Zhao Qingzhe,Liang Chao,and Mo Beixin.Identification of novel components of Arabidopsis miRNA pathway and mutant analysis[J].Journal of Shenzhen University Science and Engineering,2017,34(No.5(441-550)):464-470.[doi:10.3724/SP.J.1249.2017.05464]





Identification of novel components of Arabidopsis miRNA pathway and mutant analysis
Zhao Qingzhe1 Liang Chao2 and Mo Beixin1
1) College of Life Sciences and Oceanography, Shenzhen University, Shenzhen Key Laboratory of Microbiology and Gene Engineering, Shenzhen 518060, Guangdong Province, P.R.China
2) College of Life Sciences and Oceanography, Shenzhen University, Guangdong Provincial Key Laboratory for Plant Epigenetics, Shenzhen 518060, Guangdong Province, P.R.China
分子生物学转基因株系SUC2:amiR-SUL 拟南芥正向遗传筛选小核糖核酸AGO1蛋白EMS诱变实时荧光定量聚合酶链式反应全基因组测序
molecular biology SUC2:amiR-SUL Arabidopsis thaliana forward genetic screening system microRNA (miRNA) AGO1 protein EMS mutagenesis quantitative real-time PCR(qRT-PCR) whole genome sequencing
Q 946.2
小核糖核酸(micro ribonucleic acid, miRNA)是一类长度为20~24 核苷酸(nucleotide, nt)的非编码的核糖核酸(ribonucleic acid, RNA),在植物生长发育过程中具有重要作用.为鉴定参与植物miRNA合成、降解和运输等通路的因子,利用拟南芥转基因株系SUC2:amiR-SUL进行正向遗传筛选体系,通过对该株系进行甲基磺酸乙酯(ethylmethylsulfone, EMS)诱变筛选获得SUP-E45突变体.对该突变体进行表型观察、实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qRT-PCR)和全基因组测序实验.结果显示,突变的基因为Ago1(Argonaute 1), 该基因编码的AGO1蛋白是miRNA通路中至关重要的蛋白,成熟的miRNA与AGO1蛋白结合形成miRISC沉默复合体 (miRNA-induced silencing complex, miRISC)从而对靶基因的表达进行负调控.验证了该筛选体系的可行性.通过该筛选体系可筛选出其他参与miRNA合成、影响miRNA 活性或miRNA运输等通路的因子,为后续拟南芥miRNA通路的研究奠定了基础.
Micro ribonucleic acids (miRNAs) are 20-24 nucleotides non-coding RNAs that play key regulatory roles in developmental and physiological processes in plants. In order to screen the new components that are involved in miRNA biogenesis, turnover and movement, we establish a forward genetic screening system using Arabidopsis thaliana transgenic line (SUC2:amiR-SUL). After ethylmethylsulfone (EMS) mutagenesis on SUC2:amiR-SUL, one stable mutant line (SUP-E45) is isolated. The result of phenotype observation, quantitative real-time polymerase chain reaction (qRT-PCR) and whole genome sequencing on the SUP-E45 mutant plants indicates that the altered phenotype is caused by the mutation in Argonaute 1 (Ago1) gene. AGO1 protein encoded by ago1 gene is crucial in the miRNA pathways, which recruits mature miRNA to form miRISC silence complex (miRNAs-induced silencing complex, miRISC) to negatively regulate the expression of target genes. The screening system can be used to select other factors that participate in the processing such as miRNA synthesis, miRNA’s activity or miRNA transport. The research can lay the foundation on the subsequent Arabidopsis miRNA pathway.


