植物介导的RNA干扰引起马铃薯晚疫病菌基因的沉默

doi:10.3724/SP.J.1006.2015.00881

摘要/Abstract

摘要：

由致病疫霉(Phytophthora infestans)引起的晚疫病是最具毁灭性的马铃薯病害。为明确植物介导的RNAi沉默致病疫霉基因的有效性，本研究采用重叠延伸PCR技术克隆同时与晚疫病菌4个ces基因均同源的融合基因C₁₂₃₄，构建内含子连接的C₁₂₃₄反向重复序列植物表达载体，采用农杆菌介导法转化晚疫病易感马铃薯品种大西洋，经PCR和Southern杂交检测，获得129个转基因株系。离体叶片接种病原菌后，有97个转基因株系发病速度明显慢于野生型，接种6 d后病斑大小和霉层厚度均明显小于对照，并且叶片感病部位没有出现失绿斑，而野生型产生了明显的失绿斑。实时定量RT-PCR分析发现，发病延缓的叶片上致病疫霉4个纤维素合酶基因的表达水平明显低于野生型。本研究表明转基因植株中产生的以晚疫病菌ces基因为靶标的dsRNA能够沉默致病疫霉相应基因表达，延缓发病进程。

关键词: 植物介导的RNAi, 马铃薯, 致病疫霉, 基因沉默

Abstract:

Potato late blight caused by Phytophthora infestans is the most devastating disease in potato. The objective of the work was to test the efficiency of plant-meidated RNAi in silencing genes in P. infestans and to find a new way to breed potato resistant to late blight. Over-lap PCR was employed to amplify a fused-gene C₁₂₃₄ simultaneously homologous to four cellulose synthase genes in P. infenstans. Then, a plant expression vector containing inverted repeat of C₁₂₃₄was constructed and transferred to Atlantic, a potato variety severely susceptible to late blight by agrobacteria-mediated genetic transformation. A hundred and twenty nine regenerated lines were confirmed to be transgenic plants with PCR and Southern blot. When detached leaves were inoculated with P. Infestans, 97 out of 129 transgenic lines delayed disease symptoms with smaller lesions and less hyphae compared with the wild type at six days after inoculation. Chlorotic spots disappeared on leaves from transgenic lines while appeared severely on leaves from the wild type at the same day. mRNA accumulation of the four cellulose synthase genes from P. infestans colonized on leaves from transgenic plants with delayed symptoms was significantly lower compared with the wild type with Real-time RT-PCR. The results demonstrated that dsRNA of C₁₂₃₄ generated in transgenic potato could induce homologous genes silence and delay the process of infection in intimately touched P. infestans.

Key words: Plant-mediated RNAi, Potato, Phytophthora infestans, Gene silence

任琴,王亚军,郭志鸿,李继平,谢忠奎,王若愚,王立,惠娜娜. 植物介导的RNA干扰引起马铃薯晚疫病菌基因的沉默[J]. 作物学报, 2015, 41(06): 881-888.

REN Qin,WANG Ya-Jun,GUO Zhi-Hong,LI Ji-Ping,XIE Zhong-Kui,WANG Ruo-Yu,WANG Li,HUI Na-Na. Gene Silence of Phytophthora infestans Induced by Plant-mediated RNA Interference in Potato[J]. Acta Agron Sin, 2015, 41(06): 881-888.

参考文献

[1]Haverkort A, Boonekamp P, Hutten R, Jacobsen E, Lotz L, Kessel G, Visser R, Van der Vossen E. Societal costs of late blight in potato and prospects of durable resistance through cisgenic modification. Potato Res, 2008, 51: 47–57

[2]Haas B J, Kamoun S, Zody M C, Jiang R H, Handsaker R E, Cano L M, Grabherr M, Kodira C D, Raffaele S, Torto-Alalibo T. Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans. Nature, 2009, 461: 393–398

[3]McDonald B A, Linde C. Pathogen population genetics, evolutionary potential, and durable resistance. Annu Rev Phy opathol, 2002, 40: 349–379

[4]Fry W. Phytophthora infestans: the plant (and R gene) destroyer. Mol Plant Pathol, 2008, 9: 385–402

[5]杨艳丽, 肖浪涛, 胡先奇. 马铃薯晚疫病菌毒素与寄主品种抗性关系研究. 中国农业科学, 2009, 42: 2202–2210

Yang Y L, Xiao L Tao, Hu X Q. Study on the relationship between the toxin of Phytophthora infestans and resistance of potato. Sci Agric Sin, 2009, 42: 2202–2210 (in Chinese with English abstract)

[6]Hendrik R, Evert J. 马铃薯广谱抗病Rpi-blb1同源基因抗性被金华晚疫病菌株克服. 园艺学报, 2010, 37: 241–246

Hendrik R, Evert J. Broad spectrum resistance from Rpi-blb1 homologous R-genes has been broken by Phytophthora infestans isolates collected from Solanum stoloniferum. Acta Hort Sin, 2010, 37: 241–246 (in Chinese with English abstract)

[7]徐建飞, 辛翠花, 黄三文. 利用田间抗病基因近等混合系防治马铃薯晚疫病. 中国农业科技导报, 2006, 8: 8–13

Xu J F, Xin C H, Huang S W. Potato late blight control using R-gene polyculture. Rev China Agric Sci Technol, 2006, 8: 8–13 (in Chinese with English abstract)

[8]Zhu Y, Chen H, Fan J, Wang Y, Li Y, Chen J, Fan J, Yang S, Hu L, Leung H. Genetic diversity and disease control in rice. Nature, 2000, 406: 718–722

