RNAi技术及其在真菌基因功能研究中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2015.08.008

摘要/Abstract

摘要： RNA干扰是指在进化过程中由高度保守的外源或内源双链RNA（dsRNA）引发的转录后水平基因沉默的现象。RNAi技术作为一种高效、特异的基因阻断技术,已被广泛用于探索基因功能和传染性疾病及恶性肿瘤的治疗领域,成为2l世纪的研究热点之一。简要综述了RNAi的发现过程,作用机制,作用特点及其在真菌基因功能研究中的应用前景,为相关研究奠定理论基础。

关键词: RNA干扰, 基因沉默, 真菌, 基因功能

Abstract: RNA interference (RNAi) is a phenomenon of post-transcriptional gene silencing triggered by highly conserved exogenous or endogenous double-strand RNA (dsRNA) during the evolution. RNAi, as an efficient and specific gene blocking technology, is widely applied to gene functional studies and the areas of treating infectious diseases and malignant tumors. RNAi has become one of the hotspots in the 21st century. This paper reviewed the several aspects of RNAi, including the history of discovering it, mechanism, characteristics, and the application prospects in fungal gene functional analysis, which may provide the theoretical basis for related researches.

Key words: RNA interference, gene silencing, fungi, gene function

苏子敬, 李巧玲, 黄程, 谢成建, 杨星勇. RNAi技术及其在真菌基因功能研究中的应用[J]. 生物技术通报, 2015, 31(8): 50-58.

Su Zijing, Li Qiaoling, Huang Cheng, Xie Chengjian, Yang Xingyong. RNAi Technology and Its Application in Fungal Gene Functional Studies[J]. Biotechnology Bulletin, 2015, 31(8): 50-58.

