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作物学报 ›› 2016, Vol. 42 ›› Issue (11): 1647-1655.doi: 10.3724/SP.J.1006.2016.01647

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

RNAi介导SMV-P3基因沉默增强大豆对花叶病毒病的抗性

杨向东,牛陆,张伟,杨静,杜茜,邢国杰,郭东全,李启云,董英山*   

  1. 吉林省农业科学院农业生物技术研究所 / 吉林省农业生物技术重点实验室,吉林长春130033
  • 收稿日期:2016-02-16 修回日期:2016-06-20 出版日期:2016-11-12 网络出版日期:2016-07-04
  • 通讯作者: 董英山, E-mail: ysdong@cjaas.com, Tel: +86-431-87063008
  • 基金资助:

    本研究由国家转基因生物新品种培育科技重大专项(2016ZX08004-004)和吉林省科技发展计划项目(20150204011NY)。

RNAi-mediated SMV-P3 Silencing Increases Soybean Resistance to Soybean Mosaic Virus

YANG Xiang-Dong,NIU Lu,ZHANG Wei,YANG Jing,DU Qian,XING Guo-Jie,GUO Dong-Quan,LI Qi-Yun,DONG Ying-Shan   

  1. Jilin Academy of Agricultural Sciences, Agricultural Biotechnology Institute / Jilin Provincial Key laboratory of Agricultural Biotechnology, Changchun 130033, China
  • Received:2016-02-16 Revised:2016-06-20 Published:2016-11-12 Published online:2016-07-04
  • Contact: Dong Yingshan, E-mail: ysdong@cjaas.com, Tel: +86-431-87063008
  • Supported by:

    This study was supported by the China National Novel Transgenic Organisms Breeding Project (2016ZX08004-004) and the Science & Technology Development Project of Jilin Province in China(20150204011NY)。

摘要:

大豆花叶病毒(soybean mosaic virus, SMV)病是我国各大豆主产区最重要的病害之一,严重影响大豆产量和籽粒外观品质。培育抗病品种是防治该病最经济有效的措施。本研究基于植物介导RNA干扰(RNA interference, RNAi)技术,将编码参与SMV运动和影响宿主域范围的P3蛋白基因RNAi片段导入栽培大豆品种,研究RNAi介导SMV-P3基因沉默对大豆抗SMV的影响。Southern杂交检测结果表明,外源RNAi片段以低拷贝的形式(1~4个)整合至大豆基因组中。对T1~T3代转基因大豆喷施除草剂和PCR鉴定结果表明,外源T-DNA插入片段在转基因大豆不同代际间能够稳定遗传。对T2和T3代转基因大豆接种SMV鉴定结果表明,转基因大豆对我国大豆产区主要流行SMV株系SC-3较非转基因对照受体品种Williams 82和SN9的抗性水平显著提高,病情指数降低至4.37%~18.51%,且抗性能够稳定遗传。综上所述,RNAi介导SM-P3基因沉默能够显著提高转基因大豆对SMV的抗性水平。

关键词: 大豆, 大豆花叶病毒病, SMV-P3, RNAi介导基因沉默

Abstract:

Soybean mosaic virus (SMV) is one of the most important diseases in major soybean production areas and has severe effects on soybean production and seed quality in China. Breeding disease-resistant varieties is the most economical and effective strategy to prevent and control SMV. In this study, RNAi fragments of the gene encoding P3 protein, which is involved in SMV mobility and affecting host range, were introduced into soybean by plant-mediated RNA interference (RNAi) techniques to explore the influence of RNAi-mediated SMV-P3 silencing on soybean SMV resistance. Southern blot analysis revealed that exogenous RNAi fragments were integrated into the soybean genome at low copy numbers (1–4). T1–T3 generation transgenic soybeans were sprayed with herbicide and inserted fragments were examined using PCR. The results indicated that T-DNA insertion fragments could be stably inherited between generations of transgenic soybean. Inoculation of T2 and T3 generation transgenic soybeans with SMV suggested that transgenic soybeans exhibited significantly higher resistance to the prevailing SMV strain, SC-3, in major soybean production areas than the non-transgenic control varieties Williams 82 and SN9. The disease index was reduced by 4.37%-18.51%. Further, the resistance could be stably inherited. In conclusion, RNAi-mediated SMV-P3 silencing can significantly increase the SMV resistance of transgenic soybeans.

