CRISPR/Cas9介导靶向敲除拟南芥GGB基因突变体的鉴定

摘要

GGB是抗旱负调控基因.为了获得拟南芥ggb突变体材料,构建了以拟南芥U6启动子驱动GGB sgRNA的CRISPR/Cas9基因组编辑载体.将构建好的编辑载体利用农杆菌介导的浸花法转化野生型拟南芥.对转基因后代GGB基因的测序结果分析发现,在靶位点处有缺失4个碱基和增加1个T碱基的2种突变体产生.分别对野生型拟南芥和上述2种ggb突变体进行半定量RT-PCR分析结果显示,突变体材料中几乎检测不到GGB基因表达,说明获得了GGB基因敲除突变体.对野生型和ggb突变体叶片失水率、耐旱表型及单株种子量的测定结果表明,与野生型相比,拟南芥GGB基因突变后,叶片失水率显著减少,抗旱性明显增强,而单株种子量却并没有改变.研究表明,GGB是一种理想的作物分子育种的候选靶基因,获得的突变体为今后从农作物中克隆的G-GB同源基因进行功能互补验证提供了有用的遗传材料.%GGB is a negative regulator of drought resistance.In order to obtain the Arabidopsis ggb mutant,we used AtU6 promoter to drive the expression of AtGGB sgRNA and coresponding CRISPR/Cas9 genome edting vector was constructed and was transferred into Arabidopsis by Agrobacterium-mediated floral dip.After sequencing of GGB in T2 generation,two kinds of mutants with a deletion of 4 bases and an addition of 1 base (T) were found at the target site,respectively.Semi-quantitative RT-PCR analysis results showed that the expression of GGB gene was almost not detected in the ggb mutant,which indicated that the mutants were GGB knockout lines.Through measuring the water loss rate,drought resistant and seed yield per plant,ggb mutant exhibited decreased water loss rate,improved drought resistance,but unchanged seed yield per plant compared to wild-type.Taken together,the above results indicated that GGB is an ideal candidate target gene for crop molecular breeding and ggb mutant is useful for functional complemention of GGB homologous genes cloned from crops.