参考文献

[1] Nakayashiki H, Nguyen QB. RNA interference：roles in fungal biology[J]. Curr Opin Microbiol, 2008, 11（6）：494-502.
[2] Fire A. RNA-triggered gene silencing[J]. Trends Genet, 1999, 15（9）：358-263.
[3] 张旭, 张树彪, 金梅. RNA干扰在植物中的应用[J]. 安徽农学通报, 2011（7）：32-38.
[4] Nakayashiki H. RNA silencing in fungi：mechanisms and applications[J]. FEBS Lett, 2005, 579（26）：5950-5957.
[5] Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans[J]. Plant Cell, 1990, 2（4）：279-289.
[6] Jorgensen R, Cluster P, English J, et al. Chalcone synthase cosuppression phenotypes in petunia flowers：comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences[J]. Plant Mol Biol, 1996, 31（5）：957-973.
[7] Cogoni C, Romano N, Macino G. Suppression of gene expression by homologous transgenes[J]. Antonie Van Leeuwenhoek, Leeuw Int J G, 1994, 65（3）：205-209.
[8] Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed[J]. Cell, 1995, 81（4）：611-620.
[9] Cogoni C, Irelan JT, Schumacher M, et al. Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation[J]. Embo J, 1996, 15（12）：3153-3163.
[10] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391（6669）：806-811.
[11] 魏敏. RNA干扰及其应用[J]. 科技与企业, 2011（11）：129.
[12] 胡大林, 庄志雄, 何云. RNA干扰与基因沉默的分子机制研究进展[J]. 中国职业医学, 2005, 32（4）：46-48.
[13] Hammond SM, Boettcher S, Caudy AA, et al. Argonaute 2, a link between genetic and biochemical analyses of RNAi[J]. Science, 2001, 293（5532）：1146-1150.
[14] Dugas DV, Bartel B. MicroRNA regulation of gene expression in plants[J]. Curr Opin Plant Biol, 2004, 7（5）：512-520.
[15] Dang YK, Yang QY, Xue ZH, et al. RNA interference in fungi：pathways, functions, and applications[J]. Eukaryot Cell, 2011, 10（9）：1148-1155.
[16] Cerutti H, Ma XR, Msanne J, et al. RNA-mediated silencing in algae：biological roles and tools for analysis of gene function[J]. Eukaryot Cell, 2011, 10（9）：1164-1172.
[17] He L, Hannon GJ. MicroRNAs：Small RNAs with a big role in gene regulation[J]. Nat Rev Genet, 2004, 5（7）：522-531.
[18] 樊荣辉, 武治印, 王永强, 等. RNAi技术及其在真菌基因功能研究中的应用[J]. 菌物学报, 2010, 29（3）：315-320.
[19] Zamore PD. RNA interference：listening to the sound of silence[J]. Nat Struct Biol, 2001, 8（9）：746-750.
[20] Harborth J, Elbashir SM, Bechert K, et al. Identification of essential genes in cultured mammalian cells using small interfering RNAs[J]. J Cell Sci, 2001, 114（24）：4557-4565.
[21] Nykänen A, Haley B, Zamore PD. ATP requirements and small interfering RNA structure in the RNA interference pathway[J]. Cell, 2001, 107（3）：309-321.
[22] Randall G, Grakoui A, Rice CM. Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs[J]. Proc Natl Acad Sci USA, 2003, 100（1）：235-240.
[23] Verma NK, Dey CS. RNA-mediated gene silencing：mechanisms and its therapeutic applications[J]. J Clin Pharm Ther, 2004, 29（5）：395-404.
[24] Mahmood-Ur-Rahman, Ali I, Husnain T, et al. RNA interference：The story of gene silencing in plants and humans[J]. Biotechnology Advances, 2008, 26（3）：202-209.
[25] 侯义龙. RNA干扰及其在植物上的应用研究进展与展望[J]. 信阳师范学院学报：自然科学版, 2010, 23（2）：316-320.
[26] Brantl S. Antisense-RNA regulation and RNA interference[J]. Biochimica et Biophysica Acta（BBA）- Gene Structure and Expression, 2002, 1575（1-3）：15-25.
[27] Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR：siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs[J]. Cell, 2001, 107（3）：297-307.
[28] McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs[J]. Nat Rev Genet, 2002, 3（10）：737-747.
[29] Davenport RJ. A faster way to shut down genes[J]. Science, 2001, 292（5521）：1469-1471.
[30] 陈莉. 核酸干扰技术的应用前景[J]. 医学研究生学报, 2008, 21（10）：1009-1010.
[31] Singh N, Rajam M. RNA Interference and functional genomics in fungi[M]//Microorganisms in Sustainable Agriculture and Biotechnology. Springer Netherlands, 2012：773-792.
[32] Matityahu A, Hadar Y, Dosoretz CG, et al. Gene silencing by RNA interference in the white rot fungus Phanerochaete chrysosporium[J]. Applied and Environmental Microbiology, 2008, 74（17）：5359-5365.
[33] Liu H, Wang P, Gong G, et al. Morphology engineering of Penicillium chrysogenum by RNA silencing of chitin synthase gene[J]. Biotechnol Lett, 2013, 35（3）：423-429.
[34] Nakayashiki H, Hanada S, Quoc NB, et al. RNA silencing as a tool for exploring gene function in ascomycete fungi[J]. Fungal Genet Biol, 2005, 42（4）：275-283.
[35] Mouyna I, Henry C, Doering TL, et al. Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus[J]. FEMS Microbiol Lett, 2004, 237（2）：317-324.
