双管基因枪介导的基因瞬时表达技术在拟南芥中的应用

基因瞬时表达是植物中研究目标基因功能的常用手段。在模式植物拟南芥(Arabidopsis thaliana)中,相比原生质体和农杆菌介导的基因异源表达技术,利用粒子轰击进行基因瞬时表达一直鲜有报道。其主要原因是拟南芥叶型相对较小、基因枪操作相对烦琐以及基因表达效率差异较大。该研究通过优化双管基因枪系统,在营养生长旺盛的拟南芥莲座叶中实现GFP和GUS基因高效表达。同时,通过GUS报告基因明确了坏死诱导因子BAX、Avh238和ATR13/Rpp13激发拟南芥细胞坏死的表型。但在本氏烟(Nicotianabenthamiana)中明显诱导细胞坏死的Avrblb1/RB基因对,在拟南芥中却丧失了诱导细胞坏死的活性。由于双管基因枪系统每次轰击时设置平行对照,可有效降低转化实验中的样本变异度,为拟南芥及其突变体研究中准确评价基因功能和高通量筛选目标基因提供新的技术参考。

The Application of Double-barreled Particle Bombardment for Transient Gene Expression in Arabidopsis

Transient gene expression is a favorite tool used for functional analysis of target genes in plant. Of three techniques applied for genetic transformation in the model plant Arabidopsis thaliana, biolistic delivery system was less used than protoplast- or Agrobacterium-mediated transformation. This is mainly due to the smaller leaf size of Arabidopsis, the complicated procedure of bombardment and the limited efficiency and consistency of gene expression. Here, we report applications of an optimized double-barreled particle bombardment system for transient transformation in Arabidopsis, which displayed high expression level of GFP and GUS reporter genes in leaf epidermal cells. By introducing the parallel control in the same shoot by co-bombardment, gene expression efficiency and consistency were dramatically improved, which allows quantitative analysis of target genes with several replicates. Furthermore, cell death inducers BAX, Avh238 or ATR13/Rpp13, were co-expressed with GUS in Arabidopsis rosette leaves and led to strong necrosis phenotypes visualized by significant reduction of number of GUS spots. On the contrary, Avrblb1/RB gene pair triggered strong cell death in Nicotiana benthaminana, but not in A. thaliana. Therefore, this time-saving protocol is an alternative to quantitatively evaluate biological functions of the gene of interest and high-throughput screening of immune suppressors in Arabidopsis and its mutants.