麻疯树AP2/ERF基因家族孤儿基因Soloist的克隆与原核表达分析

doi:10.11707/j.1001-7488.20180908

摘要/Abstract

摘要： [目的]含有AP2结构域的AP2/ERF蛋白是与植物抗逆性密切相关的转录因子家族，Soloist是AP2/ERF家族中的孤儿基因，大部分植物只有1个。克隆麻疯树该基因编码框的全长cDNA序列，并对其理化性质、基因结构、启动子区域调控元件、表达特性及蛋白原核表达进行分析，为深入开展麻疯树Soloist基因的功能鉴定及其在麻疯树遗传改良中的应用提供依据。[方法]利用AP2结构域的隐马尔可夫模型结合拟南芥与水稻的Soloist蛋白序列对麻疯树蛋白质数据库进行相似性检索，经过Pfam与CDD在线工具的验证获得麻疯树AP2/ERF基因家族成员。利用不同的工具对Soloist基因进行生物信息学对比分析。通过实时荧光定量PCR检测该基因在麻疯树不同器官及低温处理下的相对表达量。通过双酶切构建该基因的原核表达载体，并转化大肠杆菌BL21（DE3）菌株进行IPTG诱导表达，分别收集不同诱导时间段的菌液，SDS-PAGE检测融合蛋白的表达情况。[结果]共鉴定到119个麻疯树AP2/ERF家族基因，其中包含1个Soloist基因，命名为JcSoloist。序列分析显示，麻疯树JcSoloist基因编码框长度为699 bp，编码232个氨基酸，预测分子量为26.3 kDa，理论等电点为9.65，包含6个外显子与5个内含子。在其基因启动子序列中鉴定到TATA框、CAAT框，以及赤霉素、水杨酸及干旱等应答元件。表达分析显示，麻疯树JcSoloist基因在麻疯树各器官中都有表达，但表达水平具有器官特异性，在根中表达量较高，而在叶片中表达量相对较低，同时，在叶片与根中都属于低温诱导表达基因，分别在低温胁迫3 h与24 h达到最大表达量，较对照提高34.38倍与3.83倍。构建了pET-32a-JcSoloist重组载体，且在原核细胞中稳定表达，SDS-PAGE结果显示融合蛋白分子量为46.0 kDa，与预期大小一致。[结论]麻疯树AP2/ERF基因家族鉴定到1个Soloist基因，包含由3条反平行β-折叠与1条α-螺旋组成的AP2结构域，与其他物种的报道一致。基因启动子区域鉴定到赤霉素、水杨酸等顺式作用元件，暗示该基因在麻疯树激素信号转导中发挥重要作用。JcSoloist基因在麻疯树叶片与根中受低温诱导表达显著，说明该基因参与麻疯树低温响应过程以及低温信号转导途径，也成为麻疯树抗冷新品种选育的候选基因。

关键词: 麻疯树, 基因家族, AP2/ERF, Soloist, 转录因子, 原核表达

Abstract: [Objective] AP2/ERF protein, with AP2 domains, is a transcription factor family associated with plant stress resistance. Soloist gene belongs to the AP2/ERF family, and most plants own only one of it. One Jatropha curcas Soloist gene, named JcSoloist, was selected and cloned, and then the physicochemical properties, gene structure, cis-acting elements in promoter, and prokaryotic expression were systematically analyzed. This study is aimed to provide a significant foundation for further studies of the functions of Soloist gene in J. curcas and genetic improvement of the species.[Method] Based on the Hidden Markov Model of conserved AP2 domain and Soloist protein sequence from Arabidopsis thaliana and Oryza sativa, J. curcas protein database was searched by BLASTP, and then the AP2/ERF family genes were obtained through Pfam and CDD verification. Contrast analyses for JcSoloist gene were conducted by various bioinformatics tools. The expression levels of JcSoloist gene in different organs and under chilling treatment were carried out using qPCR(quantitative real-time PCR). A recombinant vector of pET-32a-JcSoloist was constructed and transferred into Escherichia coli BL21 (DE3). After induced expression by IPTG, samples were collected from bacterial suspension at different induction times, and then SDS-PAGE was used to analyze the protein expression level.[Result] The results showed that 119 AP2/ERF family genes were identified depend on J. curcas genome and protein databases. The full-length coding sequence of JcSoloist is 699 bp with 6 exons and 5 introns in gene structure. The CDS encoded 232 amino acids with the molecular weight of 26.3 kDa and the pI value of 9.65. TATA-box, CAAT-box, responsive elements of gibberellin, salicylic acid and drought were identified in the promoter. qPCR analysis revealed that JcSoloist expressed in different organs, with abundant expression in root, but scarcely present in leaves. Meanwhile, JcSoloist gene was remarkably cold-induced expression in leaves and root, which reach the highest expression levels after 3 h (34.38-fold) and 24 h (3.83-fold) chilling treatment, respectively. The prokaryotic expression vector of pET-32a-JcSoloist was constructed and a stable prokaryotic expression system was obtained, SDS-PAGE results showed that the molecular weight of fusion protein was 46.0 kDa, which is in consistence with the expected weight.[Conclusion] One Soloist gene was identified in J. curcas AP2/ERF gene family, and containing three anti-paralleled beta sheets and one alpha helix in the coding AP2 domain, which is in consistence with other reported species. The identification of cis-acting elements such as gibberellin and salicylic acid, suggesting that JcSoloist may play a crucial role in J. curcas hormonal signaling transduction. The expression levels of JcSoloist gene were remarkably induced by low temperature, indicating that JcSoloist possibly participates in the pathways of chilling response or chilling signaling transduction, and involved in the formation of cold resistance in J. curcas, this becomes a candidate gene for the breeding of cold-resistance varieties in J. curcas.

Key words: Jatropha curcas, gene family, AP2/ERF, Soloist, transcription factor, prokaryotic expression

中图分类号:

S718.46

王海波, 龚明, 郭俊云, 辛胡, 唐利洲, 刘潮, 高永, 代冬琴. 麻疯树AP2/ERF基因家族孤儿基因Soloist的克隆与原核表达分析[J]. 林业科学, 2018, 54(9): 60-69.

Wang Haibo, Gong Ming, Guo Junyun, Xin Hu, Tang Lizhou, Liu Chao, Gao Yong, Dai Dongqin. Molecular Cloning and Prokaryotic Expression of Orphan Gene Soloist of AP2/ERF Gene Family in Jatropha curcas[J]. Scientia Silvae Sinicae, 2018, 54(9): 60-69.

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