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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 郭萍等 / 利用水稻原生质体快速分析 mirna 靶标 RNA November 25, 2014, 30(11): Chin J Biotech, All rights reserved 生物技术与方法 1751 利用水稻原生质体快速分析 mirna 靶标 RNA 1, 郭萍 2, 武瑶 1 1,, 李嘉 3, 方荣祥 1, 贾燕涛 ,,,. mirna RNA. 生物工程学报, 2014, 30(11): Guo P, Wu Y, Li J, et al. Efficient transient expression to analyze mirna targets in rice protoplasts. Chin J Biotech, 2014, 30(11): 摘要 : 与转基因方法相比, 基因瞬时表达系统在基因表达研究上具有快速便捷的特点为检验水稻 mirna 与靶标基因之间的调控关系, 将 MIRNA 基因与 GFP / 靶标序列融合基因 ( 或 GFP / 靶标突变序列融合基因 ) 构建在同一瞬时表达载体上, 并转化水稻原生质体, 通过观察含有 GFP / 靶标序列融合基因和 GFP / 靶标突变序列融合基因的载体之间的荧光强度差异, 以及通过 qrt-pcr 方法检测靶标和非靶标 mrna 水平差异来验证 mirna 对靶标基因的调控用 osamir156 和 osamir397 及其靶标序列对实验设计方法进行验证, 荧光显微观察和 qrt-pcr 检测证明,osamiR156 和 osamir397 能降低相应靶标序列 GFP 融合基因的转录物水平和 GFP 荧光水平此种水稻原生质体瞬时表达方法用于在体内进行大规模 mirna 靶标基因检测由于其他近缘单子叶植物很可能与水稻有近似的小 RNA 加工系统, 因此对于其他单子叶植物 mirna 功能研究也将有很好的应用前景 : mirna 靶标, 水稻原生质体, 体内荧光检测 Received: February 17, 2014; Accepted: April 8, 2014 Supported by: National Basic Research Program of China (973 Program) (No. 2011CB100703), National Natural Science Foundation of China (Nos , ). Corresponding author: Yantao Jia. Tel: ; Fax: ; jiayt@im.ac.cn (973 ) (No. 2011CB100703) (Nos , )

2 1752 ISSN CN /Q Chin J Biotech November 25, 2014 Vol.30 No.11 Efficient transient expression to analyze mirna targets in rice protoplasts Ping Guo 1,2, Yao Wu 1, Jia Li 1,3, Rongxiang Fang 1, and Yantao Jia 1 1 State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing , China 2 University of Chinese Academy of Sciences, Beijing , China 3 Beijing City University, Beijing , China Abstract: Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between mirnas and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qrt-pcr method. Two microrna genes, osamir156 and osamir397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice mirna target in vivo, and may be suitable for functional analysis of mirnas of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice. Keywords: mirna target, rice protoplast, in vivo fluorescence assay RNA (Non-coding small RNA) RNA mirnas (MicroRNAs) sirna (Short interfering RNAs) sirna sirna (Natural antisense transcript sirna, nat-sirna) sirna (Trans-acting short interfering RNAs, tasirnas) sirna (Heterochromatic sirnas) sirna (Long sirna, lsirna) mrna DNA [1] RNA mirnas 50% [2] MicroRNAs RNA RNA Dicer mirna lin-4 let-7 mirnas [3-4] mirnas RNA (RNA-induced silencing complex, RISC) RNA mrna 3 -UTR (Untranslated region) CDS (Coding regions) RNA [5] mirna mirna

