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Scientia Silvae Sinicae ›› 2018, Vol. 54 ›› Issue (9): 60-69.doi: 10.11707/j.1001-7488.20180908

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Molecular Cloning and Prokaryotic Expression of Orphan Gene Soloist of AP2/ERF Gene Family in Jatropha curcas

Wang Haibo1, Gong Ming2, Guo Junyun3, Xin Hu4, Tang Lizhou1, Liu Chao1, Gao Yong1, Dai Dongqin1   

  1. 1. Center for Yunnan Plateau Biological Resources Protection and Utilization Key Laboratory of Yunnan Province Universities of the Diversity and Ecological Adaptive Evolution for Animals and Plants on YunGui Plateau Qujing Normal University Qujing 655011;
    2. School of Life Sciences, Yunnan Normal University Kunming 650500;
    3. College of Biological Resource and Food Engineering, Qujing Normal University Qujing 655011;
    4. College of Forestry, Southwest Forestry University Kunming 650224
  • Received:2018-01-04 Revised:2018-02-24 Online:2018-09-25 Published:2018-09-10

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

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