结球甘蓝SRK-ARC1-Exo70A1互作域的确定及作用强度

doi:10.3864/j.issn.0578-1752.2016.01.002

摘要/Abstract

摘要： 【目的】深入研究甘蓝自交不亲和信号传导关键元件S-位点受体激酶SRK与臂重复蛋白ARC1及ARC1与Exo70A1间相互识别的分子机理，鉴定SRK-ARC1及ARC1-Exo70A1之间的互作区段，并分析其作用强度，明确蛋白间互作功能域。【方法】通过生物信息学分析得到蛋白功能域，根据分析结果以典型的自交不亲和结球甘蓝E1为材料分别扩增SRK、ARC1和Exo70A1含不同功能域的截短体片段，利用分子克隆技术将SRK激酶域（SRKj）及其截短体SRKjΔ1—SRKjΔ4，Exo70A1全长及其截短体Exo70A1Δ1—Exo70A1Δ3的编码序列分别亚克隆至pGADT7（AD）质粒，将ARC1及其截短体ARC1Δ1—ARC1Δ8的编码序列分别亚克隆至载体pGBKT7（BD）质粒。用PEG/LiAc法将获得的AD和BD重组质粒两两组合分别共转化到酵母AH109感受态中，观察融合菌株在SD/-Leu-Trp-His-Ade/ X-a-gal/25 mM 3-AT平板上的菌落生长情况和颜色变化情况，进一步测定其β-半乳糖苷酶活性。最后通过原核表达体外孵育检测蛋白质相互作用的方法对SRK-ARC1及ARC1-Exo70A1的相互作用进行验证。【结果】DNA测序和内切酶分析显示成功构建18个酵母双杂交表达载体，且无自激活能力。在SRK-ARC1的10个试验组合中，只有ARC1Δ4、ARC1Δ8、ARC1与SRKj组合的融合菌株在SD/-Leu-Trp-His-Ade/X-a-gal/25 mM 3-AT培养基上长出蓝色菌落，激活报告基因HIS3、ADE2和MEL1。随着SRKj或ARC1截短体片段的延长，二者的β-半乳糖苷酶活性逐渐增加，其中，ARC1Δ4与SRKj组合的β-半乳糖苷酶活性最高（酶活为15.98）。在ARC1-Exo70A1 16个试验组合中，Exo70A1Δ3与ARC1Δ1Δ3都相互作用，其融合菌株在SD/-Leu-Trp-His-Ade/X-a-gal/25 mM 3-AT培养基上长出蓝色菌落，激活报告基因HIS3、ADE2和MEL1。随着ARC1或Exo70A1 截短体片段的延长，二者的β-半乳糖苷酶活性呈现先增加后降低的趋势，其中ARC1Δ2与Exo70A1Δ3组合的β-半乳糖苷酶活性最大（酶活性为25.07）。说明ARC1的N端和 Exo70A1的N端发生了互作，而ARC1的C端、全长与Exo70A1都不发生互作。体外表达检测蛋白相互作用发现，SRKj与ARC1Δ4、ARC1Δ2与Exo70A1Δ3均可以直接发生相互作用。【结论】SRK的激酶域（SRKj）与ARC1的C端臂重复区发生互作，缩短SRK激酶域中的任何结构域或者缩短ARC1的臂重复区，二者都不会发生互作。ARC1的亮氨酸拉链和蜷曲螺旋与Exo70A1的N端结构域（去除pfamExo70A1域）介导了二者的相互作用。SRK-ARC1的作用力强度小于ARC1-Exo70A1的作用力强度。

关键词: 结球甘蓝（Brassica oleracea var. capitata L.）, 自交不亲和, SRK, ARC1, Exo70A1, 酵母双杂交, 截短体