[9]Leung H, Zhu Y, Revilla-Molina I, Fan J X, Chen H, Pangga I, Cruz C V, Mew T W. Using genetic diversity to achieve sustainable rice disease management. Plant Dis, 2003, 87: 1156–1169

[10]Eamens A, Wang M B, Smith N A, Waterhouse P M. RNA silencing in plants: yesterday, today, and tomorrow. Plant Physiol, 2008, 147: 456–468

[11]Knip M, Constantin M E, Thordal-Christensen H. Trans-kingdom cross-talk: small RNAs on the move. PLoS Genet, 2014, 10: e1004602

[12]Waterhouse P M, Graham M W, Wang M-B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA, 1998, 95: 13959–13964

[13]Shepherd D N, Martin D P, Thomson J A. Transgenic strategies for developing crops resistant to geminiviruses. Plant Sci, 2009, 176: 1–11

[14]Escobar M A, Civerolo E L, Summerfelt K R, Dandekar A M. RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis. Proc Natl Acad Sci USA, 2001, 98: 13437–13442

[15]Nowara D, Gay A, Lacomme C, Shaw J, Ridout C, Douchkov D, Hensel G, Kumlehn J, Schweizer P. HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell Online, 2010, 22: 3130–3141

[16]Yin C, Jurgenson J E, Hulbert S H. Development of a host-induced RNAi system in the wheat stripe rust fungus Puccinia striiformis f. sp. tritici. Mol Plant-Microbe Interact, 2011, 24: 554–561

[17]Koch A, Kumar N, Weber L, Keller H, Imani J, Kogel K H. Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species. Proc Natl Acad Sci USA, 2013, 110: 19324–19329

[18]Aly R, Cholakh H, Joel D M, Leibman D, Steinitz B, Zelcer A, Naglis A, Yarden O, Gal-On A. Gene silencing of mannose 6-phosphate reductase in the parasitic weed Orobanche aegyptiaca through the production of homologous dsRNA sequences in the host plant. Plant Biotechnol J, 2009, 7: 487–498

[19]Baum J A, Bogaert T, Clinton W, Heck G R, Feldmann P, Ilagan O, Johnson S, Plaetinck G, Munyikwa T, Pleau M. Control of coleopteran insect pests through RNA interference. Nature Biotechnol, 2007, 25: 1322–1326

[20]Mao Y B, Cai W J, Wang J W, Hong G J, Tao X Y, Wang L J, Huang Y P, Chen X Y. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol, 2007, 25: 1307–1313

[21]Upadhyay S K, Chandrashekar K, Thakur N, Verma P C, Borgio J F, Singh P K, Tuli R. RNA interference for the control of whiteflies (Bemisia tabaci) by oral route. J Biosci, 2011, 36: 153–161

[22]Goodwin S B, Drenth A, Fry W E. Cloning and genetic analyses of two highly polymorphic, moderately repetitive nuclear DNAs from Phytophthora infestans. Curr Genet, 1992, 22: 107–115

[23]王关林, 方宏筠. 植物基因工程(第2版). 北京: 科学出版社, 2002. pp 776–777

Wang G L, Fang H Y. Plant Genetic Engineering, 2nd Edn. Beijing: Science Press, 2002. pp 776–777 (in Chinese)

[24]Gustafson V, Mallubhotla S, MacDonnell J, Sanyal-Bagchi M, Chakravarty B, Wang-Pruski G, Rothwell C, Audy P, De Koeyer D, Siahbazi M. Transformation and plant regeneration from leaf explants of Solanum tuberosum L. cv. ‘Shepody’. Plant Cell, Tissue Organ Culture, 2006, 85: 361–366

[25]Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucl Acids Res, 1980, 8: 4321–4326

[26]Grenville-Briggs L J, van West P. The biotrophic stages of oomycete-plant interactions. Adv Appl Microbiol, 2005, 57: 217–244

[27]李先平, 何云昆, 陈善娜, 赵志坚, 李树莲, 李汝刚. 表达HarpinEa基因的转基因马铃薯的晚疫病抗性分析. 云南农业大学学报, 2002, 7(3): 252–257

Li X P, He Y K, Chen S N, Zhao Z J, Li S L, Li R G. Analysis of late blight resistance in transgenic potato expressing HarpinEa gene. J Yunnan Agric Univ, 2002, 7(3): 252–257 (in Chinese with English abstract)

[28]Grenville-Briggs L J, Anderson V L, Fugelstad J, Avrova A O, Bouzenzana J, Williams A, Wawra S, Whisson S C, Birch P R, Bulone V. Cellulose synthesis in Phytophthora infestans is required for normal appressorium formation and successful infection of potato. Plant Cell Online, 2008, 20: 720–738

[29]Koch A, Kogel K H. New wind in the sails: improving the agronomic value of crop plants through RNAi-mediated gene silencing. Plant Biotechnol J, 2014, 12: 821–831

[30]Price D R G, Gatehouse J A. RNAi-mediated crop protection against insects. Trends Biotechnol, 2008, 26: 393–400

[31]Duan C G, Wang C H, Guo H S. Application of RNA silencing to plant disease resistance. Silence, 2012, 3: 5

[32]Zhang M, Wang Q, Xu K, Meng Y, Quan J, Shan W. Production of dsRNA sequences in the host plant is not sufficient to initiate gene silencing in the colonizing oomycete pathogen Phytophthora parasitica. PloS One, 2011, 6: e28114

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