[36] Carneiro JS, de la Bastide PY, Hintz WE. Regulated gene silencing in the fungal pathogen Ophiostoma novo-ulmi[J]. Physiological and Molecular Plant Pathology, 2013, 82：28-34.
[37] Moazeni M, Khoramizadeh MR, Kordbacheh P, et al. RNA-mediated gene silencing in Candida albicans：Inhibition of hyphae formation by use of RNAi technology[J]. Mycopathologia, 2012, 174（3）：177-185.
[38] Chi JT, Chang HY, Wang NN, et al. Genomewide view of gene silencing by small interfering RNAs[J]. Proc Natl Acad Sci USA, 2003, 100（11）：6343-6346.
[39] Thompson JD. Applications of antisense and siRNAs during preclinical drug development[J]. Drug Discov Today, 2002, 7（17）：912-917.
[40] Caracuel-Rios Z, Talbot NJ. Silencing the crowd：high-throughput functional genomics in Magnaporthe oryzae[J]. Mol Microbiol, 2008, 68（6）：1341-1344.
[41] 粟挺, 刘爱玲, 陈信波. RNA干扰载体构建方法的研究进展[J]. 湖南农业科学, 2011（19）：1-4.
[42] Bachman J. Site-directed mutagenesis[M]// Lorsch J. Laboratory Methods in Enzymology：DNA. UD, USA：Academic Press, 2013, 529：241-248.
[43] Hofer U. Techniques and application RNAi ‘off-targets’ pathogen infection[J]. Nature Reviews Microbiology, 2014, 12（5）：314-315.
[44] Oliveira-Garcia E, Deising HB. Infection structure-specific expres-sion of beta-1, 3-glucan synthase is essential for pathogenicity of Colletotrichum graminicola and evasion of beta-glucan-triggered immunity in Maize[J]. Plant Cell, 2013, 25（6）：2356-2378.
[45] Panwar V, McCallum B, Bakkeren G. Host-induced gene silencing of wheat leaf rust fungus Puccinia triticina pathogenicity genes mediated by the Barley stripe mosaic virus[J]. Plant Molecular Biology, 2013, 81（6）：595-608.
[46] Wang YD, Yang PC, Cui F, et al. Altered immunity in crowded locust reduced fungal（Metarhizium anisopliae）pathogenesis [J]. PLoS Pathogens, 2013, 9（1）：e1003102.
[47] Barad S, Horowitz SB, Kobiler I, et al. Accumulation of the mycotoxin patulin in the presence of gluconic acid contributes to pathogenicity of Penicillium expansum[J]. Molecular Plant-Microbe Interactions, 2013, 27（1）：66-77.
[48] Carreras-Villasenor N, Esquivel-Naranjo EU, Manuel Villalobos-Escobedo J, et al. The RNAi machinery regulates growth and development in the filamentous fungus Trichoderma atroviride[J]. Molecular Microbiology, 2013, 89（1）：96-112.
[49] Liu H, Cottrell TR, Pierini LM, et al. RNA interference in the pathogenic fungus Cryptococcus neoformans[J]. Genetics, 2002, 160（2）：463-470.
[50] Krajaejun T, Gauthier GM, Rappleye CA, et al. Development and a application of a green fluorescent protein sentinel system for identification of RNA interference in Blastomyces dermatitidis illuminates the role of septin in morphogenesis and sporulation[J]. Eukaryot Cell, 2007, 6（8）：1299-1309.
[51] Rappleye CA, Engle JT, Goldman WE. RNA interference in Histoplasma capsulatum demonstrates a role for alpha-（1, 3）-glucan in virulence[J]. Mol Microbiol, 2004, 53（1）：153-165.
[52] Lawrence GJ, Dodds PN, Ellis JG. Transformation of the flax rust fungus, Melampsora lini：selection via silencing of an avirulence gene[J]. Plant J, 2010, 61（2）：364-369.
[53] Liu H, Wang P, Hu YH, et al. Construction of an RNAi expression vector and transformation into Penicillium chrysogenum[J]. Annals of Microbiology, 2014, 64（1）：113-120.
[54] Singh S, Braus-Stromeyer S, Timpner C, et al. Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem[J]. Appl Microbiol Biotechnol, 2010, 85（6）：1961-1976.
[55] Nguyen QB, Kadotani N, Kasahara S, et al. Systematic functional analysis of calcium-signalling proteins in the genome of the rice-blast fungus, Magnaporthe oryzae, using a high-throughput RNA-silencing system[J]. Mol Microbiol, 2008, 68（6）：1348-1365.
[56] Nguyen QB, Itoh K, Van Vu B, et al. Simultaneous silencing of endo-β-1, 4 xylanase genes reveals their roles in the virulence of Magnaporthe oryzae[J]. Mol Microbiol, 2011, 81（4）：1008-1019.
[57] Mu DS, Shi L, Ren A, et al. The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum[J]. PLoS One, 2012, 7（8）：e43737.
[58] Khatri M, Rajam MV. Targeting polyamines of Aspergillus nidulans by siRNA specific to fungal ornithine decarboxylase gene[J]. Medical Mycology, 2007, 45（3）：211-220.
[59] Whisson SC, Avrova AO, Van West P, et al. A method for double-stranded RNA-mediated transient gene silencing in Phytophthora infestans[J]. Mol Plant Pathol, 2005, 6（2）：153-163.
[60] Nunes CC, Dean RA. Host-induced gene silencing：a tool for understanding fungal host interaction and for developing novel disease control strategies[J]. Mol Plant Pathol, 2012, 13（5）：519-529.