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大豆胞囊线虫VIGS载体的构建与鉴定

大豆胞囊线虫 VIGS 载体的构建与鉴定

张怡阮九霄马晓萌傅露露乔月娥段素娟孟娉李成伟

周口师范学院生命科学系,植物遗传与分子育种重点实验室,河南周口466001

摘要:拟克隆大豆胞囊线虫肌钙蛋白(Hg-tnc)和酰胺样多肽(Hg-flp)基因的部分片段,构建胞囊线虫的病毒诱导基因沉默体系(VIGS),以期为大豆抗胞囊线虫抗性品种的培育及胞囊线虫基因功能验证的研究奠定理论基础。利用RT-PCR方法从大豆胞囊线虫中克隆出目的基因,电泳检测表明,克隆的Hg-tnc基因和Hg-flp基因部分片段分别约为1 000 bp和700 bp。将目的基因连接到烟草脆裂病毒载体pYY13上,转化大肠杆菌,利用PCR进行初步鉴定后进行序列测定。结果表明,大豆胞囊线虫Hg-tnc基因和Hg-flp基因已插入到pYY13载体上,VIGS载体构建成功。

病毒诱导的基因沉默;胞囊线虫;肌钙蛋白;酰胺样多肽;烟草脆裂病毒

S435.651A1000 -7091(2012)03 -0223 -04

Identification and Construction of the Recombinant Vector of Virus Induced Gene Silencing of Soybean Heterodera glycines IchinoheZHANG YiRUAN Jiu-xiaoMA Xiao-meng FU Lu-lu QIAO Yue-e

DUAN Su-juan MENG PingLI Cheng-wei

2012 -02 - 13

河南省教育厅自然科学研究计划项目(2011B180060);周口师范学院青年科研基金项目(zknuqn201046B);周口师范学院实验室开放项目(201115)

张怡(1985 -),女,河南洛阳人,助教,硕士,主要从事植物与微生物互作研究。

李成伟(1972 -),男,河南商丘人,教授,博士,主要从事植物与病原体互作、抗性分子育种及生物反应器制药等研究。

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@@[1] 陈井生,李肖白,李泽宇,等.不同抑制性土壤真菌菌 株对大豆胞囊线虫的抑制作用[J].河南农业科学, 2011,40(7):90 -92.

@@[ 2 ] Riggs R D, Wrathel J A. Biology and management of the soybean cyst nematode[ M ] . Minnesota, USA : APS Press, 1992 : 186.

@@[ 3 ] Fu D Q,Zhu B Z,Zhao X D,et al. Advance of the virus induced gene silencing in plant [ J ]. Chinese Biotech, 2005,1:89 -95.

@@[4] Kumagai M H, Donson J,Della-Cioppa G,et al. Cytoplas mic inhibition of carotenoid biosynthesis with virus-de rived RNA[ J]. Proc Natl Acad Sci USA, 1995,92 ( 5 ) : 1679 - 1683.

@@[5] Liu Y, Schiff M,Dinesh-Kumar S P. Virus-induced gene silencing in tomato [ J ]. Plant J,2002,31 ( 6 ) : 777 - 786.@@[6] Brigneti G,Martin-Hernandez A M,Jin H,et al. Virus-in

duced gene silencing in Solanum species [ J ]. Plant J, 2004,39 ( 2 ) :264 - 272.

@@[ 7 ] Chung E,Seong E, Kim Y C, et al. A method of high fre quency virus-induced gene silencing in chili pepper( Cap sicum annuum L. cv. Bukang) [ J ]. Mol Cells, 2004, 17 (2) :377 -380.

@@[8] Igarashi A, Yamagata K, Sugai T, et al. Apple latent spherical virus vectors for reliable and effective virus-in duced gene silencing among a broad range of plants inclu ding tobacco, tomato, Arabidopsis thaliana, cucurbits and legumes [ J ]. Virology ,2009,386 ( 2 ) :407 - 416.@@[9] Kimber M J,McKinney S,McMaster S,et al.flp gene dis ruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference[ J ]. FASEB J,2007,21(4) :1233 - 1243.

