水稻黑条矮缩病毒基因组S7编码的2个非结构蛋白在病株中的表达检测

doi:10.3969/j.issn.10017216.2012.01.003

中国水稻科学 ›› 2012, Vol. 26 ›› Issue (1): 9-15.DOI: 10.3969/j.issn.10017216.2012.01.003

水稻黑条矮缩病毒基因组S7编码的2个非结构蛋白在病株中的表达检测

吕明芳1,2羊健2 张恒木2 陈剑平2,*

¹浙江师范大学化学与生命科学学院, 浙江金华 321004； ²浙江省农业科学院病毒学与生物技术研究所/浙江省植物有害生物防控国家重点实验室培育基地/农业部植物保护与生物技术重点开放实验室/浙江省植物病毒重点开放实验室，浙江杭州 310021

收稿日期:2011-05-11 修回日期:2011-06-17 出版日期:2012-01-10 发布日期:2012-01-10
通讯作者: 陈剑平2,*
基金资助:
国家973计划资助项目（2010CB126203）；浙江省自然科学基金资助项目（Y3090657）

Detection of Two Nonstructural Proteins Encoded by Genome Segment S7 of Rice BlackStreaked Dwarf Virus in Infected Rice Plants

LU Mingfang1,2, YANG Jian2, ZHANG Hengmu2, CHEN Jianping2,*

¹College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China; ²State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control/Key Laboratory of Plant Protection and Biotechnology, Ministry of Agriculture/Zhejiang Provincial Key Laboratory of Plant Virology/Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;

Received:2011-05-11 Revised:2011-06-17 Online:2012-01-10 Published:2012-01-10
Contact: CHEN Jianping2,*

摘要/Abstract

摘要： 水稻黑条矮缩病毒（RBSDV）基因组片段S7含有2个非重叠的阅读框，所编码的蛋白分别为p7a和p7b。根据RBSDV S7序列（EU111804）设计特异性引物分别扩增编码p7a和p7b蛋白的基因片段，并亚克隆至原核表达载体pET32a（+）和pSBET上,再以大肠杆菌BL21(DE3)pLysS或BL21(DE3)pLysE为宿主菌进行高水平表达,利用纯化的蛋白免疫小鼠，制备了p7a和p7b蛋白的特异性抗血清。蛋白质印迹分析表明p7a和p7b蛋白均在水稻病株中表达，但p7a含量较为丰富，易于在病株中检测到，而p7b在病株中含量则很低；在病毒粒子中，两者均未检测到任何信号，表明两者均为RBSDV的非结构蛋白。

关键词: 水稻黑条矮缩病毒, 非结构蛋白, 原核表达

Abstract: The genome segment S7 of Rice blackstreaked dwarf virus (RBSDV) contains two nonoverlapping major open reading frames (ORFs), which encoded two proteins, p7a and p7b,respectively. Based on the sequence of S7 (EU111804), two pairs of S7specific primers were designed and respectively used to amplify the fragments encoding the p7a and p7b proteins. The expected products were subcloned into the expression vector pET32a（+） or pSBET and expressed at high level in E. coli BL21(DE3)pLysS or BL21(DE3)pLysE, respectively. An antiserum specifically against p7a or p7b proteins was prepared using the purified expression proteins to immunize mice. The results of Western blot revealed that both p7a and p7b proteins were expressed in infected plants, and p7a protein was more abundant and easily detected than p7b protein. However, both have not been detected in the viral particles, indicating that both proteins encoded by RBSDV S7 were nonstructural proteins.

Key words: Rice blackstreaked dwarf virus, nonstructural protein, prokaryotic expression

中图分类号:

吕明芳1,2羊健2 张恒木2 陈剑平2,* . 水稻黑条矮缩病毒基因组S7编码的2个非结构蛋白在病株中的表达检测[J]. 中国水稻科学, 2012, 26(1): 9-15.

LU Mingfang1,2, YANG Jian2, ZHANG Hengmu2, CHEN Jianping2,*. Detection of Two Nonstructural Proteins Encoded by Genome Segment S7 of Rice BlackStreaked Dwarf Virus in Infected Rice Plants[J]. Chinese Journal of Rice Science, 2012, 26(1): 9-15.

参考文献

\[1\]Zhang H M, Chen J P, Lei J L, et al. Sequence analysis shows a dwarf disease on rice, wheat and maize in China is caused by rice blackstreaked dwarf virus. Euro J Plant pathol, 2001, 107(5): 563567.

\[2\]Fang S, Yu J, Feng J,et al. Identification of rice blackstreaked dwarf fijivirus in maize with rough dwarf disease in China. Arch Virol, 2001, 146(1): 167170.

\[3\]Bai F W, Yan J, Qu Z C, et al. Phylogenetic analysis reveals that a dwarfing disease on different cereal crops in China is due to rice black streaked dwarf virus (RBSDV). Virus Genes, 2002, 25(2): 201206.

\[4\]Zhang H M, Chen J P, Adams M J. Molecular characterisation of segments 1 to 6 of Rice blackstreaked dwarf virus from China provides the complete genome. Arch Virol,2001,146(12): 23312339.

