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核桃DREB基因家族鉴定及干旱胁迫下的表达分析
Genome-Wide Identification of the DREB Gene Family in Juglans regia and Transcriptome Analysis under Stress Conditions
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摘要: 为分析核桃DREB基因家族成员并分析其在干旱胁迫中的表达模式，将从核桃基因组中鉴定DREB家族成员用MEGA构建进化树，通过MEME、NCBI CD-Search等工具分析蛋白保守基序、结构域及理化性质，采用TBTools进行染色体定位与共线性分析，结合转录组测序及RT-qPCR验证差异表达基因，并利用PlantCARE预测核心基因启动子顺式作用元件。结果表明：共鉴定到55个核桃DREB家族成员，聚类为6组；蛋白氨基酸数113 ～ 463，分子量
13220.24 ～ 51862.71Da，等电点4.72 ～11.2，98.2%成员不稳定系数>40，均为疏水性蛋白；基因多分布于染色体两端；干旱胁迫下根部有10个差异基因（7上调3下调）、叶中有15个（11上调4下调），其中7个基因在根和叶中均差异表达；对这7个基因进行RT-qPCR实验，验证了转录组结果的准确性；核心基因启动子含光响应、ABA响应、干旱诱导、低温响应等元件。综上所述，核桃DREB家族成员在干旱胁迫中以正调控为主，核桃DREB家族成员在干旱胁迫下以上调表达为主，可能在逆境应答中发挥正调控作用。7个核心基因在根和叶中均受干旱胁迫显著诱导，且其启动子区富含ABA响应等逆境相关元件，提示它们在核桃干旱胁迫应答网络中可能发挥关键作用。Abstract:Objective To elucidate the molecular mechanism of Juglans regia in response to drought stress, identify members of its DREB gene family, and analyze their expression patterns under abiotic stress.Method Members of the DREB family were identified from the walnut genome, and phylogenetic trees were constructed using MEGA. Conserved motifs, domains, and physicochemical properties of proteins were analyzed via tools such as MEME and NCBI CD-Search. Chromosomal localization and collinearity analysis were performed using TBTools. Walnut seedlings were subjected to PEG6000-simulated drought and heat stress treatments; differentially expressed genes (DEGs) were screened using RNA-seq (with DESeq package, criteria: p<0.05 and |log2FoldChange|≥2) and validated by RT-qPCR. Cis-acting elements in the promoters of hub genes were predicted using PlantCARE.Result A total of 55 DREB family members were identified, clustered into 6 groups. The proteins had 113 - 463 amino acids, molecular weights of13220.24 -51862.71 Da, isoelectric points of 4.72 - 11.2; 98.2% had an instability index >40, and all were hydrophobic. Genes were mostly distributed at chromosome ends. Under drought stress, 10 DEGs (7 up-regulated, 3 down-regulated) were found in roots, and 15 DEGs (11 up-regulated, 4 down-regulated) in leaves. Seven hub genes were differentially expressed under both stresses. RT-qPCR confirmed the accuracy of RNA-seq results. Promoters of hub genes contained elements related to light response, ABA response, drought induction, and low-temperature response.Conclusion Members of the walnut DREB family are predominantly positively regulated under abiotic stress; intron-less or few-intron features may enhance their stress response efficiency. Some hub genes (e.g., DREB1A, DREB1B) play key roles in low-temperature and drought stress. This study provides important genetic resources and theoretical basis for stress-resistant molecular breeding of walnuts.
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