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1 生物工程学报 Chin J Biotech 2009, November 25; 25(11): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 生物技术与方法 trna 巩菊芳 1, 曹诣斌 1, 陈祥龙 2, 齐育平 1, 杨小强 1, 金晓玲 1, , , : 利用 T7 RNA 聚合酶 (T7 RNA polymerase, T7 RNAP) 的体外转录方法因其简便高效而在 RNA 制备中得到广泛应用, 但该法由于 T7 RNAP 的启动子跨越转录起始位点而会导致转录产物多出附加序列 ; 如果去掉 T7 启动子的转录起始位点, 则会严重降低 T7 RNAP 的转录活性本实验很好地消除了以上弊端, 将 T7 RNAP 的高效转录系统与核酶高度专一的自剪切技术相结合, 成功构建了一种不影响转录效率的体外转录方法, 而且转录后核酶能够在设计的特定位点进行自剪切并释放出目的 RNA, 得到了活性达 pmol/µg 的人线粒体色氨酸 trna(hmtrna ) 该方法转录效率高重复性好, 适合 RNA 的大量精确制备 : 体外转录, 核酶, 人线粒体色氨酸 trna 3 High-level transcription of human mitochondrial trna Trp based on ribozyme and characterization on its aminoacylation activity Jufang Gong 1, Yibin Cao 1, Xianglong Chen 2, Yuping Qi 1, Xiaoqiang Yang 1, and Xiaoling Jin 3 1 College of Chemistry and Biology, Zhejiang Normal University, Jinhua , China 2 State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai , China 3 School of Environment and Art Design, Central South University of Forestry and Technology, Changsha , China Abstract: In vitro transcription systems with T7 RNA polymerase (T7 RNAP) were widely used in preparation of RNA because of their simplicity and high efficiency. The transcripts would have additional 5 sequence since T7 promoter spans the transcription start site, while deletion of the transcription start site would severely reduce the T7 RNAP transcriptional activity. We successfully developed an in vitro transcription by combining of T7 RNAP high efficient transcription system and highly specific self-splicing technology of ribozymes, in this system, ribozyme self-splices at the designed specific site and releases the aim RNA without affecting transcription efficiency of T7 RNAP, the aminoacylation activity of human mitochondrial trna Trp (HmtRNA ) is pmol/µg. This method with its high efficiency on transcription and good repeatability is Received: July 27, 2009; Accepted: October 9, 2009 Supported by: Natural Science Foundation of Zhejiang Province (Nos. Y205353, Y ). Corresponding author: Jufang Gong. Tel: ; Fax: ; E- mail: sky103@zjnu.cn 浙江省自然科学基金项目 (No. Y205353, Y ) 资助

2 : trna 1733 very suitable for preparation of accurate RNA in large scale. Keywords: transcription in vitro, ribozyme, human mitochondrial trna Trp trna [1], trna Trp (HmtRNA ) [2-5], HmtRNA, trna (H mt TrpRS) trna Trp, HmtRNA RNA, T7 RNAP,, 5 -GGG, T7 RNAP, RNA, RNA, RNA, 5 -GGG, T7 RNAP,, 5 -GGG RNA, 5 -AGA HmtRNA, T7 RNAP RNA, 5 HmtRNA, trna, HmtRNA 1 材料和方法 1.1 菌株和质粒 Escherichia coli DH5α PMD18-T TaKaRa 1.2 酶和试剂 BstN I New England Biolabs, T4 DNA dntps Ex Taq DNA TaKaRa, RiboMAX TM Large Scale RNA Production System-T7 Promega, L-[5-3 H] Amersham Pharmacia, L- Gibco BRL, Ni-NTA agarose Qiagen, HmtRNA Invitrogen, BBI, 1.3 方法 HmtRNA Haseloff [6],, pcfold RNA, 33, 22 nt 11 nt (5 5 nt 3 6 nt) 5 -ATTTCTCTGATGAGTCCGTGAGGACGAA ATAGT-3 ( ) HmtRNA ( 1) ~btc914/search/index. html HmtRNA Trp (UCA), 5 -AGAAATTTA GGTTAAATACAGACCAAGAGCCTTCAAAGCCC TCAGTAAGTTGCAATACTTAATTTCTGCCA-3, HmtRNA 1: 5 -ATTTCTCTGATGAGTCCGTGAGG ACGAAATAGTACTCACTATTAGAAATTT-3 2: 5 -CTCTAATACGACTCACTATAGGGA TTTCTCTGATGAGTCCGTGAG-3, T7 3: 5 -GCCTGGCAGAAATTAA-3, BstN I PCR, 1 PCR 1 3, HmtRNA, : 94 o C 5 min; 94 o C 30 s, 48 o C 25 s, 72 o C 20 s, 30 ; 72 o C 10 min trna 5 3 BstN I 5 -CCTGG-3, 2 PCR 2 3, 1 PCR,,,

