利用CBE系统编辑Wx基因调控稻米直链淀粉含量

doi:10.16819/j.1001-7216.2025.240705

中国水稻科学 ›› 2025, Vol. 39 ›› Issue (6): 771-778.DOI: 10.16819/j.1001-7216.2025.240705

利用CBE系统编辑Wx基因调控稻米直链淀粉含量

陈玉叶¹^,²^,^#, 焦晓真²^,^#, 王健², 王克剑², 陈峰³, 朱克明¹^,^*(), 刘朝雷²^,^*()

¹江苏大学生命科学学院，江苏镇江 212013
²中国水稻研究所水稻生物育种全国重点实验室，杭州 310006
³山东省农业科学院，济南 250100

收稿日期:2024-07-07 修回日期:2024-10-23 出版日期:2025-11-10 发布日期:2025-11-19
通讯作者: * email:uegzkg@sina.com.cn;liuchaolei@caas.cn
作者简介:第一联系人：
^#共同第一作者
基金资助:
浙江省自然科学基金资助项目(LQ20C130010);国家水稻产业技术体系（山东省农业良种工程）资助项目(2024LZGCQY007);山东省科技型中小企业创新能力提升工程资助项目(2024TSGC0841);山东省水稻产业技术体系资助项目(SDAIT-17-17)

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

CHEN Yuye¹^,²^,^#, JIAO Xiaozhen²^,^#, WANG Jian², WANG Kejian², CHEN Feng³, ZHU Keming¹^,^*(), LIU Chaolei²^,^*()

¹College of Life Sciences, Jiangsu University, Zhenjiang 212013, China
²State Key Laboratory of Rice Biology and Breeding, China National Rice Research Institute, Hangzhou 310006, China
³Shandong Academy of Agricultural Sciences, Jinan 250100, China

Received:2024-07-07 Revised:2024-10-23 Online:2025-11-10 Published:2025-11-19
Contact: * email:uegzkg@sina.com.cn;liuchaolei@caas.cn

摘要/Abstract

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

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

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.

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.

图/表 5

表1 本研究使用的引物信息

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

图1 CBE系统编辑圣稻18 Wx基因 A: Wx基因结构及靶点位置；B: 靶向Wx基因的CBE载体结构；C: 获得的圣稻18背景Wx编辑株系突变类型。蓝色小写字母和横线表示突变碱基；红色字母表示PAM序列。D：缺失；SNP：单个核苷酸的变异。

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.

图2 筛选无外源成分WxP94L株系 A: 获得的WxP94L纯合株系；B: 利用靶向Cas9、Hyg引物筛选无外源成分WxP94L株系。白色数字表示无外源成分的WxP94L单株。M: DL2000 DNA分子量标记；−: 阴性对照；+: 阳性对照。

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.

图3 野生型与WxP94L等位型农艺性状比较 A: 野生型与WxP94L成熟期植株表型，标尺=20 cm；B: 野生型与WxP94L成熟期穗表型，标尺=5 cm；C~H: 野生型与WxP94L株高、分蘖数、结实率、粒长、粒宽和千粒重比较。所有性状均无显著差异（t检验，n=10）。

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).

图4 野生型与WxP94L等位型米质性状比较 A：野生型与WxP94L米质外观比较；B~E：野生型与WxP94L直链淀粉含量、胶稠度、碱消值和蛋白质含量的比较。*和**表示分别表示在0.05和0.01水平条件下达到显著差异（t检验，n=3）。

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).

