水稻着丝粒特异组蛋白CENH3抗体的制备与应用

doi:10.16819/j.1001-7216.2017.7036 475

摘要/Abstract

摘要：

目的着丝粒是真核生物染色体的基本功能元件之一,其功能是在细胞有丝分裂和减数分裂时期精确地调控染色体配对和分离并维持染色体的稳定。着丝粒结构是由DNA和蛋白质形成的一种复合体。着丝粒特异组蛋白(centromere-specific histone H3, CENH3)是功能着丝粒是否具有活性的最基本特征。所以制备CENH3的相关抗体是进行着丝粒结构与功能研究的前提条件之一。方法通过设计短肽进行兔免疫实验,制备了水稻着丝粒特异组蛋白CENH3兔源抗体,利用ELISA和蛋白免疫荧光(immunofluorescence, IF)等检测方法对抗体有效性进行了鉴定。结果 ELISA检测显示制备的CENH3抗体有效稀释度为1: 40万,并且蛋白免疫荧光信号在水稻体细胞每条染色体的着丝粒区域均能检测到。同时,该抗体也可以应用于玉米等其他物种。通过染色质免疫沉淀(ChIP)技术获得与CENH3相结合的DNA分子,并进行PCR扩增和FISH定位分析,结果显示相应的ChIP-DNA位于水稻功能着丝粒区域。结论本研究制备的水稻CENH3兔源抗体能满足着丝粒研究中相关实验的要求,可进一步应用于着丝粒的结构与功能研究。

关键词: 水稻, 着丝粒特异组蛋白, 染色质免疫沉淀, 蛋白免疫荧光

Abstract:

【Objective】 The centromeres of eukaryotic chromosomes are essential for cell division and inheritance of genetic information. The centromere is a complex composed of centromeric proteins and DNA motifs. In particular, a centromere-specific histone 3 variant, referred to CENH3, is a key centromere-specific protein for centromeric chromatin. So the preparation of CENH3 antibody is necessary for the research of centromeric structure and function. 【Method】We designed a short-peptide for the rabbit immune experiment and prepared CENH3 antibody in rice. ELISA and protein immunofluorescence(IF) assay were performed to detect the effectiveness and availability of CENH3 antibody. 【Result】 The effective dilution of CENH3 antibody was detected at 1:400 000. Through IF analysis, obvious signals were detected in the centromeric region of each chromosome in rice and maize. DNA associated with CENH3 was selectively immunoprecipitated in chromatin immunoprecipitation assay(ChIP). After PCR amplification and FISH(fluorescence in situ hybridization) analysis, the results showed ChIP-DNA was located in functional centromere in rice. 【Conclusion】 These results demonstrated that CENH3 antibody prepared in this study can meet the requirement of related experiments in the research of centromere, and the antibody was prepared successfully.

Key words: rice, centromere-specific histone, chromatin immune-precipitation assay, immunofluorescence

中图分类号:

Q786
S511.01

薛超, 张融, 郭瑞, 刘帅, 刘晓宇, 沈明晨, 邓世峰, 龚志云. 水稻着丝粒特异组蛋白CENH3抗体的制备与应用[J]. 中国水稻科学, 2017, 31(5): 475-482.

Chao XUE, Rong ZHANG, Rui GUO, Shuai LIU, Xiaoyu LIU, Mingchen SHEN, Shifeng DENG, Zhiyun GONG. Preparation and Application of Centromere-Specific Protein CENH3 Antibody in Rice[J]. Chinese Journal OF Rice Science, 2017, 31(5): 475-482.

图/表 4

图1 水稻日本晴根尖细胞CENH3抗体的蛋白免疫荧光信号检测分析

Fig. 1. Immunofluorescence analysis of CENH3 antibody in somatic root tip cells of Nipponbare.

图2 大麦(A)、小麦(B)和玉米(C)根尖细胞CENH3抗体的蛋白免疫荧光信号检测分析

Fig. 2. Immunofluorescence analysis of CENH3 antibody in somatic root tip cells of barley(A), wheat(B) and maize(C).

图3 CENH3抗体免疫沉淀DNA的PCR结果

Fig. 3. PCR results of ChIP-DNA immunoprecipitated with CENH3 antibody.

图4 日本晴根尖细胞ChIP-DNA的荧光原位杂交信号检测结果

Fig. 4. FISH analysis of ChIP-DNA in somatic root tip cells of Nipponbare.

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