Tn5是一种细菌转座子。经改造的Tn5能够高效地切割DNA,同时连接上特定的接头序列,因而广泛应用于高通量二代测序文库构建中。CUT&Tag(Cleavage Under Target&Tagmentation)是一种改进的研究蛋白质与DNA互作的技术,具有重复性好...Tn5是一种细菌转座子。经改造的Tn5能够高效地切割DNA,同时连接上特定的接头序列,因而广泛应用于高通量二代测序文库构建中。CUT&Tag(Cleavage Under Target&Tagmentation)是一种改进的研究蛋白质与DNA互作的技术,具有重复性好、信噪比高及操作简便等优点。该技术采用pA(Protein A)或pG(Protein G)与Tn5形成融合蛋白,定位于特定抗体(用于识别目标蛋白),利用Tn5的特性,在目标位点附近打断DNA的同时引入测序接头,随后提取DNA,再进行PCR扩增即可获得测序文库。但不同类型的抗体与pA或pG的亲和力不同,因此限制了部分抗体在CUT&Tag技术中的应用。为克服这一局限,该文构建了pG与Tn5的融合蛋白表达载体,通过原核表达及亲和纯化的方式获得pG-Tn5重组蛋白;并以RNA聚合酶Ⅱ(PolⅡ)特异性抗体PolⅡSer5P(小鼠IgG1型抗体和兔IgG型抗体)为例,在模式植物拟南芥(Arabidopsis thaliana)中评估pA-Tn5与pG-Tn5在不同类型抗体的CUT&Tag测序文库构建中的效果。结果表明,IgG1型抗体与p G-Tn5的亲和力更高,构建的文库质量更好,而IgG型抗体与2种酶的亲和力相当;同时,较低起始量的植物材料也能获得较好的效果,证明了CUT&Tag的应用优势。该研究优化了CUT&Tag技术,可为后续CUT&Tag实验中针对不同抗体时Tn5融合蛋白的选择提供参考。展开更多
Plants have evolved efficient mechanisms for adapting to temperature fluctuations,known as heat stress response and heat stress memory.Although the transcriptional regulatory network of plant heat stress response has ...Plants have evolved efficient mechanisms for adapting to temperature fluctuations,known as heat stress response and heat stress memory.Although the transcriptional regulatory network of plant heat stress response has been established,little is known about the genome-wide transcriptional changes occurring within the first several minutes after heat shock.Here,we investigated the nascent RNA and mature messenger RNA(mRNA)from plant leaf tissues exposed to 5 min of heat shock treatment using global run-on sequencing and RNA sequencing methods.Only a small group of genes were up-or downregulated at both the nascent RNA and mRNA levels.Primed plants that were already exposed to mild heat stress exhibited a more drastic alteration at multiple transcriptional steps than naive plants that had not experienced heat stress.Upon heat shock,we also observed the following:(i)engaged RNA polymerase Ⅱ accumulated downstream of transcription start sites;(ii)5′pausing release was a ratelimiting step for the induction of some heat shock protein genes;(iii)numerous genes switched transcription modes;(iv)pervasive read-through was induced at terminators;and(v)heat stress memory occurs at multiple steps of the transcription cycle,such as at Pol Ⅱ recruitment,5′pausing,elongation,and termination.展开更多
基金supported by the National Natural Science Foundation of China(31900463 to M.L.and 31871289,31471165 to Z.D.)。
文摘Plants have evolved efficient mechanisms for adapting to temperature fluctuations,known as heat stress response and heat stress memory.Although the transcriptional regulatory network of plant heat stress response has been established,little is known about the genome-wide transcriptional changes occurring within the first several minutes after heat shock.Here,we investigated the nascent RNA and mature messenger RNA(mRNA)from plant leaf tissues exposed to 5 min of heat shock treatment using global run-on sequencing and RNA sequencing methods.Only a small group of genes were up-or downregulated at both the nascent RNA and mRNA levels.Primed plants that were already exposed to mild heat stress exhibited a more drastic alteration at multiple transcriptional steps than naive plants that had not experienced heat stress.Upon heat shock,we also observed the following:(i)engaged RNA polymerase Ⅱ accumulated downstream of transcription start sites;(ii)5′pausing release was a ratelimiting step for the induction of some heat shock protein genes;(iii)numerous genes switched transcription modes;(iv)pervasive read-through was induced at terminators;and(v)heat stress memory occurs at multiple steps of the transcription cycle,such as at Pol Ⅱ recruitment,5′pausing,elongation,and termination.
