钩吻GeERF4B-1转录因子的鉴定与功能分析

Identification and functional analysis of the transcriptional factor GeERF4B-1 in Gelsemium elegans

摘要:

钩吻(Gelsemium elegans)为马钱科钩吻属藤本植物，具有药用和禽畜饲用功效，但其在生长过程中易受低温危害的影响，因此挖掘低温响应基因对钩吻抗寒育种具有重要意义。乙烯响应因子(ethylene responsive factor, ERF)属于AP2/ERF转录因子超家族成员，在植物逆境胁迫应答反应中发挥关键作用。本研究基于钩吻转录组数据库挖掘到的响应低温胁迫的GeERF的unigene序列，利用逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)技术从钩吻叶片克隆获得GeERF4B-1转录因子的cDNA全长序列。生物信息学分析表明，GeERF4B-1基因开放阅读框长度为759 bp，编码252个氨基酸，蛋白相对分子量为27 kDa，预测为不稳定的碱性亲水性蛋白。系统进化树分析结果显示，GeERF4B-1属于ERF家族的B-4亚族成员。亚细胞定位实验结果表明，GeERF4B-1定位在细胞核。实时荧光定量PCR (real time quantitative PCR, RT-qPCR)分析表明，GeERF4B-1基因在钩吻根、茎、叶中均有表达，在根中的表达量最高。经4 ℃低温、茉莉酸甲酯(methyl jasmonate, MeJA)和脱落酸(abscisic acid, ABA)处理后，相比于对照，GeERF4B-1基因均被诱导上调表达，分别在24 h或48 h达到峰值，表明该基因积极响应低温、MeJA和ABA胁迫；而氯化钠(sodium chloride, NaCl)和干旱处理后，GeERF4B-1基因均被诱导下调表达。此外，构建GeERF4B-1基因的原核表达载体，诱导获得大小约52 kDa的融合蛋白。平板胁迫验证结果显示，与对照相比，转入GeERF4B-1的原核表达菌株能增强对低温的耐受性，但对盐胁迫和甘露醇胁迫较敏感。本研究为钩吻的抗逆性育种提供了潜在的基因资源和理论参考。

Abstract:

Gelsemium elegans, a vine plant of Loganiaceae, has both medicinal and forage values. However, it is susceptible to low temperatures during growth. Exploring low temperature response genes is of great significance for cold resistance breeding of G. elegans. Ethylene response factors (ERFs) are the transcription factors of the AP2/ERF superfamily and play a crucial role in plant stress response. In this study, based on the unigene GeERF involved in the response to low temperature stress in the transcriptome of G. elegans, a full-length cDNA sequence of the transcription factor GeERF4B-1 was cloned from the leaves of G. elegans by reverse transcription-polymerase chain reaction (RT-PCR). Bioinformatics analysis showed that GeERF4B-1 had an open reading frame of 759 bp, encoding a protein composed of 252 amino acid residues and with a relative molecular weight of 27 kDa. The deduced protein was predicted to be an unstable, alkaline, and hydrophilic protein. The phylogenetic tree showed that GeERF4B-1 was in the same clade as the B-4 subfamily of the ERF family. The results of the subcellular localization experiment revealed that GeERF4B-1 was located in the nucleus. Real time quantitative PCR (RT-qPCR) analysis indicated that GeERF4B-1 was expressed in the root, stem, and leaf of G. elegans, with the highest expression level in the root. Compared with the control, the treatments with a low temperature (4 ℃), methyl jasmonate (MeJA), and abscisic acid (ABA) up-regulated the expression level of GeERF4B-1, which reached the peak at 24–48 h. This result revealed that GeERF4B-1 actively responded to low temperature, MeJA, and ABA stresses. However, both sodium chloride (NaCl) and drought treatments down-regulated the expression of GeERF4B-1. In addition, a prokaryotic expression vector of GeERF4B-1 was constructed, and a fusion protein of approximately 52 kDa was yielded after induced expression. The results of the plate stress assay showed that compared with the control, the prokaryotic strain expressing GeERF4B-1 demonstrated enhanced tolerance to low temperatures and sensitivity to salt and mannitol stresses. This study provides theoretical references and potential genetic resources for breeding G. elegans varieties with stress resistance.