Liu Shuangwei, Ma Jingjing, Liu Hongmei, Guo Yingtian, Li Wei, Niu Shihui
Beijing Advanced Innovation Center for Tree Breeding By Molecular Design, National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, 100083 People's Republic of China.
Plant Methods. 2020 Apr 10;16:52. doi: 10.1186/s13007-020-00594-5. eCollection 2020.
Functional genomic studies using genetics approaches of conifers are hampered by the complex and enormous genome, long vegetative growth period, and exertion in genetic transformation. Thus, the research carried out on gene function in is typically performed by heterologous expression based on the model plant Arabidopsis. However, due to the evolutionary and vast diversification from non-flowering (gymnosperms) to flowering (angiosperms) plants, several key differences may alter the underlying genetic concerns and the analysis of variants. Therefore, it is essential to develop an efficient genetic transformation and gene function identification protocol for . .
In the present study we established a highly efficient transgene -mediated transient expression system for . . Using a β-glucuronidase gene (GUS) as a reporter gene expression, the highest transformation efficiency (70.1%) was obtained by co-cultivation with strain GV3101 at an optical density at 600 nm of 0.8, with 150 μM acetosyringone for 30 min followed by 3 days in the dark at 23 ± 1 °C. This protocol would be applied to other conifers; GUS staining was observed 24 h post-infection.
We report a simple, fast, and resilient system for transient -mediated transformation high-level expression of target genes in . , which will also improve transformation efficiency in other conifer species.
利用针叶树遗传方法进行的功能基因组学研究受到复杂庞大的基因组、漫长的营养生长期以及遗传转化难度的阻碍。因此，对[针叶树名称未给出]基因功能的研究通常是基于模式植物拟南芥进行异源表达。然而，由于从非开花（裸子植物）到开花（被子植物）植物的进化和巨大分化，一些关键差异可能会改变潜在的遗传问题和变异分析。因此，为[针叶树名称未给出]开发一种高效的遗传转化和基因功能鉴定方案至关重要。
在本研究中，我们为[针叶树名称未给出]建立了一种高效的转基因介导的瞬时表达系统。以β-葡萄糖醛酸酶基因（GUS）作为报告基因表达，与GV3101菌株在600nm光密度为0.8、150μM乙酰丁香酮共培养30分钟，然后在23±1°C黑暗中培养3天，获得了最高转化效率（70.1%）。该方案可应用于其他针叶树；感染后24小时观察到GUS染色。
我们报告了一种简单、快速且有弹性的系统，用于[针叶树名称未给出]中转基因介导的目标基因高水平表达的瞬时转化，这也将提高其他针叶树种的转化效率。