\[5\]Wang Z H, Fang S G, Xu J L, et al. Sequence analysis of the complete genome of rice blackstreaked dwarf virus isolated from maize with rough dwarf disease. Virus Genes, 2003, 27(2): 163168.

\[6\]Liu H, Wei C, Zhong Y, et al. Rice blackstreaked dwarf virus outer capsid protein P10 has selfinteractions and forms oligomeric complexes in solution. Virus Res, 2007, 127(1): 3442.

\[7\]Zhang C, Liu Y, Liu L, et al. Rice black streaked dwarf virus P91, an alphahelical protein, selfinteracts and forms viroplasms in vivo. J Gen Virol, 2008, 89(7): 17701776.

\[8\]Wang Q, Tao T, Zhang Y, et al. Rice blackstreaked dwarf virus P6 selfinteracts to form punctate, viroplasmlike structures in the cytoplasm and recruits viroplasmassociated protein P91. Virol J, 2011, 8: 24.

\[9\]Liu H, Wei C H, Zhong Y W, et al. Rice blackstreaked dwarf virus minor core protein P8 is a nuclear dimeric protein and represses transcription in tobacco protoplasts. FEBS Letters, 2007, 581: 25342540.

\[10\]欧阳元龙，吴建祥，熊如意，等. 水稻黑条矮缩病病毒外壳蛋白基因S10 的原核表达、多克隆抗体制备及应用. 中国水稻科学, 2010， 24(1)： 2530.

\[11\]羊健，张恒木，陈剑平，等. 水稻黑条矮缩病毒p8蛋白的原核表达、抗血清制备及其特性. 植物保护学报, 2007， 34（3）： 252256.

\[12\]Isogai M, Uyeda I, Lee B C. Detection and assignment of proteins encoded by rice black streaked dwarf fijivirus S7, S8, S9 and S10. J Gen Virol, 1998, 79(6): 14871494.

\[13\]Uyeda I, BongChoon L, Ando Y. Reovirus isolation and RNA extraction//Foster G D, Taylor S C. Methods in Molecular Biology, vol 81. Plant Virology Protocols: from Virus isolation to Transgenic Resistance. Totowa: Humana Press, 1998: 6575.

\[14\]林林，郑红英，陈炯，等.大蒜E病毒外壳蛋白基因的原核表达及抗血清制备. 微生物学报， 2004， 44（4）： 533535.

\[15\]梁国栋.最新分子生物学实验技术. 北京：科学出版社， 2001，312316.

\[16\]Marzachi C, Boccardo G, Nuss D L. Cloning of the maize rough dwarf virus genome: Molecular confirmation of the plantreovirus classification scheme and identification of two large nonoverlapping coding domains within a single genomic segment. Virology, 1991, 180(2), 518526.

\[17\]Isogai M, Uyeda I,Lindsten K. Taxonomic characteristics of fijiviruses based on nucleotide sequences of the oat sterile dwarf virus genome. J Gen Virol, 1998, 79(6): 14791485.

\[18\]Nakashima N, Koizumi M, Watanabe H,et al. Complete nucleotide sequence of the Nilaparvata lugens reovirus: A putative member of the genus Fijivirus. J Gen Virol, 1996, 77 (1): 139146.

\[19\]Guzmán F A, Distéfano A J, Arneodo J D, et al. Sequencing of the bicistronic genome segments S7 and S9 of Mal de Río Cuarto virus (Fijivirus, Reoviridae) completes the genome of this virus. Arch Virol, 2007, 152: 565573.

\[20\]Soo H M, Handley J A, Maugeri M M, et al. Molecular characterisation of Fiji disease fijivirus segment 9. J Gen Virol, 1998, 79 (12): 31553161.

\[21\]Harding R, Burns P, Geijskes R, et al. Molecular analysis of Fiji disease virus segments 2, 4 and 7 completes the genome sequence. Virus Genes, 2006, 32(1): 4347.

\[22\]钟永旺，周洁，庄斌全，等. 水稻黑条矮缩病毒第七号片段的cDNA 克隆及其在大肠杆菌中的表达. 微生物学报， 2003， 43（4）： 442447.

\[23\]Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eukaryotic ribosomes. Mol Cell Biol, 1987 (7): 34383445.

\[24\]Morris D R, Geballe A P. Upstream open reading frames as regulators of mRNA translation. Mol Cell Biol, 2000, 20 (23): 86358642.

\[25\]Wang L, Wessler S R. Inefficient reinitiation is responsible for upstream open reading framemediated translational repression of the maize R gene. Plant Cell, 1998， 10: 17331746.

\[26\]Imai A, Hanzawa Y, Komura M,et al. The dwarf phenotype of the Arabidopsis acl5 mutant is suppressed by a mutation in an upstream ORF of a bHLH gene. Development, 2006，133: 35753585.

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水稻黑条矮缩病毒基因组S7编码的2个非结构蛋白在病株中的表达检测

Detection of Two Nonstructural Proteins Encoded by Genome Segment S7 of Rice BlackStreaked Dwarf Virus in Infected Rice Plants

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