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玉米蔗糖非酵解型蛋白激酶2底物蛋白的筛选与鉴定
明 川, 拓昊苑, 陶 怡,等
四川农业大学 玉米研究所
摘要:
【目的】筛选玉米蔗糖非酵解型蛋白激酶2(Sucrose non-fermenting protein kinase 2,SnRK2)的底物蛋白,鉴定其在玉米脱落酸(Abscisic acid,ABA)信号转导中的作用,为进一步解析玉米中ABA介导的非生物逆境抗性机制提供参考。【方法】根据前期研究结果,通过与拟南芥SnRK2已知底物蛋白的序列比对和保守基序分析,从磷酸化水平响应ABA诱导且上调的238个蛋白中预测ZmSnRK2作用的底物蛋白,用酵母双杂交试验验证ZmSnRK2家族成员与候选蛋白的相互作用,分析其在玉米ABA信号转导中的作用。【结果】从前期鉴定的玉米磷酸化水平响应ABA诱导且上调的238个蛋白中,预测出10个ZmSnRK2候选底物蛋白,实际扩增出其中8个蛋白的编码基因以及10个ZmSnRK2基因家族成员。酵母双杂交试验结果表明,GenBank序列号为 XP_008666965.1、NP_001142137.1和P_008656995.1的候选底物蛋白,因自体磷酸化不能用酵母双杂交检测,GenBank序列号为NP_001183680.1的候选底物蛋白可以与ZmSnRK2.1、2.2、2.4、2.5、2.7、2.8、2.10、2.11蛋白相互作用,GenBank序列号为NP_001145831.1和NP_001167942.1的候选底物蛋白分别可以与ZmSnRK2.1和ZmSnRK2.10蛋白相互作用。【结论】NP_001183680.1和NP_001167942.1蛋白是ZmSnRK2家族部分成员的磷酸化底物,是玉米ABA信号转导途径的下游组分;NP_001183680.1蛋白与拟南芥丝氨酸/苏氨酸蛋白激酶同源,NP_001167942.1蛋白为包含SAM结构域的蛋白,这2个蛋白均涉及许多生理生化过程,说明ZmSnRK2下游的ABA信号转导呈发散状。
关键词:  脱落酸  玉米  信号转导  底物蛋白  蔗糖非酵解型蛋白激酶2
DOI:
分类号:
基金项目:国家自然科学基金项目(31071433)
Screening and identification of substrate proteins of sucrose non-fermenting protein kinase 2 in maize
MING Chuan, TUO Haoyuan, TAO Yi,et al
Abstract:
【Objective】This study screened the substrate proteins of sucrose non-fermenting protein kinase 2 (SnRK2) and evaluated their function in maize abscisic acid (ABA) signaling to provide reference for further elucidation of ABA-mediated abiotic tolerance mechanism.【Method】According to previous studies,the candidate substrate proteins were predicted by sequence alignment with known substrate proteins in Arabidopsis and analysis of conserved phosphorylation motifs from the proteins with up regulated phosphorylation level in response to ABA induction.The interaction between the ZmSnRK2 family members involved in ABA signaling and their candidate substrate proteins were then confirmed by yeast two-hybrid assay, and evaluated for their function in ABA signaling in maize.【Result】Ten proteins were predicated as the candidate substrates of ZmSnRK2 from the 238 proteins with up-regulated phosphorylation level in response to ABA induction identified in previous studies.The encoding sequences of eight proteins and 10 members of the ZmSnRK2 gene family were amplified.In the yeast two-hybrid assay,proteins XP_008666965.1,NP_001142137.1 and XP_008656995.1 were excluded from yeast two hybrid assay for their self phosphorylation.Interaction was detected between NP_001183680.1 and ZmSnRK2.1,2.2,2.4,2.5,2.7,2.8,2.10,2.11,NP_001145831.1 and ZmSnRK2.1,and between NP_001167942.1 and ZmSnRK2.10.【Conclusion】Proteins NP_001183680.1 and NP_001167942.1 are phosphorylated substrates of some members of the ZmSnRK2 family,and the downstream components of maize ABA signaling.Protein NP_001183680.1 is homologous to serine/threonine protein kinase in Arabidopsis,and NP_001167942.1 is a SAM domain-containing protein.These two proteins involves in different physiological and biochemical processes,suggesting that the ABA signaling pathway downstream of ZmSnRK2 is divergent.
Key words:  abscisic acid  maize  signaling  substrate protein  sucrose non-fermenting protein kinase 2