利用CRISPR/Cas9技术创制抗稻瘟病香型早籼温敏核不育系

doi:10.16819/j.1001-7216.2022.211007

中国水稻科学 ›› 2022, Vol. 36 ›› Issue (3): 248-258.DOI: 10.16819/j.1001-7216.2022.211007

利用CRISPR/Cas9技术创制抗稻瘟病香型早籼温敏核不育系

梁敏敏^#, 张华丽^#, 陈俊宇, 戴冬青, 杜成兴, 王惠梅, 马良勇^*()

中国水稻研究所水稻生物学国家重点实验室/国家水稻改良中心，杭州 310006

收稿日期:2021-10-25 修回日期:2021-12-20 出版日期:2022-05-10 发布日期:2022-05-11
通讯作者: 马良勇
作者简介:第一联系人：
#共同第一作者
基金资助:
中央级公益性科研院所基本科研业务费专项(CPSIBRF-CNRRI-202105);水稻生物学国家重点实验室开放课题(20210207);水稻生物学国家重点实验室开放课题(20190110);中国农业科学院科技创新工程资助项目。

Developing Fragrant Early indica TGMS Line with Blast Resistance by Using CRISPR/Cas9 Technology

LIANG Minmin^#, ZHANG Huali^#, CHEN Junyu, DAI Dongqing, DU Chengxing, WANG Huimei, MA Liangyong^*()

State Key Laboratory of Rice Biology and Chinese National Center for Rice Improvement, China National Rice Research Institute, Hangzhou 310006, China

Received:2021-10-25 Revised:2021-12-20 Online:2022-05-10 Published:2022-05-11
Contact: MA Liangyong
About author:First author contact:
#These authors contributed equally to this work

摘要/Abstract

摘要：

【目的】创制新型抗稻瘟病香型早籼温敏核不育系，为高产优质杂交水稻选育提供资源。【方法】利用CRISPP/Cas9技术在水稻稻瘟病基因Pi21、温敏不育基因TMS5和香味基因Badh2的第1外显子处设计靶位点，构建多基因表达载体pC1300-2×35S::g^TMS5-g^Badh2-g^Pi21，转化优质常规籼稻品种中早70，测序鉴定分析获得纯合阳性稳定株系。利用稻瘟病喷雾接种和打孔接种方法对稻瘟病基因Pi21的纯合突变株系进行稻瘟病抗性鉴定，利用GC-MS技术对Badh2纯合突变株系的香味物质2-乙酰-1-吡咯啉（2-AP）含量进行测定。【结果】在T₀转基因株系中，Pi21、TMS5和Badh2突变频率分别为87.5%、80.0%和87.5%，突变类型多为双等位突变。从T₁代中筛选不含载体骨架的纯合突变株系，获得两种三突变纯合株系。稻瘟病接种结果表明，与野生型相比，T₂代Pi21纯合变异株系的抗性显著提高。同时，接种后纯合突变体株系内相关防卫基因的表达量显著上调，ROS积累量也显著增加。tms5纯合变异株系表现出典型的温敏不育特性，TMS5基因的表达水平与野生型相比显著降低，高温下Ub_L404基因的表达水平明显高于野生型。与野生型相比，在Badh2纯合突变体植株中Badh2的表达水平显著下调，并且香味物质2-AP含量极显著增加。【结论】利用CRISPR/Cas9技术成功对Pi21、TMS5和Badh2基因同时进行定向编辑，获得了具有高抗稻瘟病的香型温敏不育系，为高抗、香型不育系材料的选育提供参考，加快高产优质杂交水稻的选育。

关键词: 水稻, CRISPR/Cas9, Badh2, Pi21, TMS5

Abstract:

