利用CRISPR/Cas9技术创制无芽鞘紫线的香型环境非敏感隐性核雄性不育种质

doi:10.16819/j.1001-7216.2025.240501

摘要/Abstract

摘要：

【目的】水稻杂种优势利用技术已发展到第三代，环境非敏感型隐性核雄性不育是第三代杂交稻的必需性状，而香味是许多水稻育种计划的重要目标。另外，水稻芽鞘紫线是极早期表现的一种明显稳定的花青素性状，有利于杂交种纯度的简单、快速和准确鉴定。本研究目的是将II-32B制备成香型、无芽鞘紫线的环境非敏感型隐性核雄性不育材料，为研发方便纯度鉴定的香型第三代杂交稻创造核心种质。【方法】利用CRISPR/Cas9技术，同时对优良水稻亲本II-32B的花粉发育相关基因OsbHLH141、香味基因OsBadh2以及芽鞘紫线基因OsMYB76进行敲除。【结果】获得了三个基因同时发生突变的材料，突变体表现为芽鞘紫线等花青素性状缺失，稻米具有香味且雄性不育。【结论】本研究成功获得了以II-32B为背景的香型、无芽鞘紫线的环境非敏感型隐性核雄性不育种质。

关键词: 环境非敏感, 隐性核雄性不育, 香味, 芽鞘紫线, CRISPR/Cas9

Abstract:

【Objective】Hybrid rice has developed into the third generation in which environment-insensitive recessive genic male sterility (EI-RGMS) is the essential trait. In many rice breeding programs, fragrance improvement is an important target. Additionally, the purple line in coleoptile is a clearly identifiable anthocyanin trait at the very early germination stage, which facilitates simple, rapid, and accurate identification of hybrid purity. The purpose of this study is to develop a fragrant EI-RGMS material lacking the coleoptile purple line based on maintainer II-32B, providing a new core germplasm for the third-generation hybrid rice.【Methods】 Using CRISPR/Cas9, we knocked out the pollen development gene OsbHLH141, fragrance gene OsBadh2, and coleoptile purple line gene OsMYB76 in the elite rice parent II-32B.【Result】 Mutant lines with all three genes knocked out showed loss of anthocyanin traits, a distinct aroma, and male sterility.【Conclusion】 This study successfully created a fragrant EI-RGMS germplasm without purple line in coleoptile in the II-32B background.

Key words: environment-insensitive, recessive genic male sterility, fragrance, purple line in coleoptile, CRISPR/Cas9

杨佳欣, 管玉圣, 杜润, 李贤勇, 蔡座坤, 王楚桃, 阳启样, 何永歆, 朱子超, 张毅. 利用CRISPR/Cas9技术创制无芽鞘紫线的香型环境非敏感隐性核雄性不育种质[J]. 中国水稻科学, 2025, 39(5): 643-649.

YANG Jiaxin, GUAN Yusheng, DU Run, LI Xianyong, CAI Zuokun, WANG Chutao, YANG Qiyang, HE Yongxin, ZHU Zichao, ZHANG Yi. Creating a Fragrant Environment-insensitive Recessive Genic Male Sterile Germplasm Lacking Coleoptile Purple Lines by CRISPR/Cas9 Gene Editing[J]. Chinese Journal OF Rice Science, 2025, 39(5): 643-649.

图/表 7

表1 本研究所用到的靶点序列

Table 1. Target sequences used in this study

靶点名称 Target name	引物序列(5΄-3΄) Sequence(5΄-3΄)
OsbHLH141 target 1	CTGGTGGAACAGAAGAGGCA TGG
OsbHLH141 target 2	TGGGAAACAACCCATAATTT TGG
OsBadh2 target 1	TTTACTGGGAGTTATGAAAC TGG
OsMYB76 target 1	AGCACGCTCAGCCGCAAGAT CGG