3 1734 ISSN CN /Q Chin J Biotech November 25, 2009 Vol.25 No.11 图 1 带有 T7 启动子和锤头核酶基因的 HmtRNA 基因重组质粒构建 Fig. 1 Construction of the recombinant plasmid of HmtRNA gene. 1 PCR 5 T7 5 -CTCT- AATACGACTCACTATAGGG-3 2 PCR T4 DNA PMD18-T, DH5α,,, Invitrogen, T7 5 -CTCTAATACGACTCACTATA-3 ( 5) 3, HmtRNA PCR, HmtRNA PCR DNA,,, PMD18-T 4,, : 5 -TGCCTGCAGGTCGACGATT-3, 2 4, 159 bp,, 60 o C, BstN I 4 h CCA, : : (25: 24: 1, V/V/V),, 139 bp µl : 1 µg BstN I HmtRNA, 40 mmol/l Tris-HCl(pH 8.0), 15 mmol/l MgCl 2, 5 mmol/l DTT, 500 mg/l BSA, 1.5 mmol/l dntps, 40 U RNasin, 200 U T7 RNAP 37 o C 4 h l0 U DNase, 37 o C 30 min DNA 8 mol/l 16%, [7] HmtRNA, 94 o C 2 min,, HmtRNA Trp (UCA) trna (H mt TrpRS) HmtRNA H mt TrpRS ( U/mg), : 50 μl 4 mmol/l ATP 1 mmol/l DTT 1 μci L-[5-3 H] 6 μmol/l, 40 mmol/l MgAc mmol/l Tris-HCl(pH 7.8) 1 g/l, 20 µl HmtRNA, l00 ml

: trna 1735 50 g/ltca( 0.05 ) 3, l0 ml 95% 1 Wallac 1490 [5-3 H]-, Line-Burk HmtRNA (pmol/ g) trna, : : (25: 24: 1, V/V/V),, 139 bp 1.7%, ( 3) 2 结果 2.

2 PCR products analysis. M: DNA marker; 1: the first PCR products of human mitochondrial trna Trp, 117 bp; 2:

4 : trna g/ltca( 0.05 ) 3, l0 ml 95% 1 Wallac 1490 [5-3 H]-, Line-Burk HmtRNA (pmol/ g) trna, : : (25: 24: 1, V/V/V),, 139 bp 1.7%, ( 3) 2 结果 2.1 带有核酶结构的野生型 HmtRNA Trp(UCA) 基因的质粒构建 71 nt HmtRNA PCR, 117 bp; PCR 140 bp PCR 1%, ( 2) PCR, 5 T7, 3 BstN I 2 PCR PMD18-T ( PMD18-T- HmtRNA ), DH5α, 图 2 第一次和第二次 PCR 扩增产物鉴定图 Fig. 2 PCR products analysis. M: DNA marker; 1: the first PCR products of human mitochondrial trna Trp, 117 bp; 2: the second PCR products of human mitochondrial trna Trp, 140 bp. 2.2 转录前 PCR 扩增及酶切结果 BstN I trna CCA,, 2 PMD18-T 4, PMD18-T- HmtRNA PCR, 159 bp,, 60 o C, BstN I 4 h CCA 图 3 PCR 产物和 BstN I 酶切结果 Fig. 3 PCR products and restriction enzyme digestion analysis. M: DNA marker; 1: products digested with BstN I, 139 bp; 2: PCR products, 159 bp. 2.3 体外转录及核酶自剪切释放目的片段 HmtRNA 4, T7, 33, 22 nt 11 nt ( 5 5 nt 3 6 nt), BstN I 2 : HmtRNA ) 71 nt, 48 nt, 2, 119 nt ( 5), 119 nt, HmtRNA ) 71 nt,,,, 85.6% trna, 71 nt 1 µg, 100 µg HmtRNA ( 6) 2.4 HmtRNA Trp(UCA) 动力学常数测定 HmtRNA, 50 µl, 200 nmol/l HmtTrpRS,

1736 ISSN1000-3061 CN11-1998/Q Chin J Biotech November 25, 2009 Vol.25 No.11 图 4 锤头状核酶结构及剪切位点示意图 Fig. 4 Schematic representation of the hammerhead ribozyme. Arrow indicates the cleavage site.

