中国番茄黄化曲叶病毒利用根吸收法诱导基因沉默(VIGS)的初步研究

生物技术通报 ›› 2014, Vol. 0 ›› Issue (1): 143-146.

中国番茄黄化曲叶病毒利用根吸收法诱导基因沉默(VIGS)的初步研究

张召军,王晓彬,王慧,刘林,张心阁,何秀霞

长春理工大学生命科学技术学院，长春 130022

收稿日期:2013-09-18 出版日期:2014-01-23 发布日期:2014-01-23
作者简介:张召军，男，生物技术专业；E-mail: zhangzhaojunk@qq.com
基金资助:
大学生创新实验项目(2012X77)，吉林省科技厅项目(20110244)

Study on Chinese Tomato Yellow Leaf Curl Virus-induced Gene Silencing(VIGS)via Root Absorption

Zhang Zhaojun, Wang Xiaobin, Wang Hui, Liu Lin, Zhang Xinge, He Xiuxia

College of Life Science，Changchun University of Science and Technology，Changchun 130022

Received:2013-09-18 Published:2014-01-23 Online:2014-01-23

摘要/Abstract

摘要： 以含硫黄素酶Su 基因的中国番茄黄化曲叶病毒卫星DNA 作为载体，通过冻融法将其转化到农杆菌中，利用根部#br#吸收法进行农杆菌侵染，对农杆菌介导的病毒诱导基因沉默体系进行了优化，探讨对病毒诱导基因沉默效率的影响。结果显示以#br#OD600 值为1.5 的含质粒pBinPLUS-2mβ-Su，pBinPLUS-1.7A 的农杆菌菌液1∶1 混合进行根部吸收法侵染22-25 d 的番茄幼苗，目#br#的基因Su 沉默导致番茄幼苗出现光漂白现象，半定量RT- PCR 检测目的基因Su mRNA 被显著降解，该体系的建立有利于对植物#br#基因进行高通量功能分析。

关键词: 病毒诱导基因沉默, 硫黄素酶Su, 基因, 根吸收法

Abstract: The tomato yellow leaf curl virus satellite DNA was used as vector which containing the Su gene, and transformed the plasmid into Agrobacterium by freeze thawing method. Agrobacterium infected plants via root absorption, and virus-induced gene silencing system was optimized, twexplore the influence to virus-induced gene silencing efficiency. The results showed infected 22-25 days tomato seedling via OD600 1.5 Agrobacterium-mediated root-absorption which containing plasmid pBinPLUS-2mβ-Su and pBinPLUS-1.7A, the silencing of Su gene resulted in photo-blcached young leaves of tomato plants. Semi-quantitative RT-PCR analysis was employed to test the effect of Su gene silencing, which showed that Su gene was degraed significantly. This gene-silencing system would benefit for the high-throughput analysis of plant gene.

Key words: Virus-induced gene silencing, Su gene, Root absorption method

张召军,王晓彬,王慧,刘林,张心阁,何秀霞. 中国番茄黄化曲叶病毒利用根吸收法诱导基因沉默(VIGS)的初步研究[J]. 生物技术通报, 2014, 0(1): 143-146.

Zhang Zhaojun, Wang Xiaobin, Wang Hui, Liu Lin, Zhang Xinge, He Xiuxia. Study on Chinese Tomato Yellow Leaf Curl Virus-induced Gene Silencing(VIGS)via Root Absorption[J]. Biotechnology Bulletin, 2014, 0(1): 143-146.

参考文献

[1] Baulcombe DC. Fast forward genetics based on virus-induced gene silencing[J].Curr Opin Plant Biol, 1999, 2(2): 109-113.
[2] Dinesh-kumar SP, Anandalakshmi R, Marathe R, et al. Virus-induced gene silencing[J].Methods Mol Biol, 2003, 236: 287-293.
[3] Fu DQ, Zhu BZ, Zhu HL, et al. Virus-induced gene silencing in tomato fruit[J].The Plant Journal, 2005, 43(2): 299-308.
[4] 陶小荣, 周雪平, 崔晓峰, 等. 病毒诱导的基因沉默及其在植物基因功能研究中的应用[J]. 生物化学与生物物理进展, 2004,31(9): 777-783.
[5] 周晓馥, 王兴智. 病毒载体诱导转录后基因沉默系统的建立及烟草基因功能的研究[J]. 分子植物育种, 2003, 1(3): 423- 424.
[6] 傅达奇, 朱本忠, 赵晓丹, 等. 病毒诱导烟草的基因沉默[J].西北植物学报, 2005, 25(8): 1517-1520.
[7] Brigneti G, Martin-Hernandez AM, et al.Virus-induced gene silencing in Solanum species[J].Plant J, 2004, 39(2): 264-272.
[8] 程维舜, 徐秋芳, 等. 适于烟草脆裂病毒诱导的本氏烟基因沉默分析的对照载体构建[J]. 浙江大学学报, 2012, 38(1): 10-20.
[9] Brigneti G, Martín-Hernández AM, Jin H, et al. Virus-induced gene silencing in Solanum species[J].Plant J, 2004, 39(2): 264-272.
[10] Chung E, Seong E, Kim YC, et al. A methodof high frequency virus-induced gene silencing in chili pepper Capsicum annuum Lcvbukang[J]. Mol Cell, 2004, 17(23): 377-380.
[11] Constantin GD, Krath BN, MacFarlane SA, et al. Virus-induced gene silencing as a tool for functional genomics in a legume species[J]. Plant J, 2004, 40(4): 622-631.
[12] 燕飞, 宋雪梅, 成卓敏. 番茄丛矮病毒p19 蛋白抑制转录后基因沉默作用机制[J]. 病毒学报, 2005, 21(5): 403-405.
[13] 杨美英, 贺红霞, 王艳, 等. 番茄茄红素β-环化酶基因高效沉默载体构建[J]. 分子植物育种, 2007, 5(1): 43-46.
[14] 陶小荣, 钱亚娟, 等. 卫星沉默载体的改良及其改变矮牵牛叶色和花色的研究[J]. 科学通报, 2006, 51(17): 2041-2044.
[15] 黄昌军, 钱亚娟, 李正和, 等. 病毒诱导的基因沉默及其在植物功能基因组研究中的应用[J]. 中国科学, 2012, 42(1): 3-15.
[16] Fu DQ, Zhu BZ, Zhu HL, et al. Enhancement of virus-induced gene silencing through low temperature and low humidity in tomato[J]. Mol Cells, 2006, 21(2): 153-160.
[17] Ryu CM, Anand A, Kang L, et al. Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species[J].Plant J, 2004, 40(2): 322-331.