参考文献 23

[1]	Tian Z X, Qian Q, Liu Q Q, Yan M X, Liu X F, Yan C J, Liu G F, Gao Z Y, Tang S Z, Zeng D L, Wang Y H, Yu J M, Gu M H, Li J Y. Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities[J]. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(51): 21760-21765.
[2]	张栋昊, 蔡妍培, 劳菲, 吴继红. 大米蛋白质与米饭食味品质关联性研究进展[J]. 食品科学, 2023, 44(9): 270-277.
	Zhang D H, Cai Y P, Lao F, Wu J H. Research progress on the relationship between rice protein and eating quality[J]. Food Science, 2023, 44(9): 270-277. (in Chinese with English abstract)
[3]	许锐, 隋勇, 李书艺, 祝振洲, 周雷, 施建斌, 蔡沙, 熊添, 蔡芳, 梅新. 稻米食味品质影响因素研究进展[J]. 食品安全质量检测学报, 2024, 15(7): 234-241.
	Xu R, Sui Y, Li S Y, Zhu Z Z, Zhou L, Shi J B, Cai S, Xiong T, Cai F, Mei X. Research progress on influencing factors of rice eating quality[J]. Journal of Food Safety and Quality, 2024, 15(7): 234-241. (in Chinese with English abstract)
[4]	Sano Y. Differential regulation of waxy gene expression in rice endosperm[J]. Theoretical and Applied Genetics, 1984, 68(5): 467-473.
[5]	Sano Y, Katsumata M, Okuno K. Genetic studies of speciation in cultivated rice: 5. Inter-and intraspecific differentiation in the waxy gene expression of rice[J]. Euphytica, 1986, 35(1): 1-9.
[6]	Zhang C Q, Zhu J H, Chen S J, Fan X L, Li Q F, Lu Y, Wang M, Yu H X, Yi C D, Tang S Z, Gu M H, Liu Q Q. Wx^lv, the ancestral allele of rice Waxy gene[J]. Molecular Plant, 2019, 12: 1157-1166.
[7]	Zhang C Q, Yang Y, Chen S J, Liu X J, Zhu J H, Zhou L H, Lu Y, Li Q F, Fan X L, Tang S Z, Gu M H, Liu Q Q. A rare Waxy allele coordinately improves rice eating and cooking quality and grain transparency[J]. Journal of Integrative Plant Biology, 2021, 63(5): 889-901.
[8]	Zhou H, Xia D, Zhao D, Li Y H, Li P B, Wu B, Gao G J, Zhang Q L, Wang G W, Xiao J H, Li X H, Yu S B, Lian X M, He Y Q. The origin of Wx^la provides new insights into the improvement of grain quality in rice[J]. Journal of Integrative Plant Biology, 2021, 63(5): 878-888.
[9]	毛慧, 彭彦, 毛毕刚, 韶也, 郑文杰, 胡黎明, 周凯, 赵炳然. 水稻直链淀粉合成调控新基因Wx410的功能与效应分析[J]. 中国水稻科学, 2022, 36(6): 579-585.
	Mao H, Peng Y, Mao B G, Shao Y, Zheng W J, Hu L M, Zhou K, Zhao B R. Function and effect analysis of a new gene Wx410 regulating amylose synthesis in rice[J]. Chinese Journal of Rice Science, 2022, 36(6): 579-585. (in Chinese with English abstract)
[10]	Bibikova M, Beumer K, Trautman J K, Carroll D. Enhancing gene targeting with designed zinc finger nucleases[J]. Science, 2003, 300(5620): 764.
[11]	Moscou M J, Bogdanove A J. A simple cipher governs DNA recognition by TAL effectors[J]. Science, 2009, 326(11): 1501.
[12]	Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339: 819-823.
[13]	Gaudelli N M, Komor A C, Rees H A, Packer M S, Badran A H, Bryson D I, Liu D R. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage[J]. Nature, 2017, 551: 464-471.
[14]	Zong Y, Song Q N, Li C, Jin S, Zhang D, Wang Y, Qiu J L, Gao C. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A[J]. Nature Biotechnology, 2018, 36(10): 950-954.
[15]	任俊, 曹跃炫, 黄勇, 董慧荣, 刘庆, 王克剑. 基因编辑技术及其水稻中的发展和应用[J]. 中国稻米, 2021, 27(4): 92-100.
	Ren J, Cao Y X, Huang Y, Dong H R, Liu Q, Wang K J. Function and effect analysis of a new gene Wx410 regulating amylose synthesis in rice[J]. China Rice, 2021, 27(4): 92-100. (in Chinese with English abstract)
[16]	Liu T T, Zou J P, Yang X, Wang K J, Rao Y C, Wang C. Development and application of prime editing in plants[J]. Rice Science, 2023, 30(6): 509-522.
[17]	Zhang J S, Zhang H, Botella J R, Zhu J K. Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties[J]. Journal of Integrative Plant Biology, 2018, 60(5): 369-375.
[18]	Huang L C, Li Q F, Zhang C Q, Chu R, Gu Z W, Tan H Y, Zhao D S, Fan X L, Liu Q Q. Creating novel Wx alleles with fine-tuned amylose levels and improved grain quality in rice by promoter editing using CRISPR/Cas9 system[J]. Plant Biotechnology Journal, 2020, 18(11): 2164-2166.
[19]	Zeng D C, Liu T L, Ma X L, Wang B, Zheng Z Y, Zhang Y L, Xie X R, Yang B W, Zhao Z, Zhu Q L, Liu Y G. Quantitative regulation of Waxy expression by CRISPR/Cas9-based promoter and 5'UTR-intron editing improves grain quality in rice[J]. Plant Biotechnology Journal, 2020, 18(12): 2385-2387.
[20]	Xu Y, Lin Q P, Li X F, Wang F Q, Chen Z H, Wang J, Li W Q, Fan F J, Tao Y J, Jiang Y J, Wei X D, Zhang R, Zhu Q H, Bu Q Y, Yang J, Gao C X. Fine-tuning the amylose content of rice by precise base editing of the Wx gene[J]. Plant Biotechnology Journal, 2021, 19: 11-13.
[21]	朱文银, 徐凤文, 姜明松, 陈峰, 徐建第, 朱其松, 杨连群, 马加清. 优质高产抗病水稻新品种圣稻18的选育及栽培技术[J]. 中国稻米, 2014, 20(2): 78-79.
	Zhu W Y, Xu F W, Jiang M S, Chen F, Xu J D, Zhu Q S, Yang L Q, Ma J Q. Breeding and cultivation techniques of shengdao 18, a new rice variety with good quality, high yield and disease resistance[J]. China Rice, 2014, 20(2): 78-79. (in Chinese with English abstract)
[22]	李然, 钱前, 高振宇. 水稻品质的遗传与育种改良研究进展[J]. 生物技术通报, 2022, 38(4): 4-19.
	Li R, Qian Q, Gao Z Y. Research progress in the inheritance and breeding improvement of rice quality[J]. Biotechnology Bulletin, 2022, 38(4): 4-19. (in Chinese with English abstract)
[23]	王才林, 陈涛, 张亚东, 朱镇, 赵凌, 林静. 通过分子标记辅助选择培育优良食味水稻新品种[J]. 中国水稻科学, 2009, 23(1): 25-30.
	Wang C L, Chen T, Zhang Y D, Zhu Z, Zhao L, Lin J. Breeding of a new rice variety with good eating quality by marker assisted selection[J]. Chinese Journal of Rice Science, 2009, 23(1): 25-30. (in Chinese with English abstract)

利用CBE系统编辑Wx基因调控稻米直链淀粉含量

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

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