Cloning of Interference Fragment of Eukaryotic Translation Initiation Factor 4E of Soybean and Construction of Its RNAi Vector

doi:10.7668/hbnxb.2014.04.001

Abstract

Abstract: Soybean transformation based on RNA interference(RNAi)can specifically silence the homologous target genes in mRNA level,providing a new strategy for disease-resistant breeding.In this study,we made the amino acid sequence alignment of eIF4E from ten different kinds of plants which have been identified to interact with the virus VPg,then we determined the conserved interval of soybean eIF4E was 62-237 amino acid sequence and cloned the 406 bp interference fragment named eIF4Ei (including 58 bp specific recombination sequence attB).Using the GATEWAY technology,the RNAi vector pB7GWIWG2(Ⅱ)-eIF4Ei was constructed,which was identified by PCR amplification,sequencing and restriction digestion.The identification of the recombinant expression vector ensured the eIF4Ei interference fragment no mutation in the process of vector construction and two ccdB sites were all replaced by eIF4Ei,in addition,the insert direction of the two interference fragments in the terminal plasmid was conversed.Overall,the RNAi vector was successfully constructed to be an inverted repeat structure,providing the basic materials for cultivating new germplasm of soybeans resistant to SMV using the soybean transformation technology based on RNAi.

Key words: Soybean mosaic virus, eIF4E gene, RNA interference, GATEWAY technology, Vector construction

CLC Number:

S565.1

GAO Le, ZHAI Rui, DING Xue-ni, LI Kai, ZHANG Hong-yun, WANG Tao, ZHI Hai-jian. Cloning of Interference Fragment of Eukaryotic Translation Initiation Factor 4E of Soybean and Construction of Its RNAi Vector[J]. journal1, 2014, 29(4): 1-6. doi: 10.7668/hbnxb.2014.04.001.

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References

[1] Zhang X,Sato S,Ye X,et al.Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene [J].Phytopathology,2011,101(11):1264-1269.
[2] Robaglia C,Caranta C.Translation initiation factors:a weak link in plant RNA virus infection [J].Trends in Plant Science,2006,11(1):40-45.
[3] Fraser R S S,Van-Loon L C.Genes for resistance to plant viruses [J].Critical Reviews in Plant Sciences,1986,3:257-294.
[4] Bonas U,Lahaye T.Plant disease resistance triggered by pathogen-derived molecules:refined models of specific recognition [J].Current Opinion in Microbiology,2002,5(1):44-50.
[5] Goldbach R,Bucher E,Prins M.Resistance mechanisms to plant viruses:an overview [J].Virus Research,2003,92(2):207-212.
[6] Soosaar J L M,Burch-Smith T M,Dinesh-Kumar S P.Mechanisms of plant resistance to viruses [J].Nature Reviews Microbiology,2005,3:789-798.
[7] Johansen I E,Lund O S,Hjulsager C K,et al.Recessive resistance in Pisum sativum and potyvirus pathotype resolved in a gene-for-cistron correspondence between host and virus [J].Journal of Virology,2001,75(14):6609-6614.
[8] Fraser R S S.The genetics of resistance to plant viruses [J].Annual Review of Phytopathology,1990,28:179-200.
[9] Wang A,Krishnaswamy S.Eukaryotic translation initiation factor 4E-mediated recessive resistance to plant viruses and its utility in crop improvement [J].Molecular Plant Pathology,2012,13(7):795-803.
[10] Truniger V,Aranda M A.Recessive resistance to plant viruses [J].Advances in Virus Research,2009,75:119-159.
[11] Maule A J,Caranta C,Boulton M I.Sources of natural resistance to plant viruses status and prospects [J].Molecular Plant Pathology,2007,8(2):223-231.
[12] Provvidenti R,Hampton R O.Sources of resistance to viruses in the potyviridae [J].Archives of Virology,1992,5:189-211.
[13] 卢航,赵小明,白雪芳,等.翻译起始因子4E在植物病毒侵染中的作用[J].中国农业科技导报,2007,9(6):35-40.
[14] Ruffel S,Gallois J L,Moury B,et al.Simultaneous mutations in translation initiation factors eIF4E and eIF(iso)4E are required to prevent pepper veinal mottle virus infection of pepper[J].The Journal of General Virology,2006,87(Pt7):2089-2098.
[15] Ruffel S,Dussault M H,Palloix A,et al.A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E(eIF4E)[J].The Plant Journal:for Cell and Molecular Biology,2002,32(6):1067-1075.
[16] Kang B C,Yeam I,Li H,et al.Ectopic expression of a recessive resistance gene generates dominant potyvirus resistance in plants [J].Plant Biotechnology Journal,2007,5(4):526-536.
[17] Nicaise V,German-Retana S,Sanjuán R,et al.The eukaryotic translation initiation factor 4E controls lettuce susceptibility to the potyvirus lettuce mosaic virus [J].Plant Physiology,2003,132(3):1272-1282.
[18] Yoshii M,Nishikiori M,Tomita K,et al.The Arabidopsis cucumovirus multiplication 1 and 2 loci encode translation initiation factors 4E and 4G [J].Journal of Virology,2004,78(12):6102-6111.
[19] Duprat A,Caranta C,Revers F,et al.The Arabidopsis eukaryotic initiation factor(iso)4E is dispensable for plant growth but required for susceptibility to potyviruses [J].The Plant Journal:for Cell and Molecular Biology,2002,32(6):927-934.
[20] 章红运.大豆花叶病毒基因组全序列测定及大豆 eIF4E 和 eIFiso4E 基因与病毒VPg基因的互作分析[D].南京:南京农业大学,2013.

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