@@[ 10] Dong Y,Burch-Smith T M,Liu Y,et al. A ligatlon-inde pendent cloning tobacco rattle virus vector for high- throughput virus-induced gene silencing identifies roles for NbMADS4-1 and-2 in floral development [J ]. Plant Physiology,2007,145:1161 -1170.

@@[11] 萨姆布鲁克J,拉塞尔DW.分子克隆实验指南[M]. 3版.北京:科学出版社,2002:27.

@@[ 12] Liu X Z,Li J Q,Zhang D S. History and status of soy bean cyst nematode in China[ J]. Int J Nematol, 1997, 7:18 -25.

@@[13] 孙玉秋,许艳丽,李春杰,等.东北地区大豆胞囊线虫 卵定殖真菌的多样性研究[J].华北农学报,2011,26 (2):233 -238.

@@[14] 卢为国,袁道华,李金英,等.大豆抗胞囊线虫基因不 同世代遗传率的变化[J].河南农业科学,2010(2):

24 - 27.

@@[ 15 ] Yamagishi N, Yoshikawa N. Virus-induced gene silen cing in soybean seeds and the emergence stage of soy

bean plants with Apple latent spherical virus vectors [ J ]. Plant Molecular Biology ,2009,71 : 15 - 24.@@[ 16 ] Zhang C,Ghabrial S A. Development of bean pod mottle virus-based vectors for stable protein expression and se quence-specific virus-induced gene silencing in soybean [ J]. Virology ,2006,344( 2 ) :401 - 411.

@@[17] Hein I,Barciszewska-paca M,Hrubikova K,et al. Virus- induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley[ J]. Plant Physiol,2005,138 ( 4 ) :2155 - 2164.@@[ 18 ] Zhu X, Dinesh-Kumar S P. Virus-induced gene silencing as a tool to identify host genes affecting viral pathogenic ity [ J ]. Methods Mol Biol,2008,451 ( 5 ) :641 - 648.@@[ 19] Senthil-kumar M ,Hema R,Anand A, et al. A systematic study to determine the extent of gene silencing in Nicoti

ana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing [ J ]. New Phytol, 2007,176 ( 4 ) : 782 -

791.

@@[ 20] MacFarlane S A, Popovich A H. Efficient expression of foreign proteins in roots from tobravirus vectors [ J ]. Vi rology ,2000,267( 1 ) :29 - 35.

@@[ 21 ] Kagawa H, Takuwa K, Sakube Y. Mutations and expres sions of the tropomyosin gene and the troponin C gene of Caenorhabditis elegans [ J ]. Cell Struct Funct, 1997,22 (1) :213 -218.

@@[ 22] Dubreuil G, Magliano M, Dubrana M P, et al. Tobacco rattle virus mediates gene silencing in a plant parasitic root-knot nematode[ J]. J Exp Bot,2009,60 ( 14 ) :4041 - 4050.

@@[23 ] Maule A G, Mousley A, Marks N J,et al. Neuropeptide signaling systems potential drug targets for parasite and pest control[ J ]. Curr Top Med Chem, 2002,2 ( 7 ) : 733 -758.

@@[24] Kimber M J, Fleming C C, Bjourson A J,et al. FMR- Famide-related peptides in potato cyst nematodes [ J ]. Mol Biochem Parasitol,2001,116( 2 ) : 199 - 208.

@@[ 25 ] Kimber M J,Fleming C C,Prior A, et al. Localisation of Globodera pallida FMRFamide-related peptide encoding genes using in situ hybridisation [ J ]. Int J Parasitol, 2002,32(9) :1095 - 1105.

