烟草NtPhyA基因的生物信息学分析和功能验证

Bioinformatic Analysis and Functional Validation of the NtPhyA Gene in Nicotiana tabacum

低温是制约烟叶生产的重要气候因素,为研究PhyA基因在烟草(Nicotiana tabacum L.)应对低温胁迫的功能,本研究通过PCR技术克隆NtPhyA基因,利用生物信息学分析基因序列和蛋白特征;构建NtPhyA过表达载体,并对该基因进行了功能验证。结果表明,NtPhyA基因编码区长度为2 394 bp,编码797个氨基酸,具有典型的PHY家族的PAS、 PHY和GAF motif特殊结构域;蛋白分析显示,NtPhyA蛋白属于不含信号肽的非分泌不稳定亲水性蛋白;亚细胞定位预测该蛋白定位于细胞质中,需转运至细胞核内调节下游基因。对获得的突变体进行低温处理后,与野生型相比,过表达NtPhyA基因的烟草株系脯氨酸含量更低,丙二醛含量和过氧化氢含量增高;转录组研究表明,NtPhyA过表达植株与野生型植株在细胞过程和应激响应上有所不同,能影响抗氧化物质合成和植物激素响应。结果表明,NtPhyA基因能参与烟草的抗寒响应生理过程,可作为提高烟草抗冷育种的候选基因,为深入解析NtPhyA基因参与烟草低温调控机制研究奠定了基础。

Low temperature is an important climatic factor that restricts tobacco leaf production.To investigate the function of the PhyA gene in tobacco(Nicotiana tabacum L.)exposed to low-temperature,in this study,NtPhyA gene was cloned by PCR and its gene se-quence and protein characteristics were analyzed by bioinformatics,and NtPhyA overexpression vector was constructed to verify the function of NtPhyA gene.The results indicate that the coding region of the NtPhyA gene was 2394 bp in length and encoded 797 amino acids with typical motifs of PAS,PHY,and GAF in the PHY family.The protein analysis revealed that the NtPhyA protein belonged to a non-secretory hydrophilic protein without a signal peptide.Subcellular localization predicted that the protein was localized in the cytoplasm and needed to be transported to the nucleus to regulate downstream genes.Compared with the wild type,the NtPhyA overex-pressed tobacco lines showed lower proline content.higher malondialdehyde content,and hydrogen peroxide content after exposure to the low temperature.Transcriptome studies showed that NtPhyA overexpressing plants differed from wild-type plants in cellular proces-ses and stress responses,and could affect the synthesis of antioxidant substances and phytohormone responses.The results indicated that the NiPhyA gene participated in the physiological response to cold tolerance in tobacco and could be used as a candidate gene to improve the breeding of cold resistance in tobacco.This study lays the foundation for an in-depth dissection of the involvement of the NtPhyA gene in the mechanism of low-temperature regulation in tobacco.