华北农学报 ›› 2025, Vol. 40 ›› Issue (3): 157-165. doi: 10.7668/hbnxb.20195636

所属专题: 植物保护 生物技术

• 资源环境·植物保护 • 上一篇    下一篇

病毒介导的甘蓝基因沉默体系的建立

王江1,2, 刘永贵2, 韩睿2, 钟雄辉2, , 颉建明1, 康俊根1,2,   

  1. 1 甘肃农业大学 园艺学院,甘肃 兰州 730070
    2 北京市农林科学院 蔬菜研究所,农业农村部华北地区园艺作物生物学与种质创制重点实验室,北京 100097
  • 收稿日期:2024-12-18 出版日期:2025-06-28
  • 通讯作者:
    康俊根(1972—),男,山西忻州人,研究员,博士,主要从事甘蓝新品种选育及抗病机理研究。
    钟雄辉(1985—),男,福建邵武人,副研究员,博士,主要从事甘蓝抗病机制研究。
  • 作者简介:

    王 江(2000—),男,山西朔州人,在读硕士,主要从事甘蓝抗病机理研究。

  • 基金资助:
    北京市自然科学基金项目(6252008); 国家自然科学基金项目(32372691); 北京市农林科学院创新能力建设专项(KJCX20230126); 北京市农林科学院蔬菜研究所改革与发展项目(KJCX202302); “十四五”重点研发计划项目(2022YFD1200502)

Establishment of Virus-Mediated Gene Silencing System in Cabbage

WANG Jiang1,2, LIU Yonggui2, HAN Rui2, ZHONG Xionghui2, , XIE Jianming1, KANG Jungen1,2,   

  1. 1 Colleage of Horticulture,Gansu Agricultural University,Lanzhou 730070,China
    2 Beijing Vegetable Research Center,Beijing Academy of Agriculture and Forestry Sciences,Key Laboratory of Biology and Genetic Improvement of Horticultural Crops,Ministry of Agriculture and Rural Affairs,Beijing 100097,China
  • Received:2024-12-18 Published:2025-06-28

摘要:

病毒诱导的基因沉默(VIGS)是一种转录后基因沉默技术,广泛应用于植物基因功能研究。然而,到目前为止,国内关于建立甘蓝VIGS基因沉默体系的报道较少。旨在甘蓝上建立以八氢番茄红素脱氢酶基因(Phytoene desaturase,PDS)作为有效视觉指示基因、病毒载体PCVA/PCVB介导的基因沉默体系。以甘蓝、大白菜和萝卜为植物材料,通过构建PCVA-PDS载体,对甘蓝PDS进行沉默。通过构建PCVA/PCVB-PDS-GFP转化农杆菌,并采用注射法侵染甘蓝和烟草叶片下表皮细胞、真空负压侵染甘蓝幼苗,结合实时荧光定量PCR技术,研究病毒介导的基因沉默体系在甘蓝中的适用性,并将该体系应用于另外2个具有代表性的十字花科作物—大白菜和萝卜。结果表明,载体PCVA/PCVB-PDS-GFP转化的农杆菌侵染甘蓝及烟草叶片细胞后,细胞膜可以观察到荧光出现;通过真空负压侵染甘蓝幼苗14 d后,新生叶片出现光漂白现象,并呈现范围逐渐扩大的趋势;实时荧光定量PCR分析显示,PDS同源基因在试验组中的相对表达量较对照组分别下降3.2,1.7倍;侵染大白菜和萝卜幼苗后,观察到大白菜和萝卜的叶脉及部分叶片出现白化现象,同时伴随一定程度的叶面卷曲。综上所述,通过对PDS基因沉默后甘蓝叶片出现光漂白现象,证明病毒介导的基因沉默体系在甘蓝植株中实现了有效的复制和传播,大白菜萝卜叶片出现白化也表明,VIGS系统同样可以应用于其他十字花科作物,进一步扩展了该沉默系统的应用范围。甘蓝VIGS沉默体系的建立,为十字花科作物相关基因功能研究提供了理论基础。

关键词: 甘蓝, 光漂白, 基因沉默, 病毒介导, 八氢番茄红素脱氢酶基因

Abstract:

Virus-induced gene silencing(VIGS)is a post-transcriptional gene silencing technique widely used in plant gene function research.However,there are few reports on the establishment of a VIGS system for cabbage in China.The aim of this study was to establish a PCVA/PCVB-mediated gene silencing system using phytoene desaturase(PDS) as an effective visual indicator gene in cabbage.Cabbage,Chinese cabbage,and radish were used as plant materials.The PDS gene was silenced by constructing the PCVA-PDS vector.The PCVA/PCVB-PDS-GFP vector was then generated and transformed into Agrobacterium tumefaciens,which was used to infect cabbage and tobacco epidermal cells via injection and to perform vacuum infiltration in cabbage seedlings.Real-time Quantitative PCR was used to study the applicability of the VIGS system in cabbage,and the system was also applied to another representative Brassicaceae crops-Chinese cabbage and radish.The results showed that green fluorescence could be observed on the cell membranes of cabbage and tobacco leaf cells after infection with Agrobacterium tumefaciens transformed with PCVA/PCVB-PDS-GFP.After 14 d of vacuum infiltration in cabbage seedlings,photobleaching appeared on the new leaves,with the affected area gradually expanding.Real-time Quantitative PCR analysis indicated that the relative expression of PDS homologous genes in the experimental groups decreased 3.2-fold and 1.7-fold compared to the control group,respectively.After infecting Chinese cabbage and radish seedlings,whitening of the leaf veins and some leaves was observed,along with some leaf curling.In conclusion,the photobleaching observed in cabbage leaves after PDS gene silencing demonstrates that the VIGS system effectively replicates and spreads within cabbage plants.The whitening of leaves in Chinese cabbage and radish also indicates that the VIGS system can be applied to other Brassicaceae crops,thus expanding the application scope of this silencing system.The establishment of the cabbage VIGS system provides a theoretical foundation for functional gene studies in Brassicaceae crops.

Key words: Cabbage, Photobleaching, Gene silencing, Virus-mediation, PDS

中图分类号: 

引用本文

王江, 刘永贵, 韩睿, 钟雄辉, 颉建明, 康俊根. 病毒介导的甘蓝基因沉默体系的建立[J]. 华北农学报, 2025, 40(3): 157-165. doi: 10.7668/hbnxb.20195636.

WANG Jiang, LIU Yonggui, HAN Rui, ZHONG Xionghui, XIE Jianming, KANG Jungen. Establishment of Virus-Mediated Gene Silencing System in Cabbage[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 157-165. doi: 10.7668/hbnxb.20195636.