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(No.5(441-550)):464-470.[doi:10.3724/SP.J.1249.2017.05464]
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]
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拟南芥microRNA通路中新因子的筛选和突变体分析
赵庆喆1,梁 超2,莫蓓莘1

1)深圳大学生命与海洋科学学院,深圳市微生物基因工程重点实验室,广东深圳518060; 2)深圳大学生命与海洋科学学院,广东省表观遗传重点实验室,广东深圳518060

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

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

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

DOI: 10.3724/SP.J.1249.2017.05464

备注

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.

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