利用CRISPR-Cas9技术编辑Badh2基因创制优质香型籼稻三系不育系

doi:10.3724/SP.J.1006.2023.22043

作物学报 ›› 2023, Vol. 49 ›› Issue (8): 2144-2159.doi: 10.3724/SP.J.1006.2023.22043

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

利用CRISPR-Cas9技术编辑Badh2基因创制优质香型籼稻三系不育系

韦新宇¹^,³(), 曾跃辉¹^,³(), 杨旺兴²^,³, 肖长春¹^,³, 候新坡²^,³, 黄建鸿¹^,³, 邹文广²^,³, 许旭明²^,³^,^*()

¹ 三明市农业科学研究院生物技术研究所, 福建三明 365500
² 三明市农业科学研究院水稻研究所, 福建三明 365500
³ 福建省(山区)作物遗传改良与创新利用重点实验室, 福建三明 365500

收稿日期:2022-07-11 接受日期:2023-02-21 出版日期:2023-08-12 网络出版日期:2023-03-03
通讯作者: 许旭明
作者简介:韦新宇, E-mail: wxy1209@163.com;
曾跃辉, E-mail: 1_zengyuehui_1@163.com第一联系人：**同等贡献
基金资助:
财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-01);福建省自然科学基金项目(2021J01535);福建省自然科学基金项目(2021J01536);三明市科技计划项目(2019-N-4)

Development of high-quality fragrant indica CMS line by editing Badh2 gene using CRISPR-Cas9 technology in rice (Oryza sativa L.)

WEI Xin-Yu¹^,³(), ZENG Yue-Hui¹^,³(), YANG Wang-Xing²^,³, XIAO Chang-Chun¹^,³, HOU Xin-Po²^,³, HUANG Jian-Hong¹^,³, ZOU Wen-Guang²^,³, XU Xu-Ming²^,³^,^*()

¹ Biotechnology Research Institute, Sanming Academy of Agricultural Sciences, Sanming 365500, Fujian, China
² Rice Research Institute, Sanming Academy of Agricultural Sciences, Sanming 365500, Fujian, China
³ Fujian Key Laboratory of Crop Genetic Improvement and Innovative Utilization for Mountain Area, Sanming 365500, Fujian, China

Received:2022-07-11 Accepted:2023-02-21 Published:2023-08-12 Published online:2023-03-03
Contact: XU Xu-Ming
About author:First author contact:**Contributed equally to this work
Supported by:
China Agriculture Research System of MOF and MARA(CARS-01);Fujian Provincial Natural Science Foundation(2021J01535);Fujian Provincial Natural Science Foundation(2021J01536);Sanming Municipal Science and Technology Project(2019-N-4)

摘要/Abstract

摘要：

稻米香味是水稻的重要食味品质之一, 其主要受第8染色体上编码甜菜碱脱氢酶基因Badh2控制, 该基因突变可导致香味物质2-乙酰-1-吡咯啉(2-AP)的含量增加从而促进香味的产生。本研究以三明市农业科学研究院自主选育的优质籼型杂交稻保持系明太B为受体, 利用CRISPR-Cas9技术对其Badh2基因进行编辑和敲除。获得2个T₀代转基因纯合突变体植株并对其衍生的48个T₁代单株进行鉴定和分析, 获得1个不含转基因载体骨架且在第2外显子插入单个碱基T的纯合突变体株系明太B-badh2。利用半定量PCR和qRT-PCR技术以及气相色谱质谱联用仪(GC-MS)检测Badh2基因相对表达量和2-AP含量; 同时采用农业行业标准(NY/T 1433-2014)推荐的48对水稻SSR引物进行指纹图谱分析。结果表明, 该株系Badh2基因RNA表达水平显著下调; 籽粒中香味物质2-AP的含量显著增加; 指纹图谱分析发现, 仅1对引物Rm571在野生型和突变体之间鉴定到等位变异, 两组材料遗传差异较小。此外, 本研究还对野生型和突变体T₂代植株表型性状、稻米蒸煮食味品质和外观品质指标进行了考察和测定分析。结果表明, 所有指标在两组材料间均无显著差异。进一步采用测交和回交转育方法并结合Badh2位点测序分析, 成功选育获得了其对应的纯合香型三系不育系明太A-badh2。通过与恢复系明恢703、明恢3009测配, 其组合产量与国家审定品种明太优703、明太优3009相近且表现出较强的超标优势。此外, 通过与香型恢复系明恢1831测配后发现其组合籽粒中香味物质2-AP含量极显著高于对照组合明太A/明恢1831。因此, 利用CRISPR-Cas9基因编辑技术, 可对水稻香味基因Badh2进行精准定向编辑和敲除, 实现对水稻香味性状的改良, 为创制香型籼稻不育系提供理论指导, 从而加快香型杂交稻育种进程。

