Developing and applying a virus-induced gene silencing system for functional genomics in walnut (Juglans regia L.) mediated by tobacco rattle virus

IF 2.6 3区生物学 Q2 GENETICS & HEREDITY

Gene Pub Date : 2024-11-13 DOI:10.1016/j.gene.2024.149087

Yaoxin Liu , Yongjie Xu , Haodong Xu , Wulamurusuli Amilijiang , Hua Wang

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引用次数: 0

Abstract

Walnut (Juglans regia L.) is a high-value tree species planted worldwide, but the incomplete less developed genetic transformation system limits its gene function analysis. In this study, virus-induced gene silencing (VIGS) mediated by tobacco rattle virus (TRV) technology was applied to walnut seedlings to degrade the transcript of target gene. Different infiltration methods were used to explore the effects of infection mode, Agrobacterium cell density, silencing fragment length, and walnut cultivars. The results showed that spray infiltration of seedlings resulted in a photobleaching phenotype of the whole plant. Leaf injection was a more effective way of infiltration. The optimal combination was the Agrobacterium cell density at OD₆₀₀ = 1.1 with target fragment = 255 bp for the treatment of walnut early-fruiting cultivar ’Xiangling.’ This combination can reach up to 48 % of gene silencing efficiency. Based on this optimized VIGS system, silencing a walnut chlorophyll synthesis-related gene, JrPOR (Protochlorophyllide reductase), to further validate the system’s effect. The results showed that the expression of JrPOR was significantly repressed, and the chlorophyll level of the silenced plants was significantly decreased compared with the control. The above results indicate that the walnut TRV-VIGS system has been successfully established and can be used for reverse genetic studies, providing an option for verifying gene function in walnut.

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开发和应用病毒诱导的基因沉默系统，在核桃（Juglans regia L.）中进行烟草瘤病毒介导的功能基因组学研究。

核桃（Juglans regia L.）是世界范围内种植的高价值树种，但遗传转化系统的不完善和不发达限制了对其基因功能的分析。本研究采用烟草鼠疫病毒（TRV）技术对核桃幼苗进行病毒诱导基因沉默（VIGS），以降解目的基因的转录本。采用不同的浸润方法探讨了感染模式、农杆菌细胞密度、沉默片段长度和核桃栽培品种的影响。结果表明，对幼苗进行喷雾浸润会导致整个植株出现光漂白表型。叶片注射是一种更有效的浸润方式。在处理核桃早果栽培品种'香玲'时，最佳组合是 OD600 = 1.1 的农杆菌细胞密度和目标片段 = 255 bp。这种组合的基因沉默效率可达 48%。在这一优化的 VIGS 系统基础上，对核桃叶绿素合成相关基因 JrPOR（原叶绿素还原酶）进行了沉默，以进一步验证该系统的效果。结果表明，与对照相比，JrPOR 的表达受到明显抑制，被沉默植株的叶绿素水平显著下降。上述结果表明，核桃 TRV-VIGS 系统已成功建立，可用于反向遗传研究，为验证核桃基因功能提供了一种选择。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Gene 生物-遗传学

CiteScore

6.10

自引率

2.90%

发文量

718

审稿时长

42 days

期刊介绍： Gene publishes papers that focus on the regulation, expression, function and evolution of genes in all biological contexts, including all prokaryotic and eukaryotic organisms, as well as viruses.