欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2011, Vol. 37 ›› Issue (03): 484-488.doi: 10.3724/SP.J.1006.2011.00484

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

应用RNA沉默技术获取抗黄瓜花叶病毒(CMV)和烟草花叶病毒(TMV)转基因烟草

牛颜冰,王德富,姚敏,闫钊,由文鑫   

  1. 山西农业大学生命科学学院，太谷 030801
  • 收稿日期:2010-07-30 修回日期:2010-11-26 出版日期:2011-03-12 网络出版日期:2011-01-17
  • 基金资助:

    本研究由国家自然科学基金项目(30571216)和山西省自然科学基金项目(20051071)资助。

Transgenic Tobacco Plants Resistant to Two Viruses via RNA Silencing

NIU Yan-Bing,WANG De-Fu,YAO Min,YAN Zhao,YOU Wen-Xin   

  1. College of Life Science, Shanxi Agricultural University, Taigu 030801, China
  • Received:2010-07-30 Revised:2010-11-26 Published:2011-03-12 Published online:2011-01-17

PDF

1649

被引次数

14

摘要： RNA沉默是迄今最为有效的抗病毒策略，利用该策略不但能获得免疫转基因植株，且所得植株不易与其他病毒基因重组或异源包壳、生物安全性较高。将本实验室已构建的携带有烟草花叶病毒(TMV)部分移动蛋白基因(ΔMP)和黄瓜花叶病毒(CMV)部分复制酶基因(ΔRep)反向重复结构的植物表达载体pBIN438-MP-Rep(i/r)，用农杆菌浸润法转化普通烟草品种K326，共得196株转化植株，经卡那霉素筛选和PCR检测发现128株为阳性转基因株；PCR-Southern和RT-PCR分析表明外源基因已整合到烟草基因组并在转录水平上得到表达；ELISA结果显示20.3%的转基因植株对CMV和TMV复合侵染表现免疫性。本结果为利用RNA沉默技术进行植物抗多种病毒育种提供重要数据，为防治其他多种病毒复合侵染提供借鉴。

关键词: RNA沉默, 黄瓜花叶病毒, 烟草花叶病毒, 双病毒抗性

Abstract: RNA silencing is a phenomenon of homologous RNA degradation induced by dsRNA, which is an effective strategy to obtain virus resistant plants so far. By means of this strategy, higher bio-security immune transgenic plants were obtained, avoiding recombining and transcapsidating with other virus genes. In this study, the recombinant plant expression vector pBIN438-MP- Rep(i/r)consisting the inverted repeat of TMV-ΔMP and CMV-ΔRep fusion fragment was transformed into tobacco cultivar K326 via Agrobacterium-mediated. The transformants were selected in the culture medium with 100 mg L–1 Kan. One hundred and ninety-six transgenic plants were obtained, One hundred and twenty-eight of which were positive plants, and the resistance to CMV and TMV was tested at the degree of virus. PCR-Southern blot and RT-PCR analysis of the transgenic plants demonstrated that the exogenous DNA was integrated into the tobacco genomic DNA and was expressed in transcriptional level. Resistance assay indicated that about 20.3% transgenic plants were immune to the co- infection with TMV and CMV. This result will provide to crops an important reference for plant anti-viral breeding and for preventing the viral co-infection.

Key words: RNA silencing, Cucumber mosaic virus, Tobacco mosaic virus, Dual-virus resistance

