小桐子植物特异性酪蛋白激酶PS-CK1-5基因的克隆及原核表达分析

Cloning and Prokaryotic Expression of Plant-specific Casein Kinase Gene PS-CK1-5 from Jatropha curcas

摘要:

酪蛋白激酶(casein kinase, CK)作为一类普遍存在的Ser/Thr蛋白激酶，通过调节靶标蛋白的活性与稳定性，在植物整个生理过程及信号转导途径中发挥重要作用。基于同源序列比对，该研究对小桐子(Jatropha curcas)酪蛋白激酶基因家族进行鉴定与表达分析。结果表明，小桐子基因组中共鉴定到7个酪蛋白激酶1基因(CK1)、5个植物特异性酪蛋白激酶1基因(PS-CK1)、3个酪蛋白激酶2α亚基基因(CK2-α)、2个酪蛋白激酶2β亚基基因(CK2-β)，4个亚家族成员在氨基酸长度、等电点及外显子数目等都有其家族特异性。蛋白的氨基酸序列比对表明，小桐子酪蛋白激酶1都包含N端保守激酶结构域，同时其内部都鉴定到典型的激酶活性环基序、ATP结合核心基序、核定位信号肽。qRT-PCR表达分析表明，小桐子JcPS-CK1-5基因在叶片与根中都属于低温诱导基因，可能参与小桐子抗冷性过程。构建其原核表达载体pET-32a-JcPS-CK1-5，并在BL21(DE3)中诱导表达，得到81.6 kD的条带，与理论融合蛋白的分子量一致。这可为小桐子CK基因的功能鉴定及逆境信号转导机制研究提供参考。

Abstract:

Casein kinase (CK), a ubiquitous Ser/Thr protein kinase, plays an important role in the whole physiological process and signal transduction pathway of plants through regulating activity and stability of target protein. The CK gene family in Jatropha curcas was identified from J. curcas genome based on the BLAST method. The results showed that a total of seven CK1s, five PS-CK1s, three CK2-αs, and two CK2-βs were identified from J. curcas genome, and the four subfamily members owned family-specificity in amino acid length, isoelectrical point, and exon number. Domain and motif analysis revealed that all of CK1 contain N-terminal conserved kinase domain, and the kinase activation loop (A-loop), ATP-binding core motif, and nuclear localization signal peptide (NLS) were identified in its interior. A plant-specific casein kinse 1-5 gene, named JcPS-CK1-5, was cloned. qRT-PCR analysis showed that JcPS-CK1-5 gene was a low temperature-induced gene in both leaves and roots, and might be involved in cold resistance of J. curcas. A prokaryotic expression vector pET-32a-JcPS-CK1-5 was constructed and transformed into Escherichia coli BL21(DE3). A band of 81.6 kD was obtained, which was consistent with the expected weight. Therefore, these would provide a reference for studying the functional identification of CK gene and the stress signal transduction mechanism in J. curcas.