Construction of RNA Interference Vector of Coat Protein Gene of Grapevine Fanleaf Virus and the Effect Evaluation in Transgenic Nicotiana benthamiana

doi:10.7668/hbnxb.2014.S1.001

Abstract

Abstract: In order to probe the efficiency of the RNAi strategy in protection plants against GFLV,we cloned a conserved segment of coat protein gene of GFLV (310 bp) by RT-PCR,constructed a RNAi plant expression vector by introducing this fragment into the destination vector of pHELLSGATE12 by gateway technology,and transformed into Nicotiana benthamiana by Agrobaterium mediated method.RT-PCR identification showed that the target gene fragment was integrated into the tobacco genome and showed expression in RNA level.We also evaluated the virus-resistance of the plants by virus inoculation.The results showed that the rate of disease occurrence in the transgenic positive lines was obviously lower than the control,indicating that the expressed dsRNAs in the transgenic plants may interfere the infection of this virus.

Key words: Grapevine fanleaf virus, RNA interference, Vector construction, Transgenosis, Nicotiana benthamiana

CLC Number:

TIAN Li-li, NIU Liang. Construction of RNA Interference Vector of Coat Protein Gene of Grapevine Fanleaf Virus and the Effect Evaluation in Transgenic Nicotiana benthamiana[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2014, 29(S1): 1-5. doi: 10.7668/hbnxb.2014.S1.001.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2014.S1.001

http://www.hbnxb.net/EN/Y2014/V29/IS1/1

References

[1] 晁无疾,乜爱华,周敏.北京地区葡萄扇叶病毒检测[J].葡萄栽培与酿酒,1997(1):1-3.
[2] 丁定群,汤浩茹,张勇,等.RNA干扰及其在果树上的应用[J].生物技术通报,2012(12):57-64.
[3] Bardonnet N,Hans F,Serghini M A,et al.Protection against virus infection in tobacco plants expressing the coat protein of grapevine fanleaf nepovirus[J].Plant Cell Reports,1994,13(6):357-360.
[4] Krastanova S,Perrin M,Barbier P,et al.Transformation of grapevine rootstocks with the coat protein gene of grapevine fanleaf nepovirus[J].Plant Cell Reports,1995,14(9):550-554.
[5] Valat L,Fuchs M,Burrus M,et al.Transgenic grapevine rootstock clones expressing the coat protein or movement protein genes of grapevine fanleaf virus:characterization and reaction to virus infection upon protolast electroporation[J].Plant Science,2006,170(4):739-747.
[6] Smith N A,Singh S P,Wang M B, et al.Total silencing by intron-spliced hairpin RNAs[J].Nature,2000,407(6802):319-320.
[7] Chen Y K,Lohuis D,Goldbach R,et al.High frequency introduction of RNA mediated resistance against cumcumber mosaic virus using inverted repeat constructs[J].Molecular Breeding,2004,14(3):215-226.
[8] Hily J M,Ravelonandro M,Damsteegt V,et al.Plum pox virus coat protein gene intron-hairpin-RNA (ihpRNA) constructs provide resistance to plum pox virus in Nicotiana benthamiana and Prunus domestica [J].Journal of the American Society for Horticultural Sciences,2007,132:850-858.
[9] Kamachi S,Mochizuki A,Nishiguchi M,et al.Transgenic nicotiana benthamiana plants resistant to cucumber green mottle Mosaic virus based on RNA silencing[J].Plant Cell Reports,2007,26(8):1283-1288.
[10] 朱俊华,朱常香,温孚江,等.正向和反向RNA重复介导的抗马铃薯Y病毒基因工程比较研究[J].植物病理学报,2004,34(2):133-140.
[11] Tougou M,Furutani N,Yamagishi N,et al.Development of resistant transgenic soybeans with inserted repeat coat protein genes of soybean dwarf virus[J].Plant Cell Reports,2006,25(11):1213-1218.
[12] 颜培强,白先权,万秀清,等.应用RNAi技术培育抗TMV病毒转基因烟草[J].遗传,2007,29(8):1018-1022.