万方数据

大豆胞囊线虫VIGS载体的构建与鉴定

作者:张怡, 阮九霄, 马晓萌, 傅露露, 乔月娥, 段素娟, 孟娉, 李成伟, ZHANG Yi, RUAN Jiu-xiao, MA Xiao-meng, FU Lu-lu, QIAO Yue-e, DUAN Su-juan, MENG Ping, LI Cheng-wei

作者单位:周口师范学院生命科学系,植物遗传与分子育种重点实验室,河南周口466001

刊名:

华北农学报

英文刊名:Acta Agriculturae Boreali-Sinica

年,卷(期):2012,27(3)

参考文献(25条)

1.陈井生;李肖白;李泽宇不同抑制性土壤真菌菌株对大豆胞囊线虫的抑制作用[期刊论文]-河南农业科学 2011(07)

2.Riggs R D;Wrathel J A Biology and management of the soybean cyst nematode 1992

3.Fu D Q;Zhu B Z;Zhao X D Advance of the virusinduced gene silencing in plant 2005

4.Kumagai M H;Donson J;Della-Cioppa G Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA 1995(05)

5.Liu Y;Schiff M;Dinesh-Kumar S P Virus-induced gene silencing in tomato[外文期刊] 2002(06)

6.Brigneti G;Martin-Hernandez A M;Jin H Virus-induced gene silencing in Solanum species[外文期刊] 2004(02)

7.Chung E;Seong E;Kim Y C A method of high frequency virus-induced gene silencing in chili pepper,(Capsicum annuum L.cv.Bukang)[外文期刊] 2004(02)

8.Igarashi A;Yamagata K;Sugai T Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco,tomato,Arabidopsis thaliana,cucurbits and legumes[外文期刊] 2009(02)

9.Kimber M J;McKinney S;McMaster S flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference[外文期刊] 2007(04)

10.Dong Y;Burch-Smith T M;Liu Y A ligatlon-independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and-2 in floral development 2007

11.萨姆布鲁克·J;拉塞尔DW分子克隆实验指南 2002

12.Liu X Z;Li J Q;Zhang D S History and status of soybean cyst nematode in China 1997

13.孙玉秋;许艳丽;李春杰东北地区大豆胞囊线虫卵定殖真菌的多样性研究[期刊论文]-华北农学报 2011(02)

14.卢为国;袁道华;李金英大豆抗胞囊线虫基因不同世代遗传率的变化[期刊论文]-河南农业科学 2010(02)

15.Yamagishi N;Yoshikawa N Virus-induced gene silencing in soybean seeds and the emergence stage of soybean plants with Apple latent spherical virus vectors 2009

16.Zhang C;Ghabrial S A Development of bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean[外文期刊] 2006(02)

17.Hein I;Barciszewska-paca M;Hrubikova K Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley[外文期刊] 2005(04)

18.Zhu X;Dinesh-Kumar S P Virus-induced gene silencing as a tool to identify host genes affecting viral pathogenicity 2008(05)

19.Senthil-kumar M;Hema R;Anand A A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing[外文期刊] 2007(04)

20.MacFarlane S A;Popovich A H Efficient expression of foreign proteins in roots from tobravirus vectors[外文期刊] 2000(01)

21.Kagawa H;Takuwa K;Sakube Y Mutations and expressions of the tropomyosin gene and the troponin C gene of Caenorhabditis elegans[外文期刊] 1997(01)

22.Dubreuil G;Magliano M;Dubrana M P Tobacco rattle virus mediates gene silencing in a plant parasitic root-

knot nematode[外文期刊] 2009(14)

23.Maule A G;Mousley A;Marks N J Neuropeptide signaling systems potential drug targets for parasite and pest control 2002(07)

24.Kimber M J;Fleming C C;Bjourson A J FMR-Famide-related peptides in potato cyst nematodes[外文期刊] 2001(02)

25.Kimber M J;Fleming C C;Prior A Localisation of Globodera pallida FMRFamide-related peptide encoding genes using in situ hybridisation[外文期刊] 2002(09)

引用本文格式:张怡.阮九霄.马晓萌.傅露露.乔月娥.段素娟.孟娉.李成伟.ZHANG Yi.RUAN Jiu-xiao.MA Xiao-meng.FU Lu-lu. QIAO Yue-e.DUAN Su-juan.MENG Ping.LI Cheng-wei大豆胞囊线虫VIGS载体的构建与鉴定[期刊论文]-华北农学报 2012(3)

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