中国水稻科学2018，Vol.32Issue(1)：35-42,8.DOI:10.16819/j.1001-7216.2018.7061
CRISPR/Cas9系统编辑水稻Wx基因
Wx Gene Editing via CRISPR/Cas9 System in Rice
摘要
Abstract
[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.关键词
水稻/CRISPR/Cas9系统/Wx/基因编辑/直链淀粉含量Key words
rice/CRISPR/Cas9 system/Wx gene/gene editing/amylose content分类
生物科学引用本文复制引用
汪秉琨,张慧,洪汝科,张锦文,杨睿,罗琼,曾千春..CRISPR/Cas9系统编辑水稻Wx基因[J].中国水稻科学,2018,32(1):35-42,8.基金项目
国家转基因生物新品种培育重大专项(2014ZX0800939B) （2014ZX0800939B）
国家现代农业产业技术体系资助项目(CARS-11-YNZQC) （CARS-11-YNZQC）
云南农业大学自然科学青年科研基金资助项目(2016ZR11). （2016ZR11）
