CRISPR/Cas9介导的二穗短柄草bdfls2敲除突变体的获得

FLS2是一类在植物中保守存在的可识别细菌鞭毛蛋白并激活位于植物先天免疫反应第一层面的重要的植物模式识别受体(pattern recognition receptors,PRRs)。为了进一步研究草坪草植物的先天免疫,本研究以冷季型草坪草模式植物二穗短柄草(Brachypodium distachyon)为材料,利用CRISPR/Cas9基因编辑技术对植物抗病免疫相关的重要基因BdFLS2进行了定向的基因编辑,获得了bdfls2敲除突变体,为草坪草植物的FLS2相关的先天免疫的进一步研究奠定了材料基础。筛选转基因阳性植株,测序分析bdfls2 突变基因,结果显示该突变体中bdfls2 基因编码序列由于碱基的缺失导致提前终止。同时该突变体在对应病原相关分子模式(PAMPs:pathogen-associated molecular pattern)flg22处理后相比于野生型,无显著的ROS爆发或防卫基因的激活,暗示二穗短柄草FLS2能够识别病原相关分子模式flg22激活分子模式触发的免疫(pattern-triggered immunity,PTI)。

Generation of bdfls2-knockout Mutant in Brachypodium distachyon Mediated by CRISPR/Cas9

The FLS2,as an important plant pattern recognition receptor(PRRs),can recognize bacterial flagellins and activate the first level of innate immune response in plants.In order to further study the innate immune of plants in lawn,having the cold-season turfgrass plant Brachypodium distachyon as materials,CRISPR/Cas9 gene editing technology was used to specifically edit the of B.distachyon’s BdFLS2 related to plant resistance and immune,and bdfls2-knockout mutants was obtained,which lays a material foundation for further studying FLS2’s innate immune.We screened transgenic positive plants,carried out sequencing analysis of mutant gene bdfls2,and the results showed that the sequence encoded by gene bdfls2 in the mutant was terminated early due to the deletion of the base.Meanwhile,the bdfls2 mutants didn’t show significant ROS burst or defense-related gene activation after treatment with pathogen-associated molecular pattern flg22 while compared with wild one;suggesting that FLS2 in B.distachyon may play an important role to recognize pathogen-associated molecular pattern flg22 and to activate pattern-triggered immunity.