[1]Prins M, Haan P D, Luyten R, Veller M V, Grinsven M Q V, Goldbach R. Broad resistance to tospovirus in transgenic tobacco plants expressing three-tospoviral gene sequence. Mol Plant-  Microbe Interact, 1995, 8: 85–91
[2]Kaniewsli W, Lawson C, Sammoms B, Haley L, Hart J, Delannay X, Tumer N E. Field resistance of transgenic Russet Burbank potato to effects of infection by potato virus X and potato virus Y. Bio/Technol, 1990, 8: 750–754
[3]Fuchs M, McFerson J R, Tricoli D M, McMaster J R, Deng R Z, Boeshore M L, Reynolds J F, Russell P F, Quemada H D, Gonsalves D. Cantaloupe line CZW-30 containing coat protein genes of cucumber mosaic virus, zucchini yellow mosaic virus, and watermelon mosaic virus-2 is resistance to these three virus in the field. Mol Breed, 1997, 3: 279–290
[4]Zhou X-P(周雪平), Li D-B(李德葆). Genetically engineering resistance to viruses and environmental risk assessment of releases of transgenic plants. Chin Bull Life Sci (生命科学), 2000, 12(1): 4–6 (in Chinese with English abstract)
[5]Fire A, Xu S, Montgomery M K, Kostas S A, Driver S E, Mello C C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391: 806–811
[6]Zhu J H, Zhu X P, Wen F J, Bai Q R, Zhu C X, Song Y Z. Effect of cDNA fragments in different length derived from potato virus Y coat protein gene on the induction of RNA-mediated virus resistance. Sci China (Ser C: Life Sci), 2004, 47: 382–388
[7]Jan F J, Fagoaga C, Pang S Z, Gonsalves D. A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing. J General Virol, 2000, 81: 2103–2109
[8]Bai Q-R(白庆荣), Zhu J-H(朱俊华), Liu X-L(刘晓玲), Zhu C-X(朱常香), Song Y-Z(宋云枝), Wen F-J(温孚江). Production of transgenic tobacco plants resistant to two viruses via RNA-mediated virus resistance. Acta Phytopathol Sin (植物病理学报), 2005, 35(2): 148–154 (in Chinese with English abstract)
[9]Zhu C-X(朱常香), Song Y-Z(宋云枝), Wen F-J(温孚江). Constructing transgenic tobacco for multiple-resistance against potato virus Y, cucumber mosaic virus and tobacco mosaic virus. Sci Agric Sin (中国农业科学), 2008, 41(4): 1040–1047 (in Chinese with English abstract)
[10]Niu Y-B(牛颜冰), Lei X-F(雷霄飞), Shen L-Y(申林炎), Wang D-F(王德富), Yao M(姚敏), Guo P-Y(郭平毅). Rapid construction of RNA silencing virus resistance vector containing TMV and CMV virus-derived genes. China Biotechnol (中国生物工程杂志), 2009, 29(2): 76–80 (in Chinese with English abstract)
[11]Sambrook J, Fristsch E F, Maniatis T, eds. Huang P-T(黄培堂) trans. Molecular Cloning: A Libratory Manual (3rd edn) (分子克隆实验指南, 第3版). Beijing: Science Press, 2002, pp 492–512 (in Chinese)
[12]Voinnet O. RNA silencing as a plant immune system against viruses. Trends Genet, 2001, 17: 449–459
[13]Tenllado F, Llave C, Díaz-Ruíz J R. RNA interference as a new biotechnological tool for the control of virus diseases in plants. Virus Res, 2004, 102: 85–96
[14]Missiou A, Kalantidis K, Boutla A, Tzortzakaki S, Tabler M, Tsagris M. Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing. Mol Breed, 2004, 14: 185–197
[15]Smith N A, Singh S P, Wang M B, Stoutjesdijk P, Green A, Waterhouse P M. Gene expression: Total silencing by intron- spliced hairpin RNAs. Nature, 2000, 407: 319–320
[16]Zamore P D. Ancient pathways programmed by small RNAs. Science, 2002, 296: 1265–1269
[17]Helliwell C A, Waterhouse P M. Constructs and methods for hairpin RNA-mediated gene silencing in plants. Methods Enzymol, 2005, 392: 24–35
[18]Chi S-Q(迟胜起), Song Y-Z(宋云枝), Zhu C-X(朱常香), Zheng C-C(郑成超), Liu X-L(刘晓玲), Wen F-J(温孚江). Effect of matrix attachment regions on the RNA-mediated resistance induced by PVYN-CP gene. Acta Phytopathol Sin (植物病理学报), 2005, 35(4): 345–351 (in Chinese with English abstract)
[1] 侯静，刘青青，徐明良. 植物抗病毒侵染的分子机制[J]. 作物学报, 2012, 38(05): 761-772.
[2] 毛建军;邱德文;杨秀芬;曾洪梅;袁京京. 蛋白激发子基因pemG1转化三生烟中及其对TMV抗性的提高[J]. 作物学报, 2008, 34(12): 2070-2076.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2