基于Pull-Down/MS技术筛选柑橘CsMYC2和CsMPK6的互作蛋白  

Screening and Identification of Interacting Proteins of CsMYC2 and CsMPK6 in Citrus Based on Pull-Down/MS Technique

作　　者：蒋政华 刘胜军 刘文洁 魏冉冉 何政辰 程超 唐政 刘保艳 谢宗周[1] 叶俊丽[1] 柴利军[1,2] 岳鹏涛 邓秀新 JIANG Zhenghua;LIU Shengjun;LIU Wenjie;WEI Ranran;HE Zhengchen;CHENG Chao;TANG Zheng;LIU Baoyan;XIE Zongzhou;YE Junli;CHAI Lijun;YUE Pengtao;DENG Xiuxin(National Key Laboratory of Germplasm Innovation&Utilization of Horticultural Crops,Department of Horticulture and Forestry,Huazhong Agricultural University,Wuhan 430070,China;Hubei Hongshan Laboratory,Wuhan 430070,China;Changjiang Survey,Planning Design and Research Co.,Ltd,Wuhan 430010,China)

机构地区：[1]华中农业大学园艺林学学院,果蔬园艺作物种质创新与利用全国重点实验室,武汉430070 [2]湖北洪山实验室,武汉430070 [3]长江勘测规划设计研究有限责任公司,武汉430010

出　　处：《园艺学报》2024年第12期2913-2927,共15页Acta Horticulturae Sinica

基　　金：中国博士后科学基金特别资助项目(2022T150248);国家自然科学基金重点项目(31930095);国家重点研发计划项目(2021YFD1200202);长江勘测规划设计研究有限责任公司自主创新项目(CX2021Z52)。

摘　　要：为探究CsMPK6-CsMYC2模块的互作网络,通过与CsMPK6-CsMYC2模块互作筛选鉴定参与茉莉酸介导的柑橘果皮着色过程的蛋白。利用Pull-Down联合质谱技术,在茉莉酸甲酯(MeJA)处理的柑橘果皮总蛋白中筛选与CsMPK6-CsMYC2模块互作的蛋白。对互作蛋白进行生信分析,发现相关蛋白参与次生代谢物的生物合成、植物丝裂原活化蛋白激酶信号通路、激素响应等生物学过程。其中,Trihelix类转录因子CsGT2和类萌发素蛋白Cs GLP对果实发育有重要作用,进一步确认Cs GT2与CsMYC2、CsGLP与CsMPK6以及CsGLP与CsMYC2的互作。分析CsGT2和CsGLP启动子的顺式作用元件,发现CsGT2和CsGLP的启动子上含有多个光、激素及逆境响应相关元件。本研究为难以通过酵母双杂交系统或IP-MS技术筛选互作蛋白的物种提供方法参考,同时也为深入研究茉莉酸介导的柑橘果实着色调控网络提供了基因储备。The purpose of this study is to explore the interaction network of CsMPK6-CsMYC2 module and identify the proteins involved in the citrus peel coloring process mediated by jasmonic acid through the interaction screening with CsMPK6-CsMYC2 module.To this purpose,Pull-Down with mass spectrometry technique(Pull-Down/MS)was performed to screen and identify the CsMPK6-CsMYC2 interacting proteins using the total proteins isolated from MeJA-treated citrus fruit.The interactive proteins were involved in secondary metabolites biosynthesis,plant mitogen-activated protein kinase signaling pathway,ribosome pathway,stress response,hormone response,abiotic stress responses and other biological processes according to bioinformatics analysis.Among them,trihelix transcription factor CsGT2 and germin-like protein CsGLP play important roles in fruit development.Therefore,the interactions of CsGT2 with CsMYC2,CsGLP with CsMPK6 and CsGLP with CsMYC2 were further confirmed.The promoters of CsGT2 and CsGLP contained multiple cis-acting elements involving in light,hormone and stress-response.This work provides a reference for species that are difficult to screen interacting proteins by using yeast two-hybrid system and IP-MS technology.Meanwhile,this work provides gene pool for further study regarding the regulatory network for the jasmonate-induced coloration in citrus fruit.

关 键 词：柑橘 着色 茉莉酸 Pull-Down/MS 互作蛋白 

分 类 号：S666[农业科学—果树学]