基于水稻原生质体的染色质免疫共沉淀技术优化及应用  

Optimization and Application of the Chromatin Immunoprecipitation Based on Rice Protoplast

作　　者：石佳 朱秀梅 薛梦雨 余超[1] 魏一鸣 杨凤环[1] 陈华民[1] SHI Jia;ZHU Xiu-mei;XUE Meng-yu;YU Chao;WEI Yi-ming;YANG Feng-huan;CHEN Hua-min(State Key Laboratory for Biology of Plant Disease and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Science,Beijing 100193)

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100193

出　　处：《生物技术通报》2022年第7期62-69,共8页Biotechnology Bulletin

基　　金：国家转基因新品种培育重大专项(2016ZX08001-002);国家自然科学基金项目(31972255)。

摘　　要：植物转录因子是植物体内调节基因表达的重要蛋白,参与多种生物学功能的调控。研究植物转录因子调控靶基因的常用方法为染色质免疫共沉淀法(chromatin immunoprecipitation,ChIP),但抗体特异性、遗传材料构建的耗时性等因素却大大限制了ChIP技术的应用范围和效果。本文通过对水稻原生质体瞬时转化、甲醛固定、免疫沉淀核酸的超声波破碎等条件的优化,建立了基于水稻原生质体的染色质免疫共沉淀技术体系(chromatin immunoprecipitation system based on rice protoplasts,ChIP-RP);并通过本技术体系验证了水稻转录因子OsNF-YA4蛋白对靶基因序列的富集作用。本技术体系将减少制备特异性抗体或构建稳定遗传材料的局限,有利于水稻转录因子直接调控靶基因的快速筛选和验证,推动水稻转录因子调控功能的分子机制解析。Transcription factors(TFs)are important regulatory proteins that control gene expression and regulate various biological functions in plant.Chromatin immunoprecipitation(ChIP)is a very widely used technique to investigate the direct target genes of TFs.However,ChIP is always limited by antibody specificity or time-consuming construction of genetic materials.In this paper,the chromatin immunoprecipitation system based on rice protoplasts(ChIP-RP)was established by optimizing the transient transformation of rice protoplasts,formaldehyde fixation and ultrasonic fragmentation of immunoprecipitated nucleic acid.Moreover,the enrichment of target genes by OsNF-YA4 protein,a transcription factor of rice,were verified via ChIP-RP method.This technical system will greatly reduce the limitations of antibody specificity or constructing stable genetic materials,facilitate rapid screening and verification of direct target genes of rice transcription factors,and promote the deciphering of the molecular mechanism of rice transcription factor regulatory functions.

关 键 词：水稻原生质体 染色质免疫共沉淀技术 转录因子 OsNF-YA4 ChIP-RP 

分 类 号：S511[农业科学—作物学]