基于CRISPR/Cas9基因编辑技术研究Badh2基因对稻米品质的影响

doi:10.13304/j.nykjdb.2023.0889

摘要/Abstract

摘要：

水稻Badh2基因的功能丧失能提高稻米中香味物质2-乙酰-1-吡咯啉（2-acetyl-1-pyrroline，2-AP）的含量，对稻米品质的改善具有重要意义。为了探究Badh2基因对稻米品质的影响，利用CRISPR/Cas9基因编辑技术以无香水稻日本晴为背景构建了Badh2基因的敲除株系，获得45 bp片段缺失的突变体badh2-1和1 bp插入的突变体badh2-2，对野生型和突变体的2-AP含量、挥发性风味物质、农艺性状、加工及外观品质、食味营养品质与食味值、RVA谱特征值进行测定分析。结果表明，与野生型相比，突变体badh2-1和badh2-2的2-AP含量显著提高；结实率、每穗粒数显著增加，但有效分蘖显著降低；外观品质有所下降，但未改变稻米品质等级；蛋白质含量、营养品质显著降低，直链淀粉含量和食味值等综合食味评分显著提高；消减值和糊化温度显著降低，使蒸煮食味变好。且2个突变体间的主要挥发性风味物质、有效分蘖、千粒重、株高、糙米率、峰值黏度也存在显著差异，突变体badh2-1的蒸煮食味品质优于突变体badh2-2。综上所述，利用CRISPR/Cas9基因编辑技术成功创制了香稻种质新资源，Badh2基因的突变会对稻米品质产生重要影响，且不同的突变方式会产生不同的影响。以上结果为后续优质香稻选育提供了参考。

关键词: 香稻, CRISPR/Cas9, 品质, 挥发性风味物质, 2-乙酰-1-吡咯啉

Abstract:

The loss of function of Badh2 gene leads to an increase of 2-acetyl-1-pyrroline （2-AP） content， which holds significant implications for enhancing rice quality. To investigate the impact of Badh2 gene on diverse aspects of rice quality， a knockout line targeted the Badh2 gene in the non-aromatic rice variety japonica cultivar Nipponbare was got using CRISPR/Cas9 gene editing technology. 2 mutants were obtained： badh2-1 with a 45 bp deletion and badh2-2 with a 1bp insertion. 2-AP content， volatile flavor substances， agronomic traits， milling and appearance quality， palatable nutritive qualities and taste values， and RVA profile characteristics of the wild type and mutant lines were determined and analyzed. The results showed that the mutants badh2-1 and badh2-2 had significantly higher 2-AP content， significant increased in seed setting and grain number per panicle， but decreased effective tillers. And the appearance quality of mutants showing decreased， but had the same rice quality grade with wild-type. The protein content and nutritional quality of mutants significantly reduced， the overall taste scores such as straight chain amylose content and taste value significantly increased. The setback and the gelatinization temperature of mutants showed a significant decrease， resulting in a better cooking taste. There were also significant differences in primary volatile flavor substances， effective tillers， thousand-grain weight， plant height， brown rice percentage， and peak viscosity between the 2 mutants， and the cooking flavor quality of badh2-1 was better than that of badh2-2. In conclusion， CRISPR/Cas9 gene editing technology could be successfully utilized in new germplasm resources of fragrant rice， the mutation of Badh2 gene had important effects on rice quality， and different mutation type should affect the quality of rice. Above results provided references for the subsequent breeding of high-quality fragrant rice.

Key words: fragrant rice, CRISPR/Cas9, quality, volatile flavor substances, 2-acetyl-1-pyrroline

中图分类号:

S511

张文婷, 李阳, 裘实, 路光明, 郭冬姝, 张保龙, 王金彦. 基于CRISPR/Cas9基因编辑技术研究Badh2基因对稻米品质的影响[J]. 中国农业科技导报, 2025, 27(5): 39-48.

Wenting ZHANG, Yang LI, Shi QIU, Guangming LU, Dongshu GUO, Baolong ZHANG, Jinyan WANG. Effects of Badh2 Gene on Rice Quality Based on CRISPR/Cas9 Gene Editing Technology[J]. Journal of Agricultural Science and Technology, 2025, 27(5): 39-48.

