葡萄类钙调磷酸酶B亚基互作蛋白激酶VvCIPK10的特性与表达

doi:10.3864/j.issn.0578-1752.2016.19.011

摘要/Abstract

摘要： 【目的】在葡萄中克隆丝苏氨酸蛋白激酶VvCIPK10，分析其激酶特性和在逆境胁迫下的表达模式，为进一步研究该基因参与逆境胁迫的分子功能，探讨葡萄抗逆分子机制提供理论依据。【方法】利用电子克隆技术获得VvCIPK10序列，设计特异引物进行RT-PCR反应，对克隆到的序列进行开放阅读框和保守结构域分析；构建原核表达载体，转化表达菌株后用IPTG进行诱导表达，收集菌体后裂解细胞，制备蛋白上样液，SDS-PAGE电泳对表达产物进行分析，同时对融合蛋白进行可溶性分析；IPTG大量诱导表达融合蛋白，收集菌体后进行超声破碎细胞，用麦芽糖结合蛋白纯化柱纯化MBP-VvCIPK10融合蛋白，SDS-PAGE电泳进行分析；纯化后的融合蛋白与体外自磷酸化缓冲液进行自磷酸化反应，反应后SDS-PAGE电泳，压磷屏检测体外自磷酸化反应；构建重组瞬时表达载体pBI221-GFP/VvCIPK10；分离拟南芥原生质体，通过PEG介导的瞬时转化方法将重组表达载体pBI221-GFP/ VvCIPK10转化至原生质体；通过基因枪介导的转化方法将重组表达载体pBI221-GFP/VvCIPK10转化至洋葱表皮细胞，培养16 h后用激光共聚焦显微镜进行荧光信号检测；选择生长相对一致且健壮的葡萄植株，于干旱、低温和盐胁迫处理后不同时间取样，同时在田间取葡萄不同组织样品，实时荧光定量PCR检测VvCIPK10在葡萄不同组织中的表达以及在不同逆境胁迫下的表达模式。【结果】PCR克隆获得葡萄VvCIPK10全长为1 357 bp，5′端非编码区为30 bp，3′端非编码区为156 bp，开放阅读框为1 171 bp，编码436个氨基酸，理论等电点为8.59，分子量为48.7 kDa。保守结构域预测分析显示该蛋白5′端具有一个激酶结构域，3′末端具有一个PPI结构域和一个NAF结构域。BLSATP分析表明葡萄VvCIPK10与桃树CIPK（XP_007205151）一致性最高（74%）。重组表达载体pMAL-C5X/VvCIPK10在大肠杆菌中经诱导表达获得与理论分子量（43 kDa+48.7 kDa）相一致的融合蛋白。MBP-VvCIPK10融合蛋白经柱纯化后获得单一的蛋白条带，VvCIPK10的自磷酸化活性依赖于Mn²⁺，不依赖于Mg²⁺和Ca²⁺，EDTA可以抑制VvCIPK10的自磷酸化活性。亚细胞定位结果显示，VvCIPK10定位在细胞核、细胞膜和细胞质。VvCIPK10在葡萄各个组织中均有表达，主要在葡萄根和叶片中大量表达，葡萄茎、花序、果实和卷须中的表达量较低。在干旱、低温和盐胁迫处理后，VvCIPK10呈现受诱导表达模式。VvCIPK10的表达在低温胁迫后6 h达到峰值，干旱和盐胁迫后2 h即达到峰值。【结论】葡萄VvCIPK10能够响应干旱、低温和盐胁迫，推测VvCIPK10在葡萄抗非生物逆境胁迫中具有重要作用。