Key words: Soybean, Soybean mosaic virus, SMV-P3, RNAi-mediated gene silencing

[1]Ross J P. Effect of soybean mosaic on component yields from blends of mosaic resistant and susceptible soybeans. Crop Sci, 1983, 23: 343–346 [2]Yang Y Q, Zheng G J, Han L, Wang D G, Yang X F, Yuan Y, Huang S H, Zhi H J. Genetic analysis and mapping of genes for resistance to multiple strains of Soybean mosaic virus in a single resistant soybean accession PI 96983. Theor Appl Genet, 2013, 126: 1783–1791 [3]Gao L, Ding X N, Li K, Liao W L, Zhong Y K, Ren R, Liu Z T, Karthikeyan A, Zhi H J. Characterization of Soybean mosaic virus resistance derived from inverted repeat SMV HC Pro genes in multiple soybean cultivars. Theor Appl Genet, 2015, 128: 1489–1505 [4]智海剑, 盖钧镒. 大豆花叶病毒及抗性遗传的研究进展. 大豆科学, 2006, 25: 174–180 Zhi H J, Gai J Y. Advances in the studies on soybean mosaic virus. Soybean Sci, 2006, 25: 174–180 [5]Yang Y Q, Lin J, Zheng G J, Zhang M C, Zhi H J. Recombinant soybean mosaic virus is prevalent in Chinese soybean fields. Arch Virol, 2014, 159: 1793–1796 [6]李延华, 吕文清. 大豆花叶病毒的显症率与大豆品种及病毒株系的关系研究. 植物保护学报, 1991, 18: 334–334 Li Y H, Lü W Q. Study on the relationship between the rate of SMV symptom appearance and soybean cultivars and SMV strains. Acta Phytophyl Sin, 1991, 18: 334–334 [7]Koo J M, Choi B K, Ahn H J, Yum H J, Choi C W. First report of an Rsv resistance-breaking isolate of Soybean mosaic virus in Korea. Plant Pathol, 2005, 54 : 573 [8]Choi B K, Koo J M, Ahn H J, Yum H J, Choi C W, Ryu K H, Chen P, Tolin S A. Emergence of Rsv-resistance breaking soybean mosaic virus isolates from Korean soybean cultivars. Virus Res, 2005, 112: 42–51 [9]Gagarinova A G, Babu M, Poysa V, Hill J H, Wang A. Identification and molecular characterization of two naturally occurring Soybean mosaic virus isolates that are closely related but differ in their ability to overcome Rsv4 resistance. Virus Res, 2008, 138: 50–56 [10]KusabaM. RNA interference in crop plant. Curr Opin Biotechno, 2004, 15: 139–143 [11]Ding S W. RNA-based antiviral immunity. Nat Rev Immunol, 2010, 10: 632–641 [12]侯静, 刘青青, 徐明良. 植物抗病毒侵染的分子机制. 作物学报, 2012, 38: 761–772 Hou J, Liu Q Q, Xu M L. Molecular mechanism of plant defense against virus attack. Acta Agron Sin, 2012, 38: 761–772 [13]Bucher E, Lohuis D, van Poppel P M J A, Geerts-Dimitriadou C, Goldbach R, Prins M. Multiple virus resistance at a high frequency using a single transgene construct. J General Virol, 2006, 87: 3697–3701 [14]Kreuze J F, Klein I S, Lazaro M U, Chuquiyuri W J C, Morgan G L, Mejía P G C, Ghislain M, Valkonen J P T. RNA silencing-mediated resistance to a crinivirus (Closteroviridae) in cultivated sweetpotato (Ipomoea batatas L.) and development of sweetpotato virus disease following co-infection with a potyvirus. Mol Plant Pathol, 2008, 9: 589–598 [15]Zhu C X, Song Y Z, Yin G H, Wen F J. Induction of RNA-mediated multiple virus resistance to Potato virus Y, Tobacco mosaic virus, and Cucumber mosaic virus. J Phytopathol, 2009, 157: 101–107 [16]Wang X Y, Eggenberger A L, Nutter F W J, Hill J H. Pathogen derived transgenic resistance to soybean mosaic virus in soybean. Mol Breed, 2001, 8: 119–127 [17]Furutani N, Hidaka S, Kosaka Y, Shizukawa Y, Kanematsu S. Coat protein gene-mediated resistance to soybean mosaic virus in transgenic soybean. Breed Sci, 2006, 56: 119–124 [18]Zhang X C, Sato S, Ye X H, Dorrance A E, Morris T J, Clemente T E, Qu F. Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene. Phytopathology, 2011, 101: 1264–1269 [19]Kim H J, Kim M J, Pak J H, Jung H W, Choi H K, Lee Y H, Baek I Y, Ko J M, Jeong S C, Pack I S, Ryu K H, Chung Y S. Characterization of SMV resistance of soybean produced by genetic transformation of SMV-CP gene in RNAi. Plant Biotechnol Rep, 2013, 7: 425–433 [20]Chen L Y, Cheng X F, Cai J Y, Zhan L L, Wu X X, Liu Q, Wu X Y. Multiple virus resistance using artificial trans-acting siRNAs. J Virol Methods, 2016, 228: 16–20 [21]Hill J H, Benner H I. Properties of Soybean mosaic virus ribonucleic acid. Phytopathology, 1980, 70: 236–239 [22]Adams M J, Antoniw J F, Beaudoin F. Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Mol Plant Pathol, 2005, 6: 471–487 [23]Jermer C E, Wang X, Tomimura K, Ohshima K, Ponz F, Walsh J A. The dual role of the Potyvirus P3 Protein of turnip mosaic virus as asymptom and avirulence determinant in Brassica. Mol Plant-Microbe Interact, 2003, 16: 777–784 [24]SuehiroN, Natsuaki T, Watanabe T, Okuda S. An important determinant of the ability of turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein. J General Virol, 2004, 85 : 2087–2098 [25]Wen R H, Saghai Maroof M A, Hajimorad M R. Amino acid changes in P3, and not the overlapping pipo-encoded protein, determine virulence of Soybean mosaic virus on functionally immune Rsv1-genotype soybean. Mol Plant Pathol, 2011, 12: 799–807 [26]Wen R H, Khatabi B, Ashfield T, Saghai Maroof M A, Hajimorad M R. The HC-Pro and P3 cistrons of an avirulent soybean mosaic virus are recognized by different resistance genes at the complex Rsv1 locus. Mol Plant-Microbe Interact, 2013, 26: 203–215 [27]Hajimorad M R, Eggenberger A L, Hill J H. Strain-specific P3 of Soybean mosaic virus elicits Rsv1-mediated extreme resistance, but absence of P3 elicitor function alone is insufficient for virulence on Rsv1-genotype soybean. Virology, 2006, 345: 156–166 [28]Chowda-Reddy R V, Sun H Y, Chen H Y, Poysa V, Ling H, Gijzen M and Wang A M. Mutations in the P3 Protein of soybean mosaic virus G2 isolates determine virulence on Rsv4-genotype soybean. Mol Plant-Microbe Interact, 2011, 24: 37–43 [29]Wang Y, Khatabi B and Hajimorad M R. Amino acid substitution in P3 of soybean mosaic virus to convert avirulence to virulence on Rsv4-genotype soybean is influenced by the genetic composition of P3. Mol Plant Pathol, 2015, 16: 301–307 [30]Wen R H, Hajimorad M R. Mutational analysis of the putative pipo of soybean mosaic virus suggests disruption of PIPO protein impedes movement. Virology, 2010, 400: 1–7 [31]Zhang L, Yang X D, Zhang Y Y, Yang J, Qi G X, Guo D Q, Xing G J, Yao Y, Xu W J, Li H Y, Li Q Y, Dong Y S. Changes in oleic acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing. Intl J Genom, 2014: 104–116 [32]Edwards K, Johnstone C, and Thompson C, A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl Acids Res, 1991, 19: 1349 [33]Tel-Zur N, Abbo S, Myslabodski D, Mizrahi Y. Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Plant Mol Biol Rep, 1999, 17: 249–254 [34]智海剑, 盖钧镒, 何小红. 大豆对SMV数量(程度)抗性的综合分级方法研究. 大豆科学, 2005, 24(2): 5–11 Zhi H J, Gai J Y, He X H. Study on methods of classification of quantitative resistance to soybean mosaic virus in soybean. Soybean Sci, 2005, 24(2): 5–11 [35]Tougou M, Furutani N, Yamagishi N, Shizukawa Y, Takahata Y, Hidaka S. Development of resistant transgenic soybeans with inverted repeat-coat protein genes of soybean dwarf virus. Plant Cell Rep, 2006, 25: 1213–1218 [36]Meyer P. Transcriptional transgene silencing and chromatin components. Plant Mol Biol, 2000, 43: 221–234 [37]杨清华. 我国大豆花叶病毒的株系分化、P3基因序列特征以及大豆对强毒株系抗病基因的标记定位. 南京农业大学博士学位论文, 江苏南京, 2009. p 80 Yang Q H. Strain differentiation and P3 sequence characteristics of soybean mosaic virus in China and gene mapping of resistance to a virulent strain in soybean. PhD Dissertation of Nanjing Agricultural University, Nanjing, China, 2009. p 80

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