表观遗传调控因子OsNRPE1b与OsRDM1蛋白互作验证
毕业论文关键词：RNA-directed DNA Methylation；OsNRPE1b；OsRDM1；Split-LUC；蛋白互作
Identification of interaction between epigenetic regulation factors OsNRPE1b and OsRDM1
Abstract: RNA-directed DNA methylation (RdDM) is an important epigenetic mechanism for silencing transposons and repeats. RdDM play an important regulatory role in maintaining genome stability and regulating gene expression in plant. Plant-specific RNA polymerase, Pol V, plays critical role in RdDM. Previous research showed that in Arabidopsis NRPE1 interacts with DDR complex, which facilitates the association of Pol V to RdDM loci. Pol V transcribes a scaffold RNA, then recruit RNA-induced silencing complex (RISC) complex and DRM2 to catalyze de novo methylation of DNA. OsNRPE1b and OsRDM1 which is part of DDR complex were homologous cloned. And Split-LUC assay was selected to identify interaction between OsNRPE1b and OsRDM1, and the result indicated that OsNRPE1b interacts with OsRDM1. It lays a foundation for understanding RdDM pathway in rice deeply.
Key words: RNA-directed DNA Methylation, OsNRPE1b, OsRDM1, Split-LUC, protein-protein interaction
表观遗传调控因子OsNRPE1b与OsRDM1蛋白互作验证
目 录
摘要3
关键词3
Abstract3
Key words3
引言3
1 材料与方法5
1.1 植物材料 5
1.2 载体与菌株 6
1.3构建OsNRPE1b-NLuc与CLuc-OsRDM1载体6
1.3.1 提取RNA6
1.3.2 逆转录6
1.3.3 克隆OsNRPE1b和OsRDM16
1.3.4 琼脂糖凝胶电泳7
1.3.5 胶回收7
1.3.6 同源重组法构建OsNRPE1b-NLuc和CLuc-OsRDM1 7
1.3.7热激法转化大肠杆菌7
1.3.8菌落PCR与菌落备份8
1.3.9 提取质粒8
1.4 烟草瞬时表达实验8
1.4.1 电击法转化农杆菌8
1.4.2农杆菌介导的瞬时表达8
1.4.3 CCD成像9
2 结果与分析9
2.1 OsNRPE1b-NLuc与CLuc-OsRDM1载体构建9
2.2 OsNRPE1b与OsRDM1存在互作12
3 讨论12
致谢13
参考文献13
表观遗传调控因子OsNRPE1b与OsRDM1蛋白互作验证
在植物中，RNA介导的DNA甲基化（RdDM）是 siRNA介导的表观遗传的主要途径。RdDM首先在植物的类病毒复制中被发现的[1]，后来发现其需要siRNAs和RNA干扰的核心蛋白[2][3]。RdDM途径在沉默转座子及DNA重复单元、调控基因表达、文持基因组稳定和保障植株正常生长发育的过程中起重要作用。[4]在拟南芥中，来源于重复序列或转座子序列的24nt siRNA触发RdDM途径的开始。24nt siRNA的合成过程如下：首先，DTF1/SAWADEE HOMEODOMAIN HOMOLOGUE 1（SHH1）蛋白能特异识别并结合组蛋白H3K9me2修饰，招募植物特有的DNA依赖的RNA聚合酶Pol IV到特定的染色质区域。[5]在DTF1与CLASSY 1（CLSY1）蛋白的帮助下，Pol IV在特定的染色质区转录出单链前体RNA，单链前体RNA随即由RNA-DEPENDENT RNA POLYMERASE 2（RDR2）合成dsRNA，再经过DICER-LIKE 3（DCL3）蛋白的作用加工成24nt的初级siRNA。[6][7]甲基化酶HUA ENHANCER 1（HEN1）在siRNA的3’端进行甲基化修饰，然后被装载到AGO蛋白，主要是AGO4或AGO6。[8]RdDM途径的进行还需要植物中另一个特有的DNA依赖的RNA聚合酶Pol V。Pol V转录非编码RNA需要由DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1（DRD1）、DEFECTIVE IN MERISTEM SILENCING 3（DMS3）和RNA-DIRECTED DNA METHYLATION 1（RDM1）组成的DDR复合体。[9]Pol V转录出的lncRNA能与siRNA碱基互补配对，从而将AGO-siRNA复合体招募到靶位点。同时Pol V可以通过其最大亚基NRPE1的C端的AGO hook基序与AGO蛋白互作。与NRPE1互作的KOW DOMAIN-CONTAINING TRANSCRIPTION FACTOR 1（KTF1）蛋白也有AGO hook基序，能与AGO蛋白相互作用。[10]INVOLVED IN DE NOVO 2（IDN2）是RdDM途径的组分，它与其同源的两个蛋白IDN2 PARALOGUE 1（IDP1）和IDP2形成复合体，参与到RdDM中。[11]Pol V转录的长非编码RNA（lncRNA）与其结合蛋白KTF1、AGO-siRNA复合体和IDN2复合体形成特定的支架平台，能招募DOMAINS REARRANGED METHYLTRANSFERASE 2（DRM2）对特定的染色质区段进行DNA甲基化修饰。