关键词: 水稻, CRISPR-Cas9, 基因编辑, 香味, Badh2, 2-乙酰-1-吡咯啉(2-AP)

Abstract:

Fragrance is one of the important traits for quality improvement in rice, which is mainly controlled by the Badh2 gene encoding a betaine aldehyde dehydrogenase on chromosome 8. The mutation of Badh2 gene can increase the content of 2-acetyl-1-pyrroline (2-AP) and promote the fragrance production in rice. In this study, the Badh2 gene in Mingtai B (MTB), an elite maintainer line of Indica hybrid rice showing high eating quality bred by Sanming Academy of Agricultural Sciences, was edited and knocked out by using CRISPR-Cas9 technology. Two T₀ transgenic lines carrying homozygous mutation on the loci of Badh2 were generated, and 48 T₁individuals derived from these two plants were genotyped by PCR amplification and sequencing analysis. A mutant line named MTB-badh2, which had a single T nucleotide insertion in the second exon of Badh2 without the vector skeleton, was finally obtained. In our study, the expression of Badh2 and the content of 2-AP were determined by semi-quantitative RT-PCR, qRT-PCR, and gas chromatography-mass spectrometry (GC-MS), respectively. Simultaneously, the 48 pairs of SSR markers recommended by “Sector Standard of Agriculture (NY/T 1433-2014)” were used to further analyze the DNA fingerprint and genetic diversity of the MTB and the MTB-badh2. The results showed that the Badh2 RNA level was significantly decreased in the MTB-badh2 compared with the wild-type MTB. In addition, the 2-AP content was dramatically increased in MTB-badh2. DNA fingerprint analysis revealed that only the Rm571 primer pair was specific for identifying allelic variation between wild type and MTB-badh2, suggesting that the genetic diversity between wild type and MTB-badh2 was very low. Furthermore, agronomic phenotype, cooking and eating quality, appearance quality of wild type, and MTB-badh2 were investigated and analyzed. There was no significant difference between MTB and MTB-badh2. The corresponding stable fragrant CMS line Mingtai A-badh2 (MTA-badh2) carrying homozygous mutant on the locus of Badh2 was successfully generated by the conventional test-crossing and back-crossing techniques combined with Badh2 sequencing analysis. Derived from this CMS line, MTA-badh2/Minghui 1831, MTA-badh2/Minghui 703, and MTA-badh2/Minghui 3009 hybrid combinations had been bred. Among them, the grain yield of MTA-badh2/Minghui 703 and MTA-badh2/Minghui 3009 were significantly increased compared with that of Mingtai you 703 and Mingtai you 3009, which were registered and released by the National Crop variety Appraisal Committee. Moreover, MTA-badh2/Minghui 1831 dramatically increased 2-AP content in the grains compared with MTA/Minghui 1831. Therefore, the CRISPR-Cas9-mediated technology could be used to edit the Badh2 and improve the fragrance in rice, and it provides the theoretical guidance for development of fragrant indica CMS line, leading to an accelerated breeding process of fragrant hybrid rice.

Key words: rice, CRISPR-Cas9, gene editing, fragrance, Badh2, 2-acetyl-1-pyrroline (2-AP)

韦新宇, 曾跃辉, 杨旺兴, 肖长春, 候新坡, 黄建鸿, 邹文广, 许旭明. 利用CRISPR-Cas9技术编辑Badh2基因创制优质香型籼稻三系不育系[J]. 作物学报, 2023, 49(8): 2144-2159.

WEI Xin-Yu, ZENG Yue-Hui, YANG Wang-Xing, XIAO Chang-Chun, HOU Xin-Po, HUANG Jian-Hong, ZOU Wen-Guang, XU Xu-Ming. Development of high-quality fragrant indica CMS line by editing Badh2 gene using CRISPR-Cas9 technology in rice (Oryza sativa L.)[J]. Acta Agronomica Sinica, 2023, 49(8): 2144-2159.