[1] Chen Xuemei. Small RNAs and Their Roles in Plant Development[M]// Annual Review of Cell and Developmental Biology. 2009:21-44.
[2] 马轩,李盛本,莫蓓莘,等.拟南芥ago1-27突变体的RNA-seq分析[J].深圳大学学报理工版,2017,34(1):27-32.
Ma Xuan, Li Shengben, Mo Beixin, et al. RNA-seq analysis on Arabidopsis ago1-27 mutant[J]. Journal of Shenzhen University Science and Engineering, 2017, 34(1): 27-32.(in Chinese)
[3] Mallory A, Vaucheret H. Form, function, and regulation of ARGONAUTE proteins[J]. The Plant Cell, 2010, 22(12): 3879-3889.
[4] Ren Guodong, Xie Meng, Dou Yongchao, et al. Regulation of miRNA abundance by RNA binding protein TOUGH in Arabidopsis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(31): 12817-12821.
[5] Han M H, Goud S, Song Liang, et al. The arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(4): 1093-1098.
[6] Dong Zhicheng, Han M H, Fedoroff N. The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(29): 9970-9975.
[7] Park W, Li Junjie, Song Rentao, et al. CARPEL FACTORY, a dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in arabidopsis thaliana[J]. Current Biology, 2002, 12(17): 1484-1495.
[8] Fang Yuda, Spector D L. Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants[J]. Current Biology, 2007, 17(9): 818-823.
[9] Gandikota M, Birkenbihl R P, Hhmann S, et al. The miRNA156/157 recognition element in the 3′ UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings [J]. The Plant Journal: for Cell and Molecular Biology, 2007, 49(4): 683-693.
[10] Chen Xuemei. A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development[J]. Science, 2004, 303(5666): 2022-2025.
[11] Carlsbecker A, Lee J Y, Roberts C J, et al. Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate[J]. Nature, 2010, 465(7296): 316-321.
[12] Shunsuke M, Koi S, Hashimoto T, et al. Non-cell-autonomous microRNA165 acts in a dose-dependent manner to regulate multiple differentiation status in the Arabidopsis root[J]. Development, 2011, 138(11): 2303-2313.
[13] Chiou T J, Aung K, Lin Shui, et al. Regulation of phosphate homeostasis by MicroRNA in Arabidopsis[J]. The Plant Cell, 2006, 18(2): 412-421.
[14] Buhtz A, Springer F, Chappell L, et al. Identification and characterization of small RNAs from the phloem of Brassica napus[J]. The Plant Journal: for Cell and Molecular Biology, 2008, 53(5): 739-749.
[15] Martin A, Adam H, Díaz-Mendoza M, et al. Graft-transmissible induction of potato tuberization by the microRNA miR172[J]. Development, 2009, 136(17): 2873-2881.
[16] 方晓峰. 拟南芥MicroRNA通路新因子的鉴定和作用机制研究[D]. 北京:北京协和医学院, 2014.
Fang Xiaofeng. Identification and Characterization of Novel Components in the Arabidopsis MicroRNA Pathway[D]. Beijing: Peking Union Medical College,2014.(in Chinese)
[17] Morel J B, Godon C, Mourrain P, et al. Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance[J]. The Plant Cell, 2002, 14(3): 629-639.
[18] Zhang Shuxin, Xie Meng, Ren Guodong, et al. CDC5, a DNA binding protein, positively regulates posttranscriptional processing and/or transcription of primary microRNA transcript[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(43): 17588-17593.
[19] Wu Xueying, Shi Yupeng, Li Jingrui, et al. A role for the RNA-binding protein MOS2 in microRNA maturation in Arabidopsis[J]. Cell Research, 2013, 23(5): 645-657.
[20] 岳路明,宋剑波,莫蓓莘,等.拟南芥AGO基因家族分析及盐胁迫下的表达验证[J].深圳大学学报理工版,2017,34(4):331-440.
Yue Luming, Song Jianbo, Mo Beixin,et al. Bioinformatical and experimental analysis of AGO genes in response to salt stress[J]. Journal of Shenzhen University Science and Engineering,2017, 34(4): 331-440.(in Chinese)


 HU Zhang-li,SHU Long-fei,and GOU De-ming.MicroRNAs quantification and related target genes for response to sulfur deprivation in Chlamydomonas reinhardtii[J].Journal of Shenzhen University Science and Engineering,2011,28(No.5(441-550)):237.
 Zhao Yunyan,Li Zhong,Chen Danni,et al.Systematic regulatory network of gamma-aminobutyric acid receptor genes[J].Journal of Shenzhen University Science and Engineering,2015,32(No.5(441-550)):128.[doi:10.3724/SP.J.1249.2015.02128]


Foundation:National Natural Science Foundation of China (31571332)
Corresponding author:Professor Mo Beixin.E-mail: bmo@szu.edu.cn
Citation:Zhao Qingzhe, Liang Chao, Mo Beixin. Identification of novel components of Arabidopsis miRNA pathway and mutant analysis[J]. Journal of Shenzhen University Science and Engineering, 2017, 34(5): 464-470.(in Chinese)
引文:赵庆喆,梁超,莫蓓莘. 拟南芥microRNA通路中新因子的筛选和突变体分析[J]. 深圳大学学报理工版,2017,34(5): 464-470.
更新日期/Last Update: 2017-09-11