图/表 9

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用途Purpose	正向引物序列 Forward primer sequence（5’-3’）	反向引物序列 Reverse primer sequence（5’-3’）
Badh2扩增 Badh2 amplification	GGCATATGGCTTCAGCTGCTCCTA	AAACTAGGAGCAGCTGAAGCCATA
Badh2-CRISPR鉴定 Badh2-CRISPR identification	GGAACTATCCTCTCCTGATGGCA	ATTTTCTTGCATCCTGCTCGTCT
Badh2定量 Badh2 qPCR	CTGAGCTGGCTAGACTAGAG	CCAACTACACCGATAGGCTC
Actin定量 Actin qPCR	GGACCCAAGAATGCTAAGCC	TGGTACCCTCATCAGGCATC
潮霉素鉴定 Hygromycin identification	ACAATCCCACTATCCTTCGCAAG	GTACTTCTACACAGCCATCGGTC
Cas9鉴定 Cas9 identification	TTTCCCCAACCTCGTGTTGTTC	GAGGTTCTTCTTGATGGAGTGG

用途Purpose	正向引物序列 Forward primer sequence（5’-3’）	反向引物序列 Reverse primer sequence（5’-3’）
Badh2扩增 Badh2 amplification	GGCATATGGCTTCAGCTGCTCCTA	AAACTAGGAGCAGCTGAAGCCATA
Badh2-CRISPR鉴定 Badh2-CRISPR identification	GGAACTATCCTCTCCTGATGGCA	ATTTTCTTGCATCCTGCTCGTCT
Badh2定量 Badh2 qPCR	CTGAGCTGGCTAGACTAGAG	CCAACTACACCGATAGGCTC
Actin定量 Actin qPCR	GGACCCAAGAATGCTAAGCC	TGGTACCCTCATCAGGCATC
潮霉素鉴定 Hygromycin identification	ACAATCCCACTATCCTTCGCAAG	GTACTTCTACACAGCCATCGGTC
Cas9鉴定 Cas9 identification	TTTCCCCAACCTCGTGTTGTTC	GAGGTTCTTCTTGATGGAGTGG

序号 Number	化合物 Compound	分子式 Molecular formula	相对含量 Relative content/%
序号 Number	化合物 Compound	分子式 Molecular formula	WT	badh2-1	badh2-2
1	邻苯二甲酸二丁酯 Dibutyl phthalate	C₁₆H₂₂O₄	—	26.29	15.73
2	棕榈酸 Hexadecanoic acid	C₁₆H₃₂O₂	27.55	—	—
3	壬醛 Nonanal	C₉H₁₈O	4.81	9.58	14.27
4	邻苯二甲酸二异丁酯 1，2-benzenedicarboxylic acid， bis（2-methylpropyl） ester	C₁₆H₂₂O₄	17.53	11.72	8.44
5	7，9-二叔丁基-1-氧杂螺（4.5）癸-6，9-二烯-2，8-二酮 7，9-di-tert-butyl-1-oxaspiro（4，5）deca-6，9-diene-2，8-dione	C₁₇H₂₄O₃	—	5.50	7.48
6	碳酸，二十烷基乙烯基酯 Carbonic acid， eicosyl vinyl ester	C₂₃H₄₄O₃	7.91	—	—
7	十二烷 Dodecane	C₁₂H₂₆	2.75	4.75	2.54
8	2，6，10-三甲基十三烷 2，6，10-trimethyltridecane	C₁₆H₃₄	1.92	3.81	6.05
9	癸酸乙酯 Decanoic acid， ethyl ester	C₁₂H₂₄O₂	—	5.38	—
10	植酮 6，10，14-trimethyl-2-pentadecanone	C₁₈H₃₆O	4.37	4.39	5.75
11	5-乙基间二甲苯 1，2，3，5-tetramethyl-benzene	C₁₀H₁₄	2.74	1.86	5.11
12	癸酸 Decanoic acid	C₁₀H₂₀O₂	1.16	4.48	3.73
13	癸醛 Decanal	C₁₀H₂₀O	1.46	1.93	4.28
14	亚油酸（Z，Z）-9，12-octadecadienoic acid	C₁₈H₃₆O₂	5.19	—	—
15	乙酸硬脂基酯 Acetic acid n-octadecyl ester	C₂₀H₄₀O₂	5.13	—	—
16	十八烷 Octadecane	C₁₈H₃₈	4.57	—	—
17	十四烷 Tetradecane	C₁₄H₃₀	2.01	5.38	3.54
18	2，6，10-三甲基十四烷 2，6，10-trimethyl-tetradecane	C₁₇H₃₆	3.81	—	—
19	顺-菖蒲烯 Cis-calamenene	C₁₅H₂₂	0.88	1.56	2.95
20	十二醛 Dodecanal	C₁₂H₂₄O	0.69	1.15	2.54
21	1-辛醇 1-octanol	C₈H₁₈O	—	1.28	2.53
22	2，3-二甲基环己醇 2，3-dimethyl-cyclohexanol	C₈H₁₆O	0.47	1.19	2.51

