浙江农业学报 ›› 2017, Vol. 29 ›› Issue (4): 625-630.DOI: 10.3969/j.issn.1004-1524.2017.04.16

• 植物保护 • 上一篇    下一篇

黄瓜花叶病毒基因沉默载体的效率和稳定性分析

向志丹, 张震霄, 李超, 杜志游*   

  1. 浙江理工大学 生命科学学院,浙江 杭州 310018
  • 收稿日期:2016-11-30 出版日期:2017-04-20 发布日期:2017-04-27
  • 通讯作者: 杜志游,E-mail:duzy@zstu.edu.cn
  • 作者简介:向志丹(1992—),女,湖北宜昌人,硕士研究生,从事分子植物病毒学研究。E-mail:wjgqzwwl@qq.com
  • 基金资助:
    国家自然科学基金(31470007)

Investigation of efficiency and stability of Cucumber mosaic virus-based gene silencing vector

XIANG Zhidan, ZHANG Zhenxiao, LI Chao, DU Zhiyou*   

  1. College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China
  • Received:2016-11-30 Online:2017-04-20 Published:2017-04-27

摘要: 病毒诱导的基因沉默(virus-induced gene silencing, VIGS)已被广泛应用于植物基因的功能研究。试验以八氢番茄红素脱氢酶(phytoene desaturase, PDS)作为指示基因,在本生烟体内分析黄瓜花叶病毒(Cucumber mosaic virus, CMV)作为VIGS载体对植物内源PDS mRNA的沉默效率。采用分子克隆的方法,将Fny-CMV 2b ORF替换为不同长度的PDS序列,并分析了其在本生烟植物上诱导的PDS沉默效率。结果表明,该重组病毒能诱导上部系统叶的光漂白表型,且沉默效率与插入片段长度呈一定的正相关性,但在顶端新生叶中均不引起明显的光漂白现象,过长的片段插入(≥518 nt)影响病毒基因组的遗传稳定性,导致插入片段的部分缺失。CMV作为 VIGS载体,最适合的插入片段约为350 nt。

关键词: 病毒诱导的基因沉默, 黄瓜花叶病毒, 2b, 八氢番茄红素脱氢酶

Abstract: Virus-induced gene silencing (VIGS) has been widely used for analysis of plant gene function. In this work, using phytoene desaturase (PDS) as an indicator, efficiency of Cucumber mosaic virus(CMV) as a VIGS vector was investigated to silence PDS mRNA in Nicotiana benthamiana. Recombinant viruses were generated by replacing the 2b ORF with a set of PDS fragments, and tested in N. benthamiana plants. Results showed that these recombinants induced photobleaching phenotype in the upper systemic leaves and silencing efficiency was correlated with the length of the PDS inserts in some extents, but they did not cause obvious photobleaching phenotype in the newly top leaves and oversized inserts (≥518 nt) reduced genetic stability of viral genome, leading to partial deletion of the inserts. Thus, the optimal insert is about 350 nt in length for CMV-based VIGS vector.

Key words: virus-induced gene silencing, Cucumber mosaic virus, 2b, phytoene desaturase

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