基于CRISPR/Cas9技术创制优质香味粳稻品系

doi:10.16819/j.1001-7216.2026.250108

摘要/Abstract

摘要：

【目的】香味是水稻重要的食味品质性状。本研究选择优质粳稻品系台20-29为研究对象，利用CRISPR/Cas9技术对其香味基因Badh2进行编辑，以期获得具有香味的优质粳稻品系。【方法】选择香味基因Badh2第3、第4外显子上的2段序列作靶位点，构建双元表达载体pHUE411-Badh2-gRNA并转入台20-29获得转基因植株。【结果】20株T₀植株经过加代、潮霉素标记检测、测序比对，共计获得22个T₁代无载体纯合突变株，包括6种突变类型，每种突变体在第3、第4外显子上存在不同程度的碱基变化。6个香味突变体bh62、bh64、bh65、bh67、bh68、bh74在穗长、粒长、粒宽、千粒重等方面与野生型台20-29基本一致，结实率有所下降，每穗粒数稍有提升，稻米品质没有明显变化。利用气相色谱-质谱技术检测突变体bh62、bh64、bh65的糙米中香味物质2-AP含量，结果表明糙米中2-AP含量较台20-29极显著提高，分别达到24.27、33.89、38.96 mg/kg。【结论】对水稻香味基因Badh2进行编辑可以高效获得具有香味的水稻优质品系。

关键词: 水稻, 潮霉素, CRISPR/Cas9, 香味基因, 2-乙酰-1-吡咯啉

Abstract:

【Objective】 Fragrance is an important trait of rice eating quality. In this study, the fragrance gene Badh2 in rice was edited using CRISPR/Cas9 technology to create a high-quality aromatic japonica rice line. 【Method】 Two target sequences located in the third and fourth exons of Badh2 were selected as target sites for constructing the binary expression vector pHUE411-Badh2-gRNA. The high-quality japonica rice line Tai 20-29 was used as the transformation material. 【Result】 Twenty T0 transgenic plants were obtained after transformation. Through propagation, PCR, and sequencing verification, six homozygous mutants with different base alterations in the third and fourth exons of Badh2 were identified. Except for a slightly lower seed setting rate and a higher grain number per panicle, the six mutants(bh62, bh64, bh65, bh67, bh68, and bh74) showed similar agronomic traits such as panicle length, grain length, grain width, and 1000-grain weight compared with the wild-type Tai 20-29. The mutants showed a significantly lower seed setting rate and a higher grain number per panicle compared to Tai 20-29. The 2-AP content in brown rice of mutants bh62, bh64, bh65, and the wild type was determined by GC-MS. The results showed that the 2-AP content in the mutants increased significantly, reaching 24.27, 33.89, and 38.96 mg/kg, respectively, which was much higher than that in Tai 20-29. 【Conclusion】 We successfully generated a high-quality aromatic japonica rice line by editing the Badh2 gene using CRISPR/Cas9 technology.

Key words: rice, hygromycin, CRISPR/Cas9, fragrance gene, 2-acetyle-1-pyrroline (2-AP)

段敏, 谢留杰, 岳雅妮, 黄善军. 基于CRISPR/Cas9技术创制优质香味粳稻品系[J]. 中国水稻科学, 2026, 40(2): 235-243.

DUAN Min, XIE Liujie, YUE Yani, HUANG Shanjun. Development of High-quality Aromatic japonica Rice Lines by Using CRISPR/Cas9 Technology[J]. Chinese Journal OF Rice Science, 2026, 40(2): 235-243.

图/表 7

图1 Badh2基因结构及gRNA靶位点位置红色碱基表示PAM序列；黑色方框表示外显子。

Fig. 1. Schematic diagram of Badh2 and target sites Red letters represent protospacer adjacent motif (PAM) sequence; Black boxes represent exons.

表1 本研究中涉及的PCR扩增引物

Table 1. Primers used in this study

引物名称 Name of prime	寡核苷酸序列（5’-3’） Oligonucleotide sequence (5’-3’)
HYG-F	GCGTCTGCTGCTCCATACAA
HYG-R	TGACATTGGGGAGTTTAGCG
Bad-IF	TAGCCTTCCAGTCTAAATGACA
Bad-IR	CAACTACACCGATAGGCTCTT

图2 部分T1代单株潮霉素标记第二次检测结果

Fig. 2. Identification result of hygromycin in T1 generation transgenic plants (partial) in the second test

图3 6种香味突变体与野生型靶位点序列比对红色横线标出PAM序列，箭头表示突变碱基位置。

Fig. 3. Comparison of target sequences between six aromatic mutants and wild type Red lines display protospacer adjacent motif (PAM) sequence. Arrows show the location of mutant bases.