【Objective】 New thermosensitive genic male sterile lines with high resistance and fragrance quality will provide resources for hybrid rice breeding. 【Method】The target genes Pi21, TMS5 and Badh2 were selected to construct the pC1300-2×35S::g^TMS5-g^Badh2-g^Pi21expression vector by CRISPR/Cas9 technology for transformation of early indica rice Zhongzao 70. The positive transgenic plants were obtained through sequencing analysis. Magnaporthe oryzae spraying inoculation and punching inoculation assays were conducted to identify the resistance to rice blast, and the content of 2-acetyl-1-pyrroline was measured by GC-MS. 【Result】In T₀ transgenic lines, the mutation rates of Pi21, TMS5 and Badh2 were 87.5%, 80.0% and 87.5%, respectively, and most of the mutant lines are biallelic mutations. Two types of the homozygous triple mutants without the vector were obtained in T₁ transgenic lines. Evaluation analysis of resistance to rice blast suggested that T₂ generation of the pi21 homozygous mutant lines showed significantly higher resistance to M. oryzae compared with the wild type. The expression levels of defense-related genes were up-regulated and the ROS accumulation increased in the homozygous mutant lines compared to the wild type after inoculation of M. oryzae. tms5 homozygous mutant lines displayed thermo-sensitive male sterility. Compared with the wild type, the expression level of TMS5 was significantly decreased and the transcription of Ub_L404 was increased in the tms5 homozygous mutants at high temperature. The expression level of Badh2 was down-regulated and the content of 2-AP increased dramatically in the Badh2 homozygous mutant lines. 【Conclusion】The homozygous triple mutant lines of Pi21, TMS5 and Badh2 were obtained by CRISPR/Cas9-mediated technology, which would provide materials for high-resistant and fragrant rice breeding.

Key words: rice, CRISPR/Cas9, Badh2, Pi21, TMS5

梁敏敏, 张华丽, 陈俊宇, 戴冬青, 杜成兴, 王惠梅, 马良勇. 利用CRISPR/Cas9技术创制抗稻瘟病香型早籼温敏核不育系[J]. 中国水稻科学, 2022, 36(3): 248-258.

LIANG Minmin, ZHANG Huali, CHEN Junyu, DAI Dongqing, DU Chengxing, WANG Huimei, MA Liangyong. Developing Fragrant Early indica TGMS Line with Blast Resistance by Using CRISPR/Cas9 Technology[J]. Chinese Journal OF Rice Science, 2022, 36(3): 248-258.

图/表 9

图1 Pi21、TMS5和Badh2基因结构和靶位点位置蓝色字母表示靶点序列，红色字母表示PAM序列。线条表示内含子，黑盒子代表外显子。

Fig. 1. Schematic diagram of the targeted sites of Pi21, TMS5 and Badh2. Blue letters are the target sequence and red letters are the protospacer adjacent motif (PAM) sequences. The lines represent introns, black boxes exons.

图2 pC1300-2×35S::gTMS5-gBadh2-gPi21 载体示意图 Hyg-潮霉素磷酸转移酶基因;LB-载体左边界;RB-载体右边界;Cas9蛋白的启动子是35S;sgRNA的启动子为U3。

Fig. 2. Schematic diagram of the pC1300-2×35S::gTMS5-gBadh2-gPi21 vector. Hyg, Hygromycin phosphotransferase gene; LB, Left border; RB, Right border; The Cas9 cassette is driven by the 35S promoter, while the sgRNA is controlled by the U3 promoter.

图3 两种三基因纯合突变体的类型蓝色字母表示靶点序列;红色字母表示碱基插入;红色连字符表示碱基缺失;+表示插入;―表示缺失;WT表示野生型。

Fig. 3. Two types of homozygous triple mutants. The initiation codon is highlighted in blue and insertions are represented by red letters. The deletions are shown by red hyphens; +, Insertion; ―, Deletion; WT, Wild-type.

表1 T0转基因株突变情况统计

Table 1. Ratios of mutant genotype and mutation types in T0 plants.

基因 Gene	株数 No. of plants	突变基因型比率 Ratio of mutation genotypes / %
基因 Gene	株数 No. of plants	纯合突变率Homozygous	杂合突变率Heterozygous	双等位突变率Bi-allele
Pi21	35	34.3 (12/35)	8.6 (3/35)	57.1 (20/35)
TMS5	35	11.4 (4/35)	0.0 (0/35)	80.0 (28/35)
Badh2	35	28.6 (10/35)	5.7 (2/35)	92.0 (23/35)

图4 稻瘟病抗性鉴定 A-纯合突变株系和野生型（WT）打孔接种;B-纯合突变株系和野生型喷雾接种;C-野生型和纯合突变株系的相对病斑面积;D-野生型和纯合突变株系的真菌生物量;E-纯合突变体及其野生型水稻Pi21相对表达量;F-野生型和纯合突变株系防卫基因的相对表达量;G-野生型和纯合突变株系ROS的积累量。数据为3次重复的平均数±标准误;**表示差异达0.01显著水平(t检验)。

Fig. 4. Identification of rice blast resistance. A, Punch inoculation of homozygous mutant lines and wild type. B, Spray inoculation of homozygous mutant lines and wild type. C, Lesion area of wild type and homozygous mutant lines. D, Relative fungal biomass of wild type and homozygous mutant lines. E. Relative expression levels of Pi21 in homozygous mutant lines and wild type; F, Relative expression levels of defense genes OsPR1a and OsPBZ1 in the wild type and the homozygous mutant lines. G, ROS accumulation in wild type and homozygous mutant lines. Data are shown as means ± SE of three biological replicates; **, Significant difference at 0.01 level(t-test).