3 郭萍等 / 利用水稻原生质体快速分析 mirna 靶标 RNA 1753 mirna mirna mirna mirna [5] mirna mirna mirna mirna RISC AGO (Argonaute proteins) RNA AGO mirna RNA 5 3 mrna mirna [6] mirna 5 RACE (Rapid Amplification of cdna Ends) mirna mirna [6] 5 RACE mirna mirna 5 RT-PCR mirna [6] mirna mirna mirna (CaMV) 35S mirna 35S 3 mirna ( mirna ) GFP pbi221 PEG ( ) GFP mirna mirna mirna RNA Real-time PCR mirna mirna 1 材料与方法 1.1 菌株质粒培养基及植物材料培养 DH5α BL21 (DH3) pbi221-egfp pcambia1300 LB [7] (Oryza sativa L. var. Nipponbare) ( =1 1) ( / 16 h/8 h) 1.2 主要试剂和仪器 T4 DNA T Promega GenBank Biosciences TRIzol Invitrogen poly(a) FirstChoice RLM-RACE Ambion R-10 R-10 Yakult PCR TOYOBO Ambion PCR Invitrogen qpcr BioRad C1000 PCR ( Leica SP8) cjb@im.ac.cn

4 1754 ISSN CN /Q Chin J Biotech November 25, 2014 Vol.30 No.11 表 1 本文所用的引物 Table 1 Primers used in this work Primer name Ter-F 35S-R PML-R PML-F BLG159-F BLG159-R Primer sequence (5 3 ) ATGACCATGATTACGATTCCCGATCTAGTA CGACGGCCAGTGCCAGCTTGCAT GAGAACACGGGGGACTCACGTGCAG GGGAAATTCGAGCTGCACGTGAGTC CGCATATGAAGCTTGCATGCCTGCAGGT CTGACGTCTACCATGATTACGAATTCCC 156-F GGGAATGATGTAGCACGG 156-R TCAGGAATTACGAAGGGTG BI156-F BI156-R MGFP-F spl-r-2 splm-r-2 GAACACGGGGGACTCACGGGAATGATGTAGCACGG GAAATTCGAGCTGCACTCAGGAATTACGAAGGGTG GAACACGGGGGACTCTAGAG GGGAAATTCGAGCTCATGACAGAAGAGAGAGAGCACAGCTCGAGTAAGATCT TCCGGACTTGTACA GGGGAAATTCGAGCTCAGGAGAGCAGGGACAGGGCGCAGCTCGAGTAAGAT CTTCCGGACTTGTACA 397-F TCCAGAGCGCACACTATT 397-R TGAGTTGCTGCATTGTTGT BI397-F BI397-R lac-r lacm-r Poly(T) adapter RP gfpqrt mir156-f mir397-f actin-f actin-r GAACACGGGGGACTCACTCCAGAGCGCACACTATT GAAATTCGAGCTGCACTGAGTTGCTGCATTGTTGT GGGGAAATTCGAGCTCTAGTTGAGTGCAGCGTTGATGAGCCTCAGCATGTAA GATCTTCCGGACTTGTAC GGGGAAATTCGAGCTCTAGTTTAAAGCTGCATTTATCAGTCTCAGCATGTAA GATCTTCCGGACTTGTAC GCGAGCACAGAATTAATACGACTCACTATAGG(T)12VN GCGAGCACAGAATTAATACGAC ACGAGCTGTACAAGTCCGG TGACAGAAGAGAGTGAGCAC TCATTGAGTGCAGCGTTGATG TGTATGCCAGTGGTCGTACCA CCAGCAAGGTCGAGACGAA 1.3 质粒的构建 pbi221-egfp 35S mirna 3 mirna GFP Ter-F PML-R 35S-R PML-F pcambia (pbi221 Hind Ⅲ EcoRⅠ pcambia1300 Hind Ⅲ/ EcoRⅠ ) Nos-T 35S PCR EcoR Ⅰ/ Hind Ⅲ pcambia1300 pcambia-pml pcambia GUS PmlⅠ BLG159F/R pcambia-pml

