Molecular Cloning and Prokaryotic Expression Analysis of Protein Kinase SnRK1 Subunit α Gene from Jatropha curcas

doi:10.13560/j.cnki.biotech.bull.1985.2018-0948

Abstract

Abstract: The SnRK1（Sucrose non-fermenting-1 related protein kinase 1）in plant is ortholog of yeast SNF1（Sucrose non-fermenting-1）and mammalian AMPK（AMP-activated protein kinase）. These evolutionarily conserved kinases are involved in metabolic sensors and cellular signaling caused by energy depletion and low-carbon status. Based on the complete genome data,bioinformatics was used to identify a protein kinase SnRK1 subunit α gene from Jatropha curcas,it was named JcSnRK1α and then cloned and characterized. The results showed that the full-length cDNA of JcSnRK1α was 1700 bp with 11 exons and 10 introns,containing a 1545 bp open reading frame（ORF）. The ORF encoded 514 amino acids with the molecular weight 58.8 kD and the pI value 8.57. Domain analysis by CDD revealed that the JcSnRK1α composed of catalytic kinase domain with T-loop region,together with an ubiquitin-associated domain（UBA）and a kinase-associated domain（KA1）. TATA box,CAAT box,heat,low temperature-,drought-,wound-,and gibberellin-responsive elements were identified in the promoter of JcSnRK1α. qRT-PCR analysis showed that JcSnRK1α expressed specifically in different organs,abundantly in stems and roots,but scarcely in leaves. Meanwhile,JcSnRK1α gene was of remarkably cold-induced expression in stems and roots,which reached the highest after 3 h and 0.5 h chilling stress,respectively,increasing 2.04 and 3.16 times compared to the control. A prokaryotic expression vector pGEX-4T-1-JcSnRK1α was constructed and transformed into Escherichia coli Rosetta,and SDS-PAGE results showed that the molecular weight was 84.8 kD,which was consistent with the expected weight. This study laid a foundation for further studies on the gene function and the mechanism in signaling transduction and stress response in J. curcas.

Key words: Jatropha curcas, protein kinase, SnRK1, gene cloning, prokaryotic expression

WANG Hai-bo, GUO Jun-yun, TIAN Xue-lian. Molecular Cloning and Prokaryotic Expression Analysis of Protein Kinase SnRK1 Subunit α Gene from Jatropha curcas[J]. Biotechnology Bulletin, 2019, 35(6): 39-47.

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