表2 野生型和tms5纯合突变体的农艺性状

Table 2. Agronomic traits of the wild-type and tms5 homozygous mutant.

株系 Line	株高 Plant height / cm	有效分蘖 Number of tillers per plant	穗长 Panicle length / cm	每穗粒数 Number of spikelets per panicle	抽穗期 Heading date / d	结实率 Seed setting rate / %
野生型 WT	100.05±0.85	17.00±1.67	23.11±0.68	166.61±22.84	52.67±1.37	87.32±0.09
tms5#3	85.15±2.02^**	27.83±4.49^**	21.36±1.05^**	145.78±11.19^**	65.33±2.07^**	0^**
tms5#20	84.10±2.27^**	28.83±3.19^**	20.85±0.96^**	141.28±16.31^**	65.50±1.87^**	0^**

图5 野生型和tms5纯合突变体植株在田间生长下的形态特征 A-野生型和纯合突变体的株型，标尺为20 cm;B-野生型和纯合突变体的花药形态，标尺为0.5 cm;C-野生型和纯合突变体的花粉育性，标尺为100 μm;D-野生型和纯合突变体的穗，标尺为5 cm;E-野生型和纯合突变体的结实率。数据为平均数±标准差，n=3;**差异达0.01显著水平（t检验）。

Fig. 5. Phenotype of the wild-type and homozygous mutant grown in field during the normal rice growing season. A, Whole plant of the wild type and homozygous mutant lines, bars=20 cm. B, Anther morphology of the wild type and homozygous mutant lines, bars=0.5 cm. C, Pollen fertility of wild type and homozygous mutant lines, bars=100 μm. D, Panicles of the wild type and homozygous mutant lines, bars=5 cm. E, Seed setting rates of the wild type and homozygous mutant lines. Data are shown as means ± SD, n=3. **Significant difference at the 0.01 levels by t-test.

图6 野生型和纯合突变体植株的形态特征及TMS5和UbL404的表达量 A-野生型和纯合突变体在冷灌处理条件下的花药形态，标尺为0.5 cm;B-野生型和纯合突变体在冷灌处理条件下的花粉育性，标尺为100 μm;C-野生型和纯合突变体在冷灌处理条件下的穗子表型，标尺为5 m;D-野生型和纯合突变体在冷灌处理条件下的结实率;E-在不同温度下水稻叶片和幼穗TMS5基因的转录水平;F-野生型和纯合突变体在不同温度下UbL404的相对表达量。数据为平均数±标准差，n=3;*和**分别表示差异达0.05和0.01显著水平（t检验）。

Fig. 6. Phenotypes and expression of TMS5 and UbL404 in wild type and homozygous mutant plants. A, Phenotypes of wild type and homozygous mutant plants under cold irrigation conditions, bars=0.5 cm. B, Anther morphology of wild type and homozygous mutant plants under cold irrigation conditions, bars=100 μm. C, Panicle of wild type and homozygous mutant plants under cold irrigation conditions, bars=100 μm. D, Seed setting rates of wild type and homozygous mutant plants under cold irrigation conditions. E, Transcription of TMS5 as estimated by qRT-PCR in leaves and young panicle of plants grown at different temperature. F, Relative expression of UbL404 as estimated by qRT-PCR in wild type and homozygous mutant lines at different temperature. Data are shown as means ± SD, n=3; *and ** represent significant difference at the 0.05 and 0.01 levels by t-test, respectively.

图7 Badh2基因的表达情况及2-AP的含量测定 A―野生型（WT）和纯合突变体Badh2基因的表达量;B―野生型和纯合突变体的2-AP含量。平均数±标准差(n=3);**表示差异达0.01显著水平（t检验）。

Fig. 7. Expression of Badh2 and content determination of 2-AP. A, Expression of Badh2 of the wild type and homozygous mutant lines. B, 2-AP levels of wild type and homozygous mutant lines. Data are shown as mean±SD (n=3); **, Significant difference at 0.01 level (t-test).

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利用CRISPR/Cas9技术创制抗稻瘟病香型早籼温敏核不育系

Developing Fragrant Early indica TGMS Line with Blast Resistance by Using CRISPR/Cas9 Technology

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