图4 6种香味突变体的农艺性状数据用平均数±标准差表示，株高、每株穗数、千粒重的样本量n=5，其余参数统计时n=15。**,*分别表示突变体与野生型相比差异达显著(P <0.01)和极显著水平(P <0.05)。

Fig. 4. Agronomic traits of six aromatic mutants and wild type Values are shown as mean±SD. Sample sizes n=5 for plant height, panicle number per plant and 1000-grain weight, n=15 for the rest factors. **and * indicate significant difference between mutants and the wild type at 0.01 and 0.05, respectively.

图5 突变体bh62与野生型台20-29成熟期时的表型对比 A，单株对比，比例尺=20 cm；B，穗型对比，比例尺=5 cm。

Fig. 5. Phenotypic comparison during mature stage between the mutant bh62 and its wild type Tai 20-29 A, Plant comparison, bar=20 cm; B, Panicle comparison, bar=5 cm.

图6 香味突变体与野生型的稻米表型（A）及糙米中2-AP含量（B）比例尺为1 cm。B图中数据测定重复3次，**表示与野生型相比，P<0.01。

Fig. 6. Rice phenotype (A) and 2-AP content of brown rice (B) in mutants and the wild type Bar=1 cm. Values are shown as mean±SD, n=3. **represents P <0.01 compared with wild type.

