Regulation of Amylose Content in Rice by Editing Wx Using CBE Base Editor

doi:10.16819/j.1001-7216.2025.240705

Abstract

Abstract:

【Objective】 The Wx gene, a key regulator of amylose synthesis in rice, encodes a granule-bound starch synthase and plays a crucial role in determining the amylose content, thereby directly influencing the quality traits of rice. 【Method】 We used the CBE base editor to introduce base substitutions within the first exon of the Wx gene in Shendao18 variety, resulting in two distinct mutation types: GCCCCC→GCCCTT and GCCCCC→GCTCTC. Both mutations led to the substitution of proline with leucine at position 94 of the Wx protein, designated as Wx^P94L-1 and Wx^P94L-2 respectively. 【Result】 Field experiments demonstrated that compared to the wild type, both Wx^P94L-1 and Wx^P94L-2 plants exhibited no significant differences in plant height, tiller number per plant, panicle length, grain morphology or 1000-grain weight. Rice quality analysis revealed that the amylose content decreased from 14.9% (wild type) to 12.5% (Wx^P94L-1) and 12.1% (Wx^P94L-2), while traits such as viscosity, alkali spreading value and protein content showed no significant change compared to the wild type. 【Conclusion】 Our study successfully generated a novel allele of the Wx gene named Wx^P94L using CBE base editor which resulted in a slight reduction in amylose content; thus providing a way for enhancing rice quality.

Key words: Wx, amylose content, base editing, CBE

摘要：

【目的】Wx基因编码颗粒淀粉合成酶，是控制直链淀粉合成的主效基因，对稻米中直链淀粉的含量起决定性作用，直接影响稻米品质性状。为了实现小幅降低稻米直链淀粉含量，并为水稻品质改良提供一种新的途径。【方法】利用单碱基编辑器CBE系统对圣稻18的Wx基因第一外显子进行碱基替换，获得了两种碱基替换类型GCCCCC→GCCCTT和GCCCCC→GCTCTC，均造成颗粒淀粉合成酶Wx第94位脯氨酸替换成亮氨酸，分别命名为Wx^P94L-1和Wx^P94L-2。【结果】田间实验表明，相比野生型，Wx^P94L-1和Wx^P94L-2植株在株高、分蘖数、穗长、粒型、千粒重等主要农艺性状上无显著差异。稻米品质分析发现，Wx^P94L-1和Wx^P94L-2植株稻米直链淀粉含量由野生型的14.9%下降至12.5%和12.1%，同时胶稠度、碱消值和蛋白含量等性状与野生型相比无显著改变。【结论】总之，本研究利用单碱基编辑器创制了Wx基因新的等位型Wx^P94L，该等位型可使稻米直链淀粉含量小幅度降低，为水稻品质改良提供了一种新途径。

关键词: Wx, 直链淀粉含量, 单碱基编辑, CBE

CHEN Yuye, JIAO Xiaozhen, WANG Jian, WANG Kejian, CHEN Feng, ZHU Keming, LIU Chaolei. Regulation of Amylose Content in Rice by Editing Wx Using CBE Base Editor[J]. Chinese Journal OF Rice Science, 2025, 39(6): 771-778.

陈玉叶, 焦晓真, 王健, 王克剑, 陈峰, 朱克明, 刘朝雷. 利用CBE系统编辑Wx基因调控稻米直链淀粉含量[J]. 中国水稻科学, 2025, 39(6): 771-778.

Figures/Tables 5

Table 1. Primers used in the study

引物名称 Primer	序列 Sequence(5’-3’)	用途 Usage
Wx-F	CTGCTCCGCCACGGGTTCCAG	靶点突变类型检测 Target mutation type detection
Wx-R	CATCCATCCAATGCGATGATC	靶点突变类型检测 Target mutation type detection
Cas9-F	GCGATGTTGACAAGCTGTTC	外源成分Cas9蛋白检测 Detection of exogenous Cas9 protein
Cas9-R	GTGTCCTTGGAGAGTTGCAG	外源成分Cas9蛋白检测 Detection of exogenous Cas9 protein
Hyg-F	GTCGTCCATCACAGTTTGC	外源成分潮霉素检测 Detection of exogenous hygromycin
Hyg-R	TCCGGAAGTGCTTGACATT	外源成分潮霉素检测 Detection of exogenous hygromycin

Fig. 1. Editing of Wx by CBE system in Shengdao 18 A, Wx gene structure and gRNA target site. B, Structure of CBE vector targeting Wx. C, Mutations in Wx editing lines in the Shengdao 18 (SD18) background. Blue lowercase letters and stripes indicate mutational bases; Red letters represent PAM sequences. D, Deletion; SNP, Single nucleotide polymorphism.

Fig. 2. Screening of transgene-free WxP94L lines A, Obtained homozygous lines of WxP94L. B, Transgene-free lines of WxP94L by amplification using Hyg F/R and Cas9 F/R primers. Transgene-free WxP94L lines were coded in white numbers. M, DL2000 DNA marker; −, Negative contral; +, Positive control.

Fig. 3. Comparison of agronomic traits in WT and WxP94L A and B, Plant and panicle morphology of WT and WxP94L at maturity. Bars = 20 cm (A), 5 cm (B). C-H, Plant height, tiller number per plant, seed setting rate, grain length, grain width and 1000-grain weight of WT and WxP94L. No significant differences were observed between the two lines for all these traits (t-test, n = 10).

Fig. 4. Comparison of rice quality traits in WT and WxP94L A, Comparison of grain appearance in WT and WxP94L. B−E, Amylose content, gel consistency, alkali spreading value, and protein content of WT and WxP94L. *and** indicate significant difference at 5% and 1%, respectively (t-test, n = 3).

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