关键词: 葡萄, 类钙调磷酸酶Ｂ亚基, 丝苏氨酸蛋白激酶, VvCIPK10, 表达分析

Abstract: 【Objective】The objective of this study is to clone serine/threonine protein kinase VvCIPK10 in grapevine, and analyze the kinase characteristics and its expression pattern under stresses. This study will provide a theoretical basis for further study on the molecular function of VvCIPK10 involved in abiotic stress, and investigate the molecular mechanism of grapevine stress resistance. 【Method】The sequences of VvCIPK10 were obtained by electronic cloning technology, and the specific primers were designed to perform RT-PCR reaction. Open reading frame and conserved structure domain of VvCIPK10 were analyzed. The prokaryotic expression vector was constructed and transformed into the expression cells, and the recombinant bacteria were induced by IPTG. The expression products were collected and the protein sample was prepared. The protein samples were analyzed by SDS-PAGE electrophoresis, and the soluble characteristics of the fusion protein were analyzed. The fusion protein was induced by IPTG. The cells were collected and broken by using ultrasonic. The MBP-VvCIPK10 fusion protein was purified by maltose binding protein purification column and analyzed by SDS-PAGE. The purified fusion protein was incubated with in vitro self-phosphorylation buffer solution. After SDS-PAGE electrophoresis, the phosphor screen was performed and the phosphorylation reaction was detected in vitro. The recombinant transient expression vector pBI221-GFP/VvCIPK10 was constructed. The recombinant expression vector pBI221-GFP/VvCIPK10 was transformed into protoplasts by PEG mediated transient transformation. The recombinant expression vector pBI221-GFP/VvCIPK10 was transformed into onion epidermal cells by gene gun mediated transformation, and the fluorescence signal was detected by laser scanning confocal microscope after 16 h of culture. The relatively consistent and robust grape plants were selected, samples were taken at different times after drought, low temperature and salt stress treatments, at the same time, different tissue samples of grapes were taken in the field, with a kit to extract total RNA. After reverse transcription, expression of VvCIPK10 was detected by real-time quantitative PCR. 【Result】The full-length of VvCIPK10 is 1 357 bp, 5′ end of non-coding region is 30 bp, 3′ end of non-coding region is 156 bp, the open reading frame is 1 171 bp. VvCIPK10 open reading frame encoded 436 amino acids, the theoretical isoelectric point is 8.59, molecular weight is 48.7 kDa. Conserved domain prediction analysis showed that the protein has a kinase domain in 5′ terminal, a PPI domain and a NAF domain in 3′ terminal. BLSATP analysis showed VvCIPK10 consistency with peach CIPK (XP_007205151) is highest (74%). Recombinant expression vector pMAL-C5X/VvCIPK10 transformation in Escherichia coli, expressed the molecular weight of fusion protein is consistent with the predicted molecular weight (43 kDa+48.7 kDa). MBP-VvCIPK10 fusion protein was purified by column. VvCIPK10 autophosphorylation activity was dependent on Mn²⁺ but not dependent on Mg²⁺ and Ca²⁺, and EDTA could inhibit the autophosphorylation activity of the VvCIPK10. Subcellular localization showed VvCIPK10 in the nucleus, cell membrane and cytoplasm. VvCIPK10 expressed in various tissues of grapevine. VvCIPK10 transcripts mainly accumulated in grapevine roots and leaves, but showed low expression levels in grapevine stem, inflorescence, fruit and tendril. After drought, low temperature and salt stress treatments, VvCIPK10 showed the induced expression model. The expression of VvCIPK10 reached peak value at 6 h after low temperature stress, and reached the peak at 2 h after drought and salt stresses. 【Conclusion】It was concluded that grapevine VvCIPK10 as a serine threonine protein kinase is able to respond to drought, low temperature and salt stress, suggesting that VvCIPK10 plays an important role in resistance to abiotic stress.

Key words: grapevine, calmodulin like B subunit, serine threonine protein kinase, VvCIPK10, expression analysis

余义和，李秀珍，郭大龙，张会灵，杨英军，李学强，张国海. 葡萄类钙调磷酸酶B亚基互作蛋白激酶VvCIPK10的特性与表达[J]. 中国农业科学, 2016, 49(19): 3798-3806.

YU Yi-he, LI Xiu-zhen, GUO Da-long, ZHANG Hui-ling, YANG Ying-jun, LI Xue-qiang, ZHANG Guo-hai. Characteristics and Expression of Calmodulin Like B Subunit Interaction Protein VvCIPK10 in Grapevine[J]. Scientia Agricultura Sinica, 2016, 49(19): 3798-3806.

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