参考文献 29

[1]	唐傲, 邵高能, 胡培松. 水稻香味基因的研究进展[J]. 中国稻米, 2009, 15(4): 1-4.
	Tang A, Shao G N, Hu P S. Research progress on aromatic gene in rice[J]. China Rice, 2009, 15(4): 1-4. (in Chinese)
[2]	张俊宝, 潘惠文, 刘海英. 水稻香味基因的遗传和研究进展[J]. 生物技术世界, 2016(3): 9, 11.
	Zhang J B, Pan H W, Liu H Y. Inheritance and research progress of rice flavor genes[J]. Biotech World, 2016(3): 9, 11. (in Chinese)
[3]	赵国超. 香稻分子育种及香味形成分子机理的初步研究[D]. 上海: 上海师范大学, 2010.
	Zhao G C. Preliminary study on molecular breeding of aromatic rice and molecular mechanism of fragrance formation[D]. Shanghai: Shanghai Normal University, 2010. (in Chinese with English abstract)
[4]	田华, 段美洋, 黎国喜, 唐湘如. 香稻香气的研究进展[J]. 种子, 2008, 27(7): 51-54.
	Tian H, Duan M Y, Li G X, Tang X R. Research progress on aroma of aromatic rice[J]. Seed, 2008, 27(7): 51-54. (in Chinese)
[5]	Bradbury L M T, Fitzgerald T L, Henry R J, Jin Q, Waters D L E. The gene for fragrance in rice[J]. Plant Biotechnology Journal, 2005, 3(3): 363-370.
[6]	Chen S H, Wu J, Yang Y, Shi W W, Xu M L. The fgr gene responsible for rice fragrance was restricted within 69 kb[J]. Plant Science, 2006, 171: 505-514.
[7]	Chen S H, Yang Y, Shi W W, Ji Q, He F, Zhang Z D, Cheng Z K, Liu X N, Xu M L. Badh2, encoding betaine aldehyde dehydrogenase, inhibits the biosynthesis of 2-acetyl-1-pyrroline, a major component in rice fragrance[J]. Plant Cell, 2008, 20: 1850-1861.
[8]	彭波, 孙艳芳, 陈报阳, 孙瑞萌, 孔冬艳, 庞瑞华, 李先文, 宋晓华, 李慧龙, 李金涛, 周棋赢, 柳琳, 段斌, 宋世枝. 水稻香味基因及其在育种中的应用研究进展[J]. 植物学报, 2017, 52(6): 797-807.
	Peng B, Sun Y F, Chen B Y, Sun R M, Kong D Y, Pang R H, Li X W, Song X H, Li H L, Li J T, Zhou Q Y, Liu L, Duan B, Song S Z. Research progress of fragrance gene and its application in rice breeding[J]. Chinese Bulletin of Botany, 2017, 52(6): 797-807. (in Chinese with English abstract)
[9]	徐华山, 李培德, 戴风美, 刘凯, 杨国才, 李三和, 闸文俊, 周雷, 陈志军. 基因组编辑技术研究进展及其在植物中的应用[J]. 安徽农学通报, 2015, 21(18): 15-19.
	Xu H S, Li P D, Dai F M, Liu K, Yang G C, Li S H, Zha W J, Zhou L, Chen Z J. Research progress of genome editing technology and its application in plants[J]. Anhui Agricultural Science Bulletin, 2015, 21(18): 15-19. (in Chinese)
[10]	段敏, 谢留杰, 高秀莹, 唐海娟, 黄善军, 潘晓飚. 利用CRISPR/Cas9技术创制广亲和水稻温敏雄性不育系[J]. 中国水稻科学, 2023, 37(3): 233-243.
	Duan M, Xie L J, Gao X Y, Tang H J, Huang S J, Pan X B. Creation of thermo-sensitive genic male sterile rice lines with wide compatibility based on CRISPR/Cas9 technology[J]. Chinese Journal of Rice Science, 2023, 37(3): 233-243. (in Chinese with English abstract)
[11]	邵高能, 谢黎虹, 焦桂爱, 魏祥进, 圣忠华, 唐绍清, 胡培松. 利用CRISPR/CAS9技术编辑水稻香味基因Badh2[J]. 中国水稻科学, 2017, 31(2): 216-222.
	Shao G N, Xie L H, Jiao G A, Wei X J, Sheng Z H, Tang S Q, Hu P S. CRISPR/CAS9-mediated editing of the fragrant gene Badh2 in rice[J]. Chinese Journal of Rice Science, 2017, 31(2): 216-222. (in Chinese with English abstract)
[12]	周文甲, 田晓杰, 任月坤, 魏祥进, 高扬, 谢黎虹, 刘华招, 卜庆云, 李秀峰. 利用CRISPR/Cas9创造早熟香味水稻[J]. 土壤与作物, 2017, 6(2): 146-152.
	Zhou W J, Tian X J, Ren Y K, Wei X J, Gao Y, Xie L H, Liu H Z, Bu Q Y, Li X F. Breeding of early-maturatity and fragrant rice via CRISPR/Cas9 mediated genome editing[J]. Soils and Crops, 2017, 6(2): 146-152. (in Chinese with English abstract)
[13]	Chen Y Y, Wang Z P, Ni H W, Xu Y, Chen Q J, Jiang L J. CRISPR/Cas9-mediated base-editing system efficiently generates gain- of function mutations in Arabidopsis [J]. Science China Life Sciences, 2017, 60(5): 520-523.
[14]	张丽, 朱勇良, 叶亚新, 谢裕林, 胡翠英, 程宏英, 金琎, 乔中英, 李庆魁. 不同农艺措施对苏香粳3号中2-乙酰-1-吡咯啉(2-AP)含量的影响[J]. 江苏农业科学, 2016, 44(3): 78-82.
	Zhang L, Zhu Y L, Ye Y X, Xie Y L, Hu C Y, Cheng H Y, Jin J, Qiao Z Y, Li Q K. Effects of different agronomic measures on the content of 2- acetyl-1-pyrroline (2-AP) in Suxiangjing No.3[J]. Jiangsu Agricultural Sciences, 2016, 44(3): 78-82. (in Chinese)
[15]	黄永相, 郭建夫, 李伟, 李东, 祝敏丹. 一种水稻香味快速检测方法: CN109490478A[P]. 2019-03-19.
	Huang Y X, Guo J F, Li W, Li D, Zhu M D. A rapid detection method for rice fragrance: CN109490478A[P]. 2019-03-19.
