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斜卧青霉转录调控蛋白Hac1染色质免疫共沉淀分析方法的建立
房艳华, 韦小敏, 李 肖,等
西北农林科技大学 资源环境学院
摘要:
【目的】对斜卧青霉转录调控因子Hac1进行染色质免疫共沉淀(CHIP)试验,为其调控机制及生物学功能研究奠定基础。【方法】以斜卧青霉为材料,DTT诱导转录调控因子Hac1表达,用交联Buffer处理菌丝,使Hac1与靶基因交联,超声破碎染色质后,进行解交联染色体检验,研究不同交联时间(5,10,20,30,40 min)、不同菌丝用量(10 mL CHIP缓冲液中分别添加0.25,0.50,1.00,1.50 g菌丝)及不同超声功率(15,20,25,30 W)、超声时间(1,3,5 s)、超声间隔时间(10,20,30,40 s)、超声次数(15,25,30,40次)对试验效果的影响。在优化的条件下对菌丝进行处理,获得合格的DNA片段,进行CHIP试验,采用随机PCR方法检测染色质免疫共沉淀效果。【结果】适用于斜卧青霉Hac1的最佳染色质免疫共沉淀试验的条件为:交联时间控制在20 min以内;最佳菌丝用量为10 mL CHIP缓冲液中加入1 g菌丝;25 W超声功率,工作时间5 s,间隔时间40 s,超声30次。在优化的条件下富集DNA片段,进行随机PCR,结果在500~750 bp间可见明显的扩增条带。【结论】优化了斜卧青霉转录调控因子Hac1 CHIP条件,成功进行了CHIP试验。
关键词:  斜卧青霉  染色质免疫共沉淀  交联  菌悬液浓度  超声条件
DOI:
分类号:
基金项目:国家自然科学基金项目(31200058)
Establishment of chromatin immunoprecipitation protocol for analysis of transcriptional regulation protein Hac1 in Penicillium decumbens
FANG Yanhua, WEI Xiaomin, LI Xiao,et a;
Abstract:
【Objective】The objective of this study was to establish chromatin immunoprecipitation protocol for analysis of transcriptional regulation factor Hac1 in Penicillium decumbens,which would lay foundation for further study on the regulation mechanism and biological function of Hac1.【Method】In this study,DTT was used to induce the expression of Hac1.Mycelium were treated by crosslinking buffer and ultra-sonication was used for disruption of chromatin. The effects of crosslinking time (5,10,20,30,and 40 min),hyphae dosage added to 10 mL CHIP buffer (0.25,0.50,1.00,and 1.50 g),ultra-sonication power (15,20,25,and 30 W),working time (1,3,and 5 s),interval time (10,20,30,and 40 s)and working times (15,25,30,and 40) on CHIP were determined.Mycelium were then treated under the optimal conditions and the DNA collected by chromatin immunoprecipitation was determined by random PCR.【Result】The obtained optimal conditions of ultra-sonication were crosslinking time within 20 min,dosage for hyphae added to 10 mL CHIP buffer 1 g,power 25 W,working time 5 s,interval time 40 s and working for 30 times.According to these conditions,DNA fragments (500-750 bp) collected from chromatin immunoprecipitation were successfully detected by random PCR.【Conclusion】The chromatin immunoprecipitation protocol for analysis of Hac1 in Penicillium decumbens was successfully established.
Key words:  Penicillium decumbens  chromatin immunoprecipitation  crosslinking  fungus suspension concentration  ultrasonic conditions.