5 1736 ISSN CN /Q Chin J Biotech November 25, 2009 Vol.25 No.11 图 4 锤头状核酶结构及剪切位点示意图 Fig. 4 Schematic representation of the hammerhead ribozyme. Arrow indicates the cleavage site. 图 5 转录后核酶自剪切产物电泳图 Fig. 5 Transcription products. 1: control group; 2: trial group. trna 0.1 ~1.8 μmol/l HmtRNA HmtTrpRS pmol/µg 3 讨论 T7 RNAP, RNA [8-9], T7,, RNA [10], RNA RNA, T7 RNAP T7 [11], T , 2, ,, 图 6 野生型人线粒体 trna Trp 结构图 Fig. 6 Wild-type human mitochondria trna Trp., G( 4), T7 RNAP, T7 RNAP N T [12-14], +1 G, T7 RNAP 1/2 [15], +3 G, 3/4 [16], 5 -GGG T7 RNAP T G, T7 RNAP, trna 5

6 : trna 1737, trna,, RNA RNA, ( ),,,,, RNA,,,,, HmtRNA Trp(UCA) 3, 1, 2 HmtRNA Trp(UCA),,,, 1 µg, 100 µg HmtRNA RNA, 100, 85.6%, 100%, [17],, 50 o C 37 o C,,, Mg 2+,, RNA,, trna,, RNA REFERENCES [1] Filosto M, Tomelleri G, Tonin P. Neuropathology of mitochondrial diseases. Biosci Rep, 2007, 27: [2] Maniura-Weber K, Taylor RW, Johnson MA, et al. A novel point mutation in the mitochondrial trna(trp) gene produces a neurogastrointestinal syndrome. Eur J Hum Genet, 2004, 12(6): [3] Ribas de Pouplana L, Schimmel P. A view into the origin of life: aminoacyl-trna synthetases. Cell Mol Life Sci, 2000, 57(6): [4] Tulinius M, Moslemi AR, Darin N, et al. Leigh syndrome with cytochrome-c oxidase deficiency and a single T insertion nt 5537 in the mitochondrial trna Trp gene. Neuropediatrics, 2003, 34(2): [5] Valente L, Piga D, Lamantea E, et al. Identification of novel mutations in five patients with mitochondrial encephalomyopathy. Biochim Biophys Acta, 2009, 1787(5): [6] Haseloff J, Gerlach WL. Simple RNA enzyme with new and highly specific endoribonuclease activity. Nature, 1988, 334(6183): [7] Sambrook J, Russell DW. Molecular Cloning. 3rd ed. Beijing: Science Press, J, DW.. 3. :, [8] Landick R. Active-site dynamics in RNA polymerases. Cell, 2004, 116(3): [9] Gamer M, Fröde D, Biedendieck R, et al. A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium. Appl Microbiol Biotechnol, 2009, 82: [10] Huang YX, Zhang L, Zhu SG. Construction of high-level transcription system in vitro by PCR. Prog Biochem Biophys, 1993, 20(6): ,,. PCR., 1993, 20(6): [11] Klement JF, Moorefield MB, Jorgensen E, et al. Discrimination between bacteriophage T3 and T7 promoters by the T3 and T7 RNA polymerases depends primarily upon a three base-pair region located 10 to 12 base-pairs upstream from the start site. J Mol Biol, 1990, 215(1): [12] Filippova SE, Ivanovskaia MG, Romanova EA, et al. Point contacts of T7 RNA polymerase in the promotor complex, as determined with phosphate-activated oligonucleotide derivatives. Mol Biol (Mosk), 2002, 36(4): [13] Yin YW, Steitz TA. Structural basis for the transition from

1738 ISSN1000-3061 CN11-1998/Q Chin J Biotech November 25, 2009 Vol.25 No.11 initiation to elongation transcription in T7 RNA polymerase. Science, 2002, 298(5597): 1387 1395.

Constuction of bacteriophone T7 late promoters with point mutations and characterization by in vitro transcripition properties. Nucleic Acids Res, 1987, 15(13): 5413 32.

7 1738 ISSN CN /Q Chin J Biotech November 25, 2009 Vol.25 No.11 initiation to elongation transcription in T7 RNA polymerase. Science, 2002, 298(5597): [14] Nayak D, Guo Q, Sousa R. Functional architecture of T7 RNA polymerase transcription complexes. J Mol Biol, 2007, 371(2): [15] Chapman KA, Burgess RR. Constuction of bacteriophone T7 late promoters with point mutations and characterization by in vitro transcripition properties. Nucleic Acids Res, 1987, 15(13): [16] Ikeda RA, CM Ligman, Warshamana S. T7 promoter contacts essential for promoter activity in vivo. Nucleic Acids Res, 1992, 20 (10): [17] Liu CH, Sun LQ, Tian B. Ribozyme which specifically cleave 12-lipoxygenase mrna: in vitro activity and its dynamics. Chin J Biotech, 2000, 16(1): ,,. 12 mrna., 2000, 16(1): 科学出版社科学出版中心生命科学分社新书推介中国生物入侵研究 ( 中英版 ) 万方浩郭建英张峰等著中文版英文版年 10 月出版 2009 年 10 月出版

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