[16]	张涛, 郑家奎, 徐建第, 蒋开锋, 吴先军, 汪旭东. 香稻品种的遗传多样性研究[J]. 中国农业科学, 2008, 41(3): 625-635.
	Zhang T, Zheng J K, Xu J D, Jiang K F, Wu X J, Wang X D. Genetic diversity of aromatic rice varieties based on markers of functional genes and SSR[J]. Scientia Agricultura Sinica, 2008, 41(3): 625-635. (in Chinese with English abstract)
[17]	余亚莹, 邵高能, 圣忠华, 蒋汉伟, 贺记外, 孙园园, 蔡怡聪, 胡培松, 唐绍清. 国内外香稻资源遗传多样性研究[J]. 植物分类与资源学报, 2015, 37(6): 871-880.
	Yu Y Y, Shao G N, Sheng Z H, Jiang H W, He J W, Sun Y Y, Cai Y C, Hu P S, Tang S Q. Genetic diversity of global aromatic rice varieties[J]. Plant Diversity and Resources, 2015, 37(6): 871-880. (in Chinese with English abstract)
[18]	Shi W W, Yuang Y, Chen S H, Xu M L. Discovery of a new fragrance allele and the development of functional markers for the breeding of fragrant rice varieties[J]. Molecular Breeding, 2008, 22(2): 185-192.
[19]	Kovach M J, Calingacion M N, Fitzgerald M A, McCouch S R. The origin and evolution of fragrance in rice (Oryza sativa L.)[J]. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(34): 14444-14449.
[20]	Shao G N, Tang A, Tang S Q, Luo J, Jiao G A, Wu J L, Hu P S. A new deletion mutation of fragrant gene and the development of three molecular markers for fragrance in rice[J]. Plant Breeding, 2011, 130(2): 172-176.
[21]	祁永斌, 王建军, 张礼霞, 王林友, 宋建. 一种CRISPR/Cas9编辑水稻香味基因Badh2的方法: CN110592135A[P]. 2019-12-20.
[22]	Amarawathi Y, Singh R, Singh A K, Singh V P, Mohapatra T, Sharma T R, Singh N K. Mapping of quantitative trait loci for basmati quality traits in rice (Oryza sativa L.)[J]. Molecular Breeding, 2008, 21(1): 49-65.
[23]	赵志鹏, 李刚, 吴书俊, 陆家安. 香稻研究进展[J]. 上海农业学报, 2009, 25(2): 110-114.
	Zhao Z P, Li G, Wu S J, Lu J A. Progress of study on aromatic rice[J]. Acta Agriculturae Shanghai, 2009, 25(2): 110-114. (in Chinese with English abstract)
[24]	张羽, 冯志峰, 吴升华, 陈雪燕. 陕西汉中地区水稻主要推广品种的香味分析[J]. 陕西农业科学, 2008, 54(3): 18-20.
	Zhang Y, Feng Z F, Wu S H, Chen X Y. Flavor analysis of main rice varieties in Hanzhong, Shaanxi Province[J]. Shaanxi Journal of Agricultural Sciences, 2008, 54(3): 18-20. (in Chinese)
[25]	胡翠英, 朱勇良, 叶亚新, 乔中英, 程宏英. Nafion/PDDAC固相微萃取富集测定大米中2-乙酰基吡咯啉[J]. 苏州科技学院学报: 自然科学版, 2014(4): 42-45, 61.
	Hu C Y, Zhu Y L, Ye Y X, Qiao Z Y, Cheng H Y. Preconcentration and determination of 2-acetyl pyrrolidine in rice based on Nafion and PDDAC coated solid-phase microextraction[J]. Journal of Suzhou University of Science and Technology: Natural Science Edition, 2014(4): 42-45, 61. (in Chinese with English abstract)
[26]	齐金岗, 陈颖, 刘开强, 王小姣, 李林娟, 赵丽冰, 李孝琼, 郭嗣斌. 利用CRISPR-Cas9技术编辑OsBADH2基因改良优质恢复系桂恢852的香味品质[J]. 南方农业学报, 2024, 55(7): 2058-2068.
	Qi J G, Chen Y, Liu K Q, Wang X J, Li L J, Zhao L B, Li X Q, Guo S B. Improving the aroma quality of high-quality restorer line Guihui 852 by editing OsBADH2 gene with CRISPR-Cas9 technology[J]. Journal of Southern Agriculture, 2024, 55(7): 2058-2068. (in Chinese with English abstract)
[27]	孙慧宇, 宋佳, 王敬国, 刘化龙, 孙健, 莫天宇, 徐善斌, 郑洪亮, 邹德堂. 利用CRISPR/Cas9技术编辑Badh2基因改良粳稻香味[J]. 华北农业学报, 2019, 34(4): 1-8.
	Sun H Y, Song J, Wang J G, Liu H L, Sun J, Mo T Y, Xu S B, Zheng H L, Zou D T. Editing Badh2 gene to improve the fragrance of japonica rice by CRISPR/Cas9 Technology[J]. Acta Agriculturae Boreali-Sinica, 2019, 34(4): 1-8. (in Chinese with English abstract)
[28]	Shan Q W, Zhang Y, Chen K L, Zhang K, Gao C X. Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology[J]. Plant Biotechnology Journal, 2015, 13(6): 791-800
[29]	张江丽, 李苏洁, 李娟, 普世皇, 普玉娇, 张亮, 谭亚玲, 陈丽娟, 谭学林, 金寿林, 文建成. 不同来源水稻种质资源香味基因badh2位点的鉴定[J]. 分子植物育种, 2015, 13(4): 727-733.
	Zhang J L, Li S J, Li J, Pu S H, Pu Y J, Zhang L, Tan Y L, Chen L J, Tan X L, Jin S L, Wen J C. Identification of the fragrant gene badh2 locus in rice germplasm resources original from different area[J]. Molecular Plant Breeding, 2015, 13(4): 727-733. (in Chinese with English abstract)