Abstract: 【Objective】 The study was for further study on the mechanism of mutual recognition among S receptor kinase (SRK), ARM-repeat containing 1 (ARC1), and Exo70A1, which were the key signal elements in self-incompatible response, and to identify the interaction domains and compare their interaction strength.【Method】According to a bioinformatics analysis, we obtained the different functional domains of three proteins. And on this basis, the truncated fragments containing different functional domains were amplified from Brassica oleracea var. capitata L. E3. Then the encoding sequences of SRKj with subfragments (SRKjΔ1-SRKjΔ4) and the full length Exo70A1 with subfragments (Exo70A1Δ1-Exo70A1Δ3) were separately subcloned into the vector pGADT7 to generate the AD recombinant plasmids, after which the encoding sequence of ARC1 with subfragments (ARC1Δ1-ARC1Δ3) were respectively constructed into vector pGBKT7 to generate the BD recombinant bait plasmids. These recombinant plasmids were respectively cotransformed into the yeast competent cells of strain AH109, and then were planted on SD/-Leu-Trp-His-Ade/X-a-gal/25 mM 3-AT nutritional media to detect the growth and color change. A β-galactosidase assay was conducted. Finally, anin vitro binding assay was performed to confirm the interaction between SRK-ARC1 and ARC1-Exo70A1.【Result】A DNA sequence and restriction enzyme analysis suggested the recombinant plasmids were correct. The yeast AH109 cells were without an autonomous activation effect on the reporter gene MEL1. In the combinations of SRKj with ARC1Δ4, ARC1Δ8, and ARC1, the yeast AH109 cells could grow and turn blue on SD/-Leu-Trp-His-Ade/X-a-gal/25 mM 3-AT nutritional media with a transcription activation of the reporter genes HIS3, ADE2 and MEL1. With the extension of the amino acid sequence, a greater β-galactosidase activity was induced, in which the combinations of SRKj with ARC1 showed relatively high levels of β-galactosidase activity (15.98). In the combinations of ARC1-Exo70A1, Exo70A1Δ3 could interact with ARC1Δ1-ARC1Δ3. Their yeast cells could grow and turn blue on SD/-Leu-Trp-His–Ade/ X-a-gal/25 mM 3-AT nutritional media, activating the reporter genes HIS3, ADE2, and MEL1. With the extension of truncated ARC1 or Exo70A1, β-galactosidase activity showed a lower trend after the first increase. Meanwhile, ARC1Δ2 with Exo70A1Δ3 exhibited the highest levels of β-galactosidase activity in all combinations (25.07). In vitro binding assay further showed that SRKj could interact with ARC1Δ4, and ARC1Δ2 also could interact with Exo70A1Δ3. 【Conclusion】The kinase domain of SRK(SRKj) and the C-terminal ARM of ARC1 were the core interaction domains of SRK-ARC1. No interactions were detected for either truncating SRKj functional domains or ARM of ARC1. The Leucine zipper with coiled-coil of ARC1 and N terminal of Exo70A1 mediated the interaction between ARC1 and Exo70A1. The interaction strength of SRK-ARC1 was less than that of ARC1-Exo70A1.

Key words: Brassica oleracea var. capitata L., self-incompatible, SRK, ARC1, Exo70A1, yeast two-hybrid, truncation

施松梅，高启国，廉小平，毕云龙，刘晓欢，蒲全明，刘贵喜，柳菁. 结球甘蓝SRK-ARC1-Exo70A1互作域的确定及作用强度[J]. 中国农业科学, 2016, 49(1): 14-26.

SHI Song-mei, GAO Qi-guo, LIAN Xiao-ping, BI Yun-long, LIU Xiao-huan, PU Quan-ming, LIU Gui-xi, LIU Jing, REN Xue-song, YANG Xiao-hong, ZHU Li-quan, WANG Xiao-jia. Identification of Interaction Domain of SRK-ARC1-Exo70A1 and Interaction Strength Analysis in Brassica oleracea var. capitata L.[J]. Scientia Agricultura Sinica, 2016, 49(1): 14-26.

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