表2 本研究所用到的引物序列

Table 2. Primer sequences used in this study

引物名称 Primer name	正向引物序列(5΄-3΄) Forward primer sequence(5΄-3΄)	反向引物序列(5΄-3΄) Reverse primer sequence(5΄-3΄)	目的 Purpose
Cas393F/R	TCGAGAACGGTCGTAAGAGGA	TGTAACGCTTCCTGTCGATGGT	转基因检测Transgenic detection
141F10/R10	CTCCAGTTAGGGTACCATGGTACT	CGGAATTCCTCACTGGCCTCATCGATGA	测序Sequencing
APCSF1/R1	CGGAATTCCAGGACTTGTTTGGAGCTTGC	GGGGTACCTGCTCGTCTGGGACCAGTAT	测序Sequencing
OsC1F4/R3	CAGTCTCACACCGCACAG	ACGGAAACCCGCAACTGC	测序Sequencing
141F8/R8	CCATGGTACTCAATCTCATC	GTCCTGAACTTCACATTTAG	分子标记检测Molecular marker detection
141F9/R9	GGTGATGCCATTGAGTATAT	GAGCTGATTGTCTTGATCAT	分子标记检测Molecular marker detection
APMF3/R3	CATGTATACCCCATCAATGG	GCTCAAAGTGTCTTGATCAC	分子标记检测Molecular marker detection
CMDF1/R1	CGAACAGACAATGAAATCAAG	CACGACGGAGCTGGACGAC	分子标记检测Molecular marker detection

图1 OsbHLH141、OsBadh2及OsMYB76基因结构和靶点位置黑色序列为靶点序列，红色字母表示PAM序列。白色区域表示非翻译区，黑色区域表示外显子。

Fig.1. Schematic diagram of targeted sites of OsbHLH141, OsBadh2 and OsMYB76 The black sequences are target sequences and the red underlined sequences are PAM sequences. The white regions are UTRs, and the black regions are exons.

图2 T0代植株突变检测 T0代测序结果，下划线部分为靶点，红色字体部分为PAM序列。

Fig. 2. Mutation detection of T0 generation plants Sequencing results of mutation of T0 generation, the underlined sequences are targets, and the red font parts are PAM sequences.

图3 F2代单株检测 A：F2代植株转基因成分检测，M为2000 bp 标记，“+”为阳性对照，“−”为阴性对照；B：F2代无花青素色植株分子标记检测，M为50 bp 标记，W代表野生型对照，红色箭头为OsbHLH141大片段缺失杂合突变和OsBadh2大片段双等位突变。

Fig. 3. Detection of F2 single plants A, Transgenic component detection of F2. M, 2000 bp marker; "+" is the positive control, and "−" is the negative control; B, Molecular marker assistant selection of F2 without anthocyanine pigment. M is 50 bp marker, W is the wild-type control, and the red arrows are the large fragment heterozygous mutations of OsbHLH141 and the large fragment heterozygous biallelic mutations of OsBadh2.

图4 F2代植株三基因突变单株芽鞘紫线和育性观察 A：野生型和突变体的芽鞘对比，标尺=2 mm；B：野生型和突变体的去壳颖花对比，标尺= 50 μm；C：野生型与突变体的花粉碘染对比，标尺= 100 μm；D：野生型和突变体植株对比，标尺= 10 cm。

Fig. 4. Observation of coleoptile purple line and fertility of a single plant with three mutated genes in F2 A, Comparison of coleoptile between wild-type and mutant, Bar=2 mm; B, Comparison of spikelets with glume removed between wild-type and mutant, Bar = 50 μm; C, Comparison of pollen iodine staining between wild-type and mutant, Bar = 100 μm; D, Comparison between wild-type and mutant plants, Bar= 10 cm.

表3 农艺性状的比较

Table 3. Comparison of agronomic traits between wild type and mutant

材料 Material	株高 Plant height (cm)	有效穗数 Effective panicle number	穗长 Panicle length (cm)	每穗粒数 No. of grains per panicle	千粒重 1000-grain weight (g)	结实率 Seed setting rate (%)
野生型 WT	98.04±2.25	14.80±1.48	23.22±0.55	201.20±16.44	22.89±0.26	94.49±1.28
突变体Mutant	90.77±1.48***	12.33±1.03*	22.20±0.66*	191.83±12.45	/	/

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