小桐子SnRK1蛋白激酶α亚基基因的克隆及原核表达分析

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

植物蔗糖非发酵-1 型相关蛋白激酶1(Sucrose non-fermenting-1 related protein kinase 1,SnRK1)与酵母SNF1 及哺乳动物AMPK 同属进化上保守的SNF1 蛋白激酶家族,参与响应由胞内低能量/低糖状态所引起的信号转导过程。基于小桐子基因组数据,利用生物信息学方法鉴定到小桐子SnRK1 蛋白激酶α亚基基因,并克隆到该基因的全长cDNA 序列,命名为JcSnRK1α。结果表明,该cDNA 序列全长1 700 bp,完整开放阅读框1 545 bp,编码514 个氨基酸,分子量为58.8 kD,理论等电点为8.57。基因结构显示,其包含11 个外显子与10 个内含子。具有包含T-loop 区域的激酶结构域、自抑制调控结构域UBA 及激酶相关结构域KA1。另外,在该基因启动子序列中鉴定到TATA 框、CAAT 框及响应高温、低温、干旱、创伤、赤霉素等应答元件。qRTPCR分析显示,小桐子JcSnRK1α基因具有器官表达特异性,在茎与根中表达量较高,而在叶片中表达量相对较低,同时,在茎与根中都属于低温诱导表达基因,分别在低温胁迫3 h 与0.5 h 达到最大表达量,较对照提高2.04 与3.16 倍。构建其原核表达载体pGEX-4T-1-JcSnRK1α,并在Rosetta 中诱导表达,得到84.8 kD 的蛋白条带,与理论融合蛋白的分子量一致。本研究为开展小桐子JcSnRK1α基因的功能鉴定以及其在信号转导与逆境应答中的机制奠定了基础。

The SnRK1(Sucrose non-fermenting-1 related protein kinase 1)in plant is ortholog of yeast SNF1(Sucrose nonfermenting- 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 kinaseassociated 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.