Abstract

Transient gene expression utilizing syringe mediated agro infiltration offers a simple yet efficient technique for various transgenic applications. Although soybean is commonly used as a model plant for functional genomic studies, till date there have been no reports available on a high throughput agro infiltration protocol for transient gene expression. In the present study, we developed a simple transient expression system, named Agrobacterium-mediated disc assay for transient expression (AGRO DATE) in mature soybean leaves involving the use of an optimized infiltration buffer [10 mM 2-(N-morpholino) ethane sulfonic acid sodium salt, 10 mM MgCl₂, 0.2 mM acetosyringone, 400 mg L⁻¹, l-cysteine, 0.5 mM dithiothreitol and 0.01% Tween 20] under vacuum using a needle-less syringe. This not only limits the genetic pollution but can also be conducted without the intervention of any specialized equipments. Model protein β-glucuronidase (GUS) was used for optimizing various parameters and the synergized composition delivered 58% transformation efficiency within 4 days of infiltration. We demonstrated the versatile applicability of the method for examining reporter genes (GUS and bar), over expression (GmIPK1) and antisense suppression of GmMIPS in soybean. In addition, AGRODATE offers a viable approach for application of agro infiltration in other recalcitrant plant species and may become a useful tool for the research community.

Graphic abstract

中文翻译：

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'AGRODATE'：一种快速的农杆菌介导的瞬时表达工具，用于叶盘中的基因功能分析

摘要

利用注射器介导的农业渗透的瞬时基因表达为各种转基因应用提供了一种简单而有效的技术。尽管大豆通常用作功能基因组研究的模型植物，但迄今为止，尚无关于瞬时基因表达的高通量农业浸润方案的报道。在本研究中，我们开发了一种简单的瞬时表达系统，称为农杆菌介导的圆盘测定法，用于成熟大豆叶片中的瞬时表达（AGRO DATE），涉及使用优化的浸润缓冲液[10 mM 2-（N-吗啉代）乙烷磺酸酸钠盐，10毫摩尔MgCl ₂，0.2mM的乙酰丁香酮，400毫克的L ^-1，升-半胱氨酸，0.5 mM二硫苏糖醇和0.01％Tween 20]在真空下使用无针注射器。这不仅限制了遗传污染，而且无需任何专业设备的干预即可进行。模型蛋白β-葡萄糖醛酸苷酶（GUS）用于优化各种参数，增效组合物在渗透后4天内转化率达到58％。我们证明了该方法的通用性，可用于检查报告基因（GUS和bar），过表达（GmIPK1）和反义抑制GmMIPS在大豆中。此外，AGRODATE提供了一种在其他顽固植物物种中应用农业渗透的可行方法，并且可能成为研究界的有用工具。

图形摘要