序号 Number	化合物 Compound	分子式 Molecular formula	相对含量 Relative content/%
序号 Number	化合物 Compound	分子式 Molecular formula	WT	badh2-1	badh2-2
1	邻苯二甲酸二丁酯 Dibutyl phthalate	C₁₆H₂₂O₄	—	26.29	15.73
2	棕榈酸 Hexadecanoic acid	C₁₆H₃₂O₂	27.55	—	—
3	壬醛 Nonanal	C₉H₁₈O	4.81	9.58	14.27
4	邻苯二甲酸二异丁酯 1，2-benzenedicarboxylic acid， bis（2-methylpropyl） ester	C₁₆H₂₂O₄	17.53	11.72	8.44
5	7，9-二叔丁基-1-氧杂螺（4.5）癸-6，9-二烯-2，8-二酮 7，9-di-tert-butyl-1-oxaspiro（4，5）deca-6，9-diene-2，8-dione	C₁₇H₂₄O₃	—	5.50	7.48
6	碳酸，二十烷基乙烯基酯 Carbonic acid， eicosyl vinyl ester	C₂₃H₄₄O₃	7.91	—	—
7	十二烷 Dodecane	C₁₂H₂₆	2.75	4.75	2.54
8	2，6，10-三甲基十三烷 2，6，10-trimethyltridecane	C₁₆H₃₄	1.92	3.81	6.05
9	癸酸乙酯 Decanoic acid， ethyl ester	C₁₂H₂₄O₂	—	5.38	—
10	植酮 6，10，14-trimethyl-2-pentadecanone	C₁₈H₃₆O	4.37	4.39	5.75
11	5-乙基间二甲苯 1，2，3，5-tetramethyl-benzene	C₁₀H₁₄	2.74	1.86	5.11
12	癸酸 Decanoic acid	C₁₀H₂₀O₂	1.16	4.48	3.73
13	癸醛 Decanal	C₁₀H₂₀O	1.46	1.93	4.28
14	亚油酸（Z，Z）-9，12-octadecadienoic acid	C₁₈H₃₆O₂	5.19	—	—
15	乙酸硬脂基酯 Acetic acid n-octadecyl ester	C₂₀H₄₀O₂	5.13	—	—
16	十八烷 Octadecane	C₁₈H₃₈	4.57	—	—
17	十四烷 Tetradecane	C₁₄H₃₀	2.01	5.38	3.54
18	2，6，10-三甲基十四烷 2，6，10-trimethyl-tetradecane	C₁₇H₃₆	3.81	—	—
19	顺-菖蒲烯 Cis-calamenene	C₁₅H₂₂	0.88	1.56	2.95
20	十二醛 Dodecanal	C₁₂H₂₄O	0.69	1.15	2.54
21	1-辛醇 1-octanol	C₈H₁₈O	—	1.28	2.53
22	2，3-二甲基环己醇 2，3-dimethyl-cyclohexanol	C₈H₁₆O	0.47	1.19	2.51

序号 Number	化合物 Compound	分子式 Molecular formula	相对含量 Relative content/%
序号 Number	化合物 Compound	分子式 Molecular formula	WT	badh2-1	badh2-2
23	3-甲基十三烷 3-methyl-tridecane	C₁₄H₃₀	1.82	1.18	1.45
24	壬酸 Nonanoic acid	C₉H₁₈O₂	1.06	—	—
25	十六烷基硫醇 Tert-hexadecanethiol	C₁₆H₃₄S	0.98	—	—
26	甲基 2-乙基己基邻苯二甲酸酯 Methyl 2-ethylhexyl phthalate	C₁₇H₂₄O₄	—	1.69	—
27	棕榈酸异丁酯 Hexadecanoic acid， 2-methylpropyl ester	C₂₀H₄₀O₂	—	1.53	0.87
28	十六醇 1-hexadecanol	C₁₆H₃₄O	—	1.44	1.38
29	香叶基丙酮 Geranylacetone	C₁₃H₂₂O	—	1.33	1.93
30	十一醛 Undecanal	C₁₁H₂₂O	0.47	—	1.81
31	十九烷 Nonadecane	C₁₉H₄₀	0.70	0.86	1.75
32	2-甲基-1-十六烷醇 2-methyl-1-hexadecanol	C₁₇H₃₆O	—	0.61	1.71
33	三（1-氯-2-丙基）磷酸酯 1-chloro-， 2-propanol， phosphate （3：1）	C₉H₁₈C_l3O₄P	—	1.10	1.64