Tn5转座酶融合蛋白在CUT&Tag实验中的优化及评价  

Optimization and Evaluation of Tn5 Transposase Fusion Protein in CUT&Tag

作　　者：刘晟宇 刘晓斌 朱家富 苏京 董志诚 刘敏 Shengyu Liu;Xiaobin Liu;Jiafu Zhu;Jing Su;Zhicheng Dong;Min Liu(Innovative Center of Molecular Genetics and Evolution,Guangzhou Key Laboratory of Crop Gene Editing,Guangdong Provincial Key Laboratory of Plant Adaptation and Molecular Design,School of Life Sciences,Guangzhou University,Guangzhou 510006,China)

机构地区：[1]广州大学生命科学学院,广东省植物适应性与分子设计重点实验室,广州市作物基因编辑应用重点实验室,分子遗传与进化创新研究中心,广州510006

出　　处：《植物学报》2023年第4期602-611,共10页Chinese Bulletin of Botany

基　　金：国家自然科学基金(No.31871289,No.31900463);广州大学研究生创新能力培养资助计划(No.2021GDJC-M23)。

摘　　要：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。Tn5 is a bacterial transposon.The engineered Tn5 can efficiently tag DNA while adding the adapter sequences.Therefore,it has been widely used in the preparation of high-throughput sequencing libraries.Cleavage Under Target&Tagmentation(CUT&Tag)is an improved technology for studying the interaction between protein and DNA,which has the advantages of good repeatability,high signal-to-noise ratio,and easy operation.This technology uses Protein A(pA)or Protein G(pG)and Tn5 to form a fusion protein,which can locate specific antibodies(the antibody is used to identify the target protein)and break the DNA near the target site while introducing sequencing adapters.Then,DNA was extracted,followed by PCR amplification to obtain the sequencing library.However,different types of antibodies have different affinities for pA and pG,thus limiting the application of CUT&Tag for some antibodies.To overcome this limitation,the expression vector of pG-Tn5 was constructed by recombination,and pG-Tn5 recombinant protein was obtained by prokaryotic expression and affinity purification.We used RNA polymerase II(Pol II)-specific antibodies(Pol II Ser5P,mouse IgG1 and rabbit IgG)to compare the efficiency of pA-Tn5 and pG-Tn5 in library preparation of CUT&Tag in Arabidopsis.The results showed that the IgG1 antibody had higher affinity for pG-Tn5,and the quality of the constructed library was better when pG-Tn5 was used.The rabbit IgG antibody has comparable affinities to the two enzymes.A lower starting amount of plant material can be applicable in CUT&Tag.This study provides a reference for the selection of Tn5 fusion proteins against different antibodies in future CUT&Tag experiments.

关 键 词：CHIP CUT&Tag RNA聚合酶Ⅱ Tn5 

分 类 号：Q78[生物学—分子生物学]