Chinese Journal OF Rice Science >
Wx Gene Editing via CRISPR/Cas9 System in Rice
These authors contributed equally to this work:WANG Bingkun, ZHANG Hui;
Received date: 2017-05-21
Revised date: 2017-08-24
Online published: 2018-01-10
【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.
Key words： rice; CRISPR/Cas9 system; Wx gene; gene editing; amylose content
Bingkun WANG, Hui ZHANG, Ruke HONG, Jinwen ZHANG, Rui YANG, Qiong LUO, Qianchun ZENG . Wx Gene Editing via CRISPR/Cas9 System in Rice[J]. Chinese Journal OF Rice Science, 2018 , 32(1) : 35 -42 . DOI: 10.16819/j.1001-7216.2018.7061
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