5 郭萍等 / 利用水稻原生质体快速分析 mirna 靶标 RNA 1755 PCR 35S 5 3 AatⅡ NdeⅠ T T-PML T-PML AatⅡ NdeⅠ pbi221-egfp Aat Ⅱ/ NdeⅠ pbi221-pml mirbase osamir156 ( 156F/R osamir156 PCR BI156F/R PCR 15 bp pbi221-pml PCR pbi221-pml PmlⅠ pbi156 pbi221-egfp MGFP-F osamir156 spl-r-2 PCR GFP ORF 3 GFPspl GFPspl pbi156 BamH Ⅰ/ SacⅠ pbi156spl MGFP-F splm-r-2 GFP GFPsplm pbi156 BamH Ⅰ/ SacⅠ pbi156splm 397F/R osamir397 BI397F/R pbi221-pml PmlⅠ pbi397 pbi221-egfp MGFP-F lac-r osamir397 GFP GFPlac MGFP-F lacm-r osamir397 GFP GFPlacm pbi397 BamH Ⅰ/ SacⅠ pbi397lac pbi397lacm 1.4 原生质体的制备 [8] 18 d 0.5 mm 10 ml 1.5% R % R mmol/l D- 20 mmol/l KCl 20 mmol/l MES [2-(N- ) ph 5.7] 10 mmol/l CaCl 2 5 mmol/l β- 0.1%BSA 50 μg/ml r/min 4 h (200 ) 50 ml 150 r/min 5 min ( ) W5 [154 mmol/l NaCl 125 mmol/l CaCl 2 5 mmol/l KCl, 2 mmol/l MES ph 5.7)] 150 r/min 5 min 1.5 原生质体的转化 MMg (4 mmol/l MES 0.6 mol/l 15 mmol/l MgCl 2 ) 10 6 /ml 100 μl 20 μg 110 μl 40% PEG (0.6 mol/l 100 mmol/l CaCl 2, 40% V/V PEG4000) min 2 ml W5 150 r/min 3 min 500 μl W GFP 荧光显微观察 h 500 μl 150 r/min cjb@im.ac.cn

6 1756 ISSN CN /Q Chin J Biotech November 25, 2014 Vol.30 No.11 3 min 450 μl 10 μl 10 ( NA 0.4) 20% 488 nm nm Hz LAS AF Lite [( )/ ] 1.7 RNA 的提取和实时定量 PCR 分析 8 10 h 150 r/min 3 min 450 μl 500 μl TRIzol 5 min 100 μl 5 min r/min 15 min 300 μl 250 μl 1 μl r/min 45 min RNA 70% 5 min 5 min RNase RNA Shi [9] 1 μg RNA DNaseⅠ poly(a) ATP 37 1 h RNA 3 poly(a) 65 5 min (SuperScript Ⅱ Reverse Transcriptase) 0.5 μg poly(t) adapter (3 RACE adapter in FirstChoice RLM-RACE kit) qpcr osamir156 PCR mir156-f RP PCR 64 bp osamir397 mir397-f RP PCR 65 bp GFP gfpqrt RP TOYOBO SYBR Green (Realtime PCR Master mix) 20 μl 0.5 μl cdna 1 mmol/l 1 mmol/l s s s 40 2 结果 2.1 瞬时表达载体的构建 mirna mirna 35S mirna mirna GFP 3 mirna mrna GFP ( GFPc ) mirna GFP GFPnc GFPc GFPc GFPnc mirna GFPc GFPnc mirna osamir156 osamir397 mirna osamir156 OsSPL14 [10] osamir397 OsLAC

7 郭萍等 / 利用水稻原生质体快速分析 mirna 靶标 RNA 1757 [11] 1 osamir156 GFPc GFPnc pbi156spl pbi156splm osamir397 pbi397lac pbi397lacm 2.2 显微观察蛋白水平差异 pbi156spl pbi156splm 12 h GFP 2 (A D) pbi156spl pbi156splm pbi397lac pbi397lacm GFP 10 2B pbi156splm ( / ) 39% pbi156spl (14%) 2.8 pbi156splm pbi156spl (P<0.01 2C) pbi397lacm 31% pbi397lac ( 18% ) (P<0.01 2E 2F) GFP mirna 图 1 Fig. 1 瞬时共表达 mirna 和融合靶标序列的 GFP 基因载体构建图谱 Schematic diagram of constructs for coexpression of mirna and GFP gene fused with target sequences. cjb@im.ac.cn