[1]	WANG Xiaoya, BU Ruifang, HU Haiyan, LONG Qiang, SUN Lianxuan, QI Lu, LIU Zheng, LI Xiaoyao, LI Chengwei. *Cloning and Function Analysis of TaJIP2* Gene Negatively Regulating Growth, Maturity and Yield in Triticum aestivum L.** [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(6): 35-44.
[2]	LI Lun, KE Jie, HUANG Gang, LIU Hongshi, SONG Yanni, ZHANG Yingjie, CHEN Lihong. *Enhanced Oxidative Stress Tolerance of Transgenic Tobacco Plants by Overexpression of Brachypodium distachyon BdDREB1-like* Gene** [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(2): 1-8.
[3]	WANG Shuang, YANG Rui, BAI Wei. Construction and Expression Analysis of RNA Interference Vector of WRKY Transcriptional Factor OsWRKY76 in Rice [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(6): 9-13.
[4]	HAN Xiaodong, GUO Rongqi, GAO Yang, YU Xiumin, SU Jie, ZHANG Ziyi, LI Guojing, WANG Ruigang. *The Responses of VHA-c1* Gene in Arabidopsis thaliana to Abiotic Stresses** [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(4): 62-66.
[5]	TONG Wenyan, HU Mengke, XU Linna, QIAO Huicong, LI Fen. Construction and Induced Expression of Prokaryotic Expression Vector of T₁₀₈₄ Deletion and Substitution Mutations in NtTkr Tail of Nicotiana tabacum [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(3): 38-42.
[6]	JIA Huili, GUO Jicheng, WU Xinming, WANG Yunqi, LIU Jianning, FANG Zhihong, SHI Yonghong, DONG Kuanhu. Cloning，Expression Analysis and Vector Construction of CkCDPK Gene from Caragana korshinskii [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(3): 43-51.
[7]	WANG Hongle, QI Lianfen, YANG Chaosha, WU Zhiming, LI Yadong. *The Agglutinin Gene Cloning of Pinellia pedatisecta* and Prokaryotic Expression** [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(1): 109-114.
[8]	CAI Man, LI Weihua, LIU Yantao, YU Yu, KONG Xianhui, WANG Juan, WANG Xuwen, LIU Li. *Expression Vector Construction,Transformation and Cold Resistance of GhSAD2* Gene from Upland Cotton** [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(6): 60-66.
[9]	YUE Yongli, YU Haiquan. *Construction of Red Fluorescent Protein Driven by Mouse FABP4* Promoter and Expression in Bovine Cells** [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(6): 26-30.
[10]	TIAN Lili, NIU Liang. *Construction of Apple stem pitting virus* Resistant RNAi Vector and Genetic Transformation into the Rootstock of M26** [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(4): 100-105.
[11]	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.
[12]	WANG Qing-yun, WANG Run-qing, FANG Cong-yan, HOU Pei, SU Liang, LI Jian-ping, SONG Mei-fang, YANG Jian-ping, LI Xue-mei, WU Da-fu. Cloning,Construction of Overexpression Vector and Bioinformatics Analysis of OsPP6C Gene from Oryza sativa [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2013, 28(5): 59-65.
[13]	LI Zhen, ZHANG Gui-lin, WANG Qing-guo, LIU Xu, LI Hai-qing, YAO Fang-yin, LIU Wei. Construction of RNA Interference Vectors for Rice Black-streaked Dwarf Virus and Genetic Transformation [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2012, 27(4): 144-148.
[14]	ZHU Yun, FAN Na-na, YU Yao-hua, ZHANG Bai-shun, YANG Yu, CHAO Xiang-jian, GUO Bao-tai, JIN Li-ping, GUO Xiao, LI Guang-eun. RNA Interference Analysis of Receptor-like Kinase Gene （StRLK） in Potato [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2012, 27(4): 18-22.
[15]	ZHENG Xing, ZHANG Cai-ying, CHANG Wen-suo, LI Xi-huan. Over-expression Vector Construction and Transgenic Soybean(Glycine maxL) Plants Identification of Transcription Factor Gene GmPTF1 [J]. ACTA AGRICULTURAE BOREALI-SINICA, 2011, 26(6): 27-31.

Please choose a citation manager

Content to export