CRISPR/Cas9系统编辑水稻Wx基因

【目的】 直链淀粉含量与稻米品质密切相关。Wx基因是控制水稻直链淀粉合成的主效基因,通过对Wx基因定点编辑以获得稳定遗传、直链淀粉含量适宜的突变体。【方法】 构建CRISPR/Cas9表达载体pGK03-Wx-gRNA (靶点1和2分别在Wx基因第1和第2外显子),利用工程菌EHA105遗传转化超级稻楚粳27,潮霉素筛选获得转化株系,对转化株系及其后代进行分子检测、测序、基因表达和遗传稳定性分析以及直链淀粉含量测定。【结果】 获得9个独立的T₀代转化株系,靶点1(L1~L5) 5个株系,突变频率100%,靶点2(L6~L9) 4个株系,突变频率75%。由T₀代突变体衍生出T₁和T₂代株系,测序发现T₀、T₁和T₂代株系出现缺失(单、双、多碱基缺失)和单碱基插入两种突变类型;T₀至T₁代部分株系(L1、L2、L3和L6)发生再编辑,T₁至T₂代遗传稳定。与野生型相比,突变株系RNA水平Wx基因表达量显著下降(P<0.01),稻米直链淀粉含量显著降低(P<0.01),从17.5%降到1.93%。【结论】 利用CRISPR/Cas9系统成功编辑水稻Wx基因,获得了稳定遗传、低直链淀粉含量的突变体,为稻米品质改良提供了材料。

Wx Gene Editing via CRISPR/Cas9 System in Rice

【Objective】Amylose content is closely related to rice quality. The objective of this research is to knockout Wx gene for obtaining rice mutants with suitable amylose content.【Method】Vector pGK03-Wx-gRNA, a CRISPR/Cas9 system, targeted either the first (target 1) or the second (target 2) exon of Wx gene was constructed and transformed into super-rice Chujing 27 to edit the Wx gene. Independent transformants were obtained via Agrobacterium-mediated transformation with hygromycin selection. The transgenic lines of T0, T1 and T2 generations were identified by DNA sequencing, in addition to qPCR and amylose content assay of T1. 【Result】Nine T0 independent transgenic lines were generated, the L1-L5 from target 1 and L6-L9 from target 2. Eight lines (L1-L8) were mutants and L9 was not confirmed as mutant by sequencing. Therefore, the mutation rate was 100% for target 1, and 75% for target 2. T1 and T2 generations of the eight lines (L1-L8) were further sequenced, which revealed two types of mutation: base deletion (single base, double and up to fourteen bases), and base insertion. Some lines (L1, L2, L3 and L6) were reedited at T1 generation when compared to its T0 generation, while the rest lines were stable at the mutation sites. All the eight mutants were stable when comparing their target gene sequence at T1 and T2 generations. qPCR results indicated that Wx gene expression at RNA level was considerably reduced in the mutant. The seed amylose content was reduced from 17.5% to 1.93%(P<0.01). 【Conclusion】The Wx gene was successfully edited by CRISPR/Cas9 system in rice, the inheritable mutants with average amylose content at 1.93% were obtained, which would provide valuable germplasm for rice quality improvement.