaCl,300 mmol·L~(-1))、低温(4℃)、脱落酸(ABA,100μmol·L~(-1))和赤霉素(GA,100μmol·L~(-1))等非生物胁迫和激素的不同程度诱导,随着处理时间的延长,整体上均呈先增加后降低的趋势。本试验为巴哈雀稗抗逆分子机理研究与抗性增强的转基因材料培育奠定了理论基础。
DREBs play important roles in regulating the expression of downstream genes in response to a variety of abiotic stresses. In order to study the function and mechanism of DREB2 gene in Paspalum notatum and further enrich the database of Paspalum notatum genome, monoclonal technique was used to clone the CDS region of PnDREB2 gene(GenBank No. MH150946) from the genome of Paspalum notatum. The analysis of nucleotide sequence indicated that PnDREB2 gene contained a 774 bp intact open reading frame, which encoded a peptide of 257 amino acids, and the predicted molecular weight and isoelctric point of the polypeptide were 28.09 kD and 5.25, respectively. The amino acid sequence analysis indicated that the predicted protein contained a typical AP2 structural domain, belonging to the subtype 1 of Subgroup A-2 in transcription factors of DREB2. Evolution and clustering analysis revealed that PnDREB2 gene of Paspalum notatum has the closest genetic relationship with Sorghum bicolor, Saccharum spontaneum, Setaria italica, Hemarthria compressa and Zea mays, and the homologies up to 91.10%, 89.50%, 88.2%, 87.6% and 87.6%, respectively. The results of realtime quantitative PCR and bioinformatics analysis suggested that PnDREB2 gene was constitutively expressed in various tissues of Paspalum notatum with higher level in stem, and then in young ear, but lower in leaf. Its expression product was located in cytoplasm, with 26 phosphorylation sites, representing the structural and functional specificity. Meanwhile, the expression of PnDREB2 gene was induced by drought(PEG-6000, 20%) and high temperature(40℃), andalso induced by high-salt(NaCl, 300 mmol·L~(-1)), low temperature(4℃), abscisic acid(ABA, 100 μmol·L~(-1)) and gibberellic acid(GA, 100 μmol·L~(-1)), with the increase of treatment time, the expression trend was first increasing and then decreasing. This work could lay a theoretical foundation for the study of resistance molecular mechanism of Paspalum notatum and for the develepment of genetically modified materials with enhanced stress resistance.
引文
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