8 1758 ISSN CN /Q Chin J Biotech November 25, 2014 Vol.30 No.11

郭萍等/利用水稻原生质体快速分析 mirna 靶标 RNA 图 2 原生质体中 GFP 显微荧光观察及定量统计 Fig. 2 Microscopic observation and quantitative statistics of protoplast GFP fluorescence.

Bottom panels are enlarged sections of square shown in top panel.

9 郭萍等/利用水稻原生质体快速分析 mirna 靶标 RNA 图 2 原生质体中 GFP 显微荧光观察及定量统计 Fig. 2 Microscopic observation and quantitative statistics of protoplast GFP fluorescence. A and D: GFP excitation of rice protoplasts transducted by pbi156spl, pbi156splm, pbi397lac and pbi397lacm. Bottom panels are enlarged sections of square shown in top panel. B and D: fluorescent ratio of rice protoplasts transducted by pbi156spl, pbi156splm, pbi397lac and pbi397lacm. C and F: relative intensity of fluorescence of rice protoplasts transducted by pbi156spl, pbi156splm, pbi397lac and pbi397lacm. Each column represents an average of ten replicates, and bars indicate SDs. cjb@im.ac.cn 1759

1760 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech November 25, 2014 Vol.30 No.11 2.

pbi397lac pbi397lacm osamir397 pbi221-pml ( 3B) 图 3 qrt-pcr 检测转化原生质体中 mirna 和 GFP 基因的表达水平 Fig. 3 Quantification of mirna and GFP transcripts in rice protoplasts by qrt-pcr.

10 1760 ISSN CN /Q Chin J Biotech November 25, 2014 Vol.30 No qrt-pcr 检测 RNA 水平差异 GFP qpcr mirna GFP RNA RNA A poly(t) adapter mirna poly(t) adapter qpcr mirna GFP gfpqrt RP GFP mrna osamir156 osamir397 actin pbi156spl pbi156splm osamir156 pbi221-pml ( 3A) pbi397lac pbi397lacm osamir397 pbi221-pml ( 3B) 图 3 qrt-pcr 检测转化原生质体中 mirna 和 GFP 基因的表达水平 Fig. 3 Quantification of mirna and GFP transcripts in rice protoplasts by qrt-pcr. (A) Relative expression level of osamirna156 in protoplasts transfected with pbi156spl, pbi156splm, and control plasmid. (B) Relative expression level of osamirna397 in protoplasts transfected with pbi156spl, pbi156splm, and control plasmid. (C) Relative expression level of GFP in protoplasts with pbi156spl or pbi156splm, respectively. (D) Relative expression level of GFP in protoplasts with pbi397lac or pbi397lacm, respectively. Each column represents three replicates, and bars indicate SDs.

11 郭萍等 / 利用水稻原生质体快速分析 mirna 靶标 RNA 1761 osamir156 pbi156spl GFP pbi156splm 58% (P<0.05 3C) osamir397 pbi397spl GFP pbi397lacm 71% (P<0.05 3D) GFP GFP mirna mirna 3 讨论 MicroRNAs RNA DNA [12] [13-17] mirbase 592 mirna mirna RNA mirna [18-19] microrna mirna mirna mirna mirna 5 RACE RNA [20] mirna [10] mirna [11] mirna [20] mirna AGO mirna AGO [21] RNA RNA [22] microrna mirna 40% mirna GFP/ GFP mirna qpcr mrna GFP mirna mirna mrna DCL AGO RDR [23] mirna cjb@im.ac.cn