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链接本文: https://zwxb.chinacrops.org/CN/10.3724/SP.J.1006.2023.22043

https://zwxb.chinacrops.org/CN/Y2023/V49/I8/2144

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	用途Usage
Target1-Fwd	GGCATGGCCACGGCGATCCCGCAG	靶序列1构建 Construction of target site 1
Target1-Rev	AAACCTGCGGGATCGCCGTGGCCA	靶序列1构建 Construction of target site 1
Target 2-Fwd	GGCAAGGCGAGATCCCGGCGGGCA	靶序列2构建 Construction of target site 2
Target 2-Rev	AAACTGGCCACGGCGATCCCGCAG	靶序列2构建 Construction of target site 2
HPT-F	ATTTGTGTACGCCCGACAGT	鉴定HPT PCR detection in HPT
HPT-R	GTGCTTGACATTGGGGAGTT	鉴定HPT PCR detection in HPT
Cas9 identify-F	AGAACCTCTCCGATGCTATCC	鉴定Cas9 PCR detection in Cas9
Cas9 identify-R	AGCAAGAGGACCAACGTAG	鉴定Cas9 PCR detection in Cas9
Badh2Seq-F	CAAGGCAGCACAGAACAGA	Badh2基因靶点测序检测 Sequencing for target in Badh2
Badh2Seq-R	GTAGTCACCACCCTACCTTG	Badh2基因靶点测序检测 Sequencing for target in Badh2
Badh2-Q-F	TCCGGGCCAAGTACCTCC	Badh2基因qPCR检测 qPCR detection in Badh2
Badh2-Q-R	CGTCCATGTCCCATGCTG	Badh2基因qPCR检测 qPCR detection in Badh2
Actin-F	ACCTTCAACACCCCTGCTAT	内参基因Actin qPCR检测 qPCR detection in Actin (as internal standard)
Actin-R	CACCATCACCAGAGTCCAAC

杂交组合 Cross combination	播始历期 Duration from seeding to heading (d)	株高 Plant height (cm)	穗长 Length of main panicle (cm)	有效穗 Panicle number per plant	穗粒数 Grain number per panicle	结实率 Seed-settingrate (%)	千粒重 1000-grain weight (g)	产量 Grain yield (kg hm^-2)	比CK增产 Compared with control (%)
明太A-badh2/明恢703 MTA-badh2/Minghui 703	74.7±0.6 a	113.2±2.5 a	25.5±0.3 A	12.7±0.6 a	210.0±5.0 A	78.1±0.1 a	25.5±0.2 A	10877.0±196.0 A	6.6
明太优703 Mingtaiyou 703	74.7±0.6 a	114.1±1.4 a	25.3±0.2 A	13.0±0.0 a	212.7±5.0 A	77.4±0.4 a	25.3±0.2 A	10843.8±224.2 A	6.3
天优华占(CK) Tianyouhuazhan (CK)	74.3±0.6 a	110.9±2.2 a	23.8±0.3 B	13.3±0.6 a	193.3±4.2 B	76.6±0.5 b	23.3±0.2 B	10205.7±85.0 B	0.0
明太A-badh2/明恢3009 MTA-badh2/Minghui 3009	75.0±0.0 a	118.4±2.1 a	24.5±0.3 A	12.7±0.6 a	196.3±8.1 A	76.9±0.4 a	25.9±0.4 A	9567.2±139.4 A	6.0
明太优3009 Mingtaiyou 3009	75.3±0.6 a	119.1±2.0 a	24.9±0.3 A	12.3±0.6 a	195.3±6.5 A	77.1±0.7 a	25.5±0.7 A	9549.5±156.7 A	5.8
天优华占(CK) Tianyouhuazhan (CK)	75.6±0.6 a	111.9±2.4 b	23.0±0.4 B	13.0±0.0 a	171.7±3.1 B	75.3±0.2 b	23.5±0.2 B	9025.5±116.0 B	0.0

利用CRISPR-Cas9技术编辑Badh2基因创制优质香型籼稻三系不育系

Development of high-quality fragrant indica CMS line by editing Badh2 gene using CRISPR-Cas9 technology in rice (Oryza sativa L.)

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