12 1762 ISSN CN /Q Chin J Biotech November 25, 2014 Vol.30 No.11 REFERENCES [1] Voinnet O. Post-transcriptional RNA silencing in plant-microbe interactions: a tuch of robustness and versatility. Curr Opin Plant Biol, 2008, 11(4): [2] Gaur RK, Rossi JJ. Regulation of gene expression by small RNAs. New York: Cold Spring Harbor Laboratory Press, 2009: 9. [3] Lee RC, Feinbaum RL, Ambros V. The C. elegas heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 1993, 75(5): [4] Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature, 2000, 403(6772): [5] Li YF, Zheng Y, Addo-Quaye C, et al. Transcriptome-wide identification of microrna targets in rice. Plant J, 2010, 62(5): [6] Llave C, Xie ZX, Kasschau KD. Cleavage of scarecrow-like mrna targets directed by a class of Arabidopsis mirna. Science, 2002, 297(5589): [7] Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press, 2002: [8] Bart R, Chern M, Park CJ, et al. A novel system for gene silencing using sirnas in rice leaf and stem-derived protoplasts. Plant Methods, 2006, 2: 13. [9] Shi R, Chiang VL. Facile means for quantifying microrna expression by real-time PCR. BioTechniques, 2005, 39(4): [10] Jiao YQ, Wang YH, Xue DW, et al. Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nat Genet, 2010, 42(6): [11] Zhang YC, Yu Y, Wang CY, et al. Overexpression of microrna Osmir397 improves rice yield by increasing grain size and promoting panicle branching. Nat Biotechnol, 2013, 31(9): [12] Cuperus JT, Fahlgren N, Carrington JC. Evolution and functional diversification of MIRNA genes. Plant Cell, 2011, 23(2): [13] Neilson JR, Zheng GXY, Burge CB, et al. Dynamic regulation of mirna expression in ordered stages of cellular development. Genes Dev, 2007, 21(5): [14] Zhu QH, Spriggs A, Matthew L, et al. A diverse set of micrornas and microrna-like small RNAs in developing rice grains. Genome Res, 2008, 18(9): [15] Fritz JH, Girardin SE, Philpott DJ. Innate immune defense through RNA interference. Sci STKE, 2006, 2006(339): pe27. [16] Nonogaki H. MicroRNA gene regulation cascades during early stages of plant development. Plant Cell Physiol, 2010, 51(11): [17] Khraiwesh B, Zhu JK, Zhu JH. Role of mirnas and sirnas in biotic and abiotic stress responses of plants. Biochim Biophys Acta, 2011, 1819(2): [18] Brennecke J, Stark A, Russell RB, et al. Principles of MicroRNA-target recognition. Plos Biol, 2005, 3(3): [19] Bartel DP, Chen CZ. Micromanagers of gene expression: the potentially widespread influence of metazoan micrornas. Nat Rev Genet, 2004, 5(5): [20] Llave C, Xie ZX, Kasschau KD, et al. Cleavage of Scarecrow-like mrna targets directed by a class of Arabidopsis mirna. Science, 2002, 297(5589): [21] Kapoor M, Arora R, Lama T, et al. Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA Polymerase gene families and their expression analysis during reproductive development and stress in rice. BMC Genomics, 2008, 9(1): [22] Finnegan EJ, Matzke MA. The small RNA world. J Cell Sci, 2003, 116(23): [23] Qian YX, Cheng Y, Cheng X, et al. Identification and characterization of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families in maize. Plant Cell Rep, 2011, 30(7): ( 本文责编陈宏宇 )

2004级研究生现代免疫学实验技术结业报告

2004级研究生现代免疫学实验技术结业报告 PCR PCR PCR RT-PCR PCR (Polymerase Chain Reaction,PCR) 80 DNA, DNA PCR PCR PCR DNA PCR -- -- DNA DNA 93 DNA PCR DNA DNA ( ) DNA 55 DNA DNA -- TaqDNA dntp DNA -- -- PCR PCR DNA DNA Y (1 X) n Y DNA X (Y)