定向敲除SD1基因提高水稻的抗倒性和稻瘟病抗性

doi:10.16819/j.1001-7216.2023.221113

中国水稻科学 ›› 2023, Vol. 37 ›› Issue (4): 359-367.DOI: 10.16819/j.1001-7216.2023.221113

定向敲除SD1基因提高水稻的抗倒性和稻瘟病抗性

李刚¹, 高清松², 李伟², 张雯霞², 王健¹, 程保山¹, 王迪¹, 高浩¹, 徐卫军¹, 陈红旗³(), 纪剑辉²()

¹江苏徐淮地区淮阴农业科学研究所/淮安市农业生物技术重点实验室，江苏淮安 223301
²淮阴师范学院/江苏省环洪泽湖生态农业与生物技术重点实验室, 江苏淮安 223300
³中国水稻研究所水稻生物育种全国重点实验室, 杭州 311401

收稿日期:2022-11-24 修回日期:2023-02-08 出版日期:2023-07-10 发布日期:2023-07-17
通讯作者: *email: chqhzfy@126.com;jijianhui@hytc.edu.cn
基金资助:
淮安市自然科学基金项目(HAB202076);江苏省重点研发计划资助项目(BE2021334);江苏省重点研发计划资助项目(BE2021323);国家自然科学基金资助项目(32070345);江苏省自然科学优秀青年基金项目(BK20180107)

Directed Knockout of SD1 Gene Improves Lodging Resistance and Blast Resistance of Rice

LI Gang¹, GAO Qingsong², LI Wei², ZHANG Wenxia², WANG Jian¹, CHEN Baoshan¹, WANG Di¹, GAO Hao¹, XU Weijun¹, CHEN Hongqi³(), JI Jianhui²()

¹Huaiyin Institute of Agricultural Science in Xuhuai Region of Jiangsu/Huai'an Key Laboratory of Agricultural Biotechnology, Huai'an 223001, China
²Jiangsu Key Laboratory for Eco-Agricultural Biotechnology Around Hongze Lake, Huaiyin Normal University, Huai’an 223300, China
³State Key Laboratory of Rice Biological Breeding Biology, China National Rice Research Institute, Hangzhou 311401, China

Received:2022-11-24 Revised:2023-02-08 Online:2023-07-10 Published:2023-07-17
Contact: *email: chqhzfy@126.com;jijianhui@hytc.edu.cn

摘要/Abstract

摘要：

【目的】 为改良高产粳稻品种淮119株高偏高及易感稻瘟病等不利性状，利用CRISPR/Cas9基因编辑技术对淮119中SD1基因进行定向敲除，为淮119后代品种改良奠定基础。【方法】 利用CRISPR/Cas9系统，以SD1基因为靶基因，构建基因敲除载体，以农杆菌介导转化淮119，获得无转基因插入的纯合突变株，进一步对纯合突变株株高、农艺性状、稻瘟病抗性及氮素吸收利用能力等进行综合分析。【结果】 利用农杆菌转化淮119，鉴定获得1株无转基因载体序列插入的纯合突变株。大田种植发现，野生型淮119出现60%面积以上的倒伏，而sd1纯合突变株群体由于株高变矮，从而有效避免了生育后期的倒伏。此外，用不同浓度的GA（0.01~1.00 μmol/L）处理，野生型苗高均显著高于突变株，表明sd1突变导致对外源GA敏感性降低。稻瘟病抗性鉴定结果表明，sd1基因突变不仅有效降低株高，同时增强了对稻瘟病的抗性水平。不利因素是，SD1基因的敲除降低了淮119的氮素吸收利用效率。【结论】 淮119中SD1基因定向敲除获得的无转基因插入纯合突变株，不仅株高显著下降，且稻瘟病抗性得到增强。

关键词: CRISPR/Cas9, 基因编辑, SD1, 稻瘟病, 抗倒性, 水稻

Abstract:

【Objective】 Huai 119 is a high-yielding japonica rice variety. To improve its unfavorable traits, especially high plant height and poor resistance to rice blast, we used the CRISPR/Cas9 gene editing technology to knock out its ‘Green Revolution’ gene SD1.【Method】 We selected the SD1 gene as the target to construct the CRISPR/Cas9 gene knockout vector, which was then transformed into Huai 119 by Agrobacterium-mediated transformation method, and the homozygous sd1 knockout line without transgenic insertion was obtained. Subsequently, we compared and analyzed the plant height, rice blast resistance, and nitrogen uptake and utilization efficiency of the sd1 and wild-type lines.【Result】 We successfully isolated a homozygous sd1 knockout line without transgenic insertion in the background of Huai 119. In field paddy, it was found that over 60% of the planting area of wild-type Huai 119 was lodging, while the sd1 homozygous mutant population effectively avoided lodging in the later stages of growth due to its shorter plant height. In addition, after treatment with different concentrations of GA (0.01-1.00 μmol/L), the plant height increase of wild-type plants was significantly greater than that of the sd1 line, indicating that the sensitivity of sd1 line to exogenous GA treatment was reduced. The identification of rice blast resistance shows that knocking out SD1 also contributed to the improvement of rice blast resistance of Huai 119. However, the absorption and utilization efficiency of nitrogen decreased due to the knockout of SD1. 【Conclusion】 The knockout of SD1 gene in the rice variety Huai 119 not only improves lodging resistance by reducing plant height, but also enhances the resistance to rice blast.

Key words: CRISPR/Cas9, gene editing, SD1, rice blast, lodging resistance, rice

李刚, 高清松, 李伟, 张雯霞, 王健, 程保山, 王迪, 高浩, 徐卫军, 陈红旗, 纪剑辉. 定向敲除SD1基因提高水稻的抗倒性和稻瘟病抗性[J]. 中国水稻科学, 2023, 37(4): 359-367.

LI Gang, GAO Qingsong, LI Wei, ZHANG Wenxia, WANG Jian, CHEN Baoshan, WANG Di, GAO Hao, XU Weijun, CHEN Hongqi, JI Jianhui. Directed Knockout of SD1 Gene Improves Lodging Resistance and Blast Resistance of Rice[J]. Chinese Journal OF Rice Science, 2023, 37(4): 359-367.

图/表 9

表1 本研究中使用的引物序列

Table 1. Primer sequences used in this study.

引物名称 Primer name	序列(5’-3’) Primer sequence（5'-3'）
SD1-P1	TGTGGAGCCATTCGTGTGGCCGAA
SD1-P2	AAACTTCGGCCACACGAATGGCTC
SD1-F	CACAGCCGCACCAACCAC
SD1-R	GTGGAAGCCGAAGGAGAGG
HPTF	GGGTGTCACGTTGCAAGACC
HPTR	ATGCCTCCGCTCGAAGTAGC
sgRNA-F	TCCCAGTCACGACGTTGTAA
sgRNA-R	GGCCATTTGTCTGCAGAAT
Cas9-F	CACCATCTACCACCTGAGAA
Cas9-R	CGAAGTTGCTCTTGAAGTTG
Actin-F	CCCCTCCTGAAAGGAAGTA
Actin-R	GGTCCGAAGAATTAGAAGCA

图1 SD1基因敲除靶点设计、T-DNA结构和突变鉴定 A―SD1基因结构及基因敲除靶点；B―脱靶序列分析；C―基因编辑载体T-DNA区段结构；D―测序结果及相应氨基酸序列。

Fig. 1. Target site for SD1 gene knock-out design, T-DNA structure and mutation identification. A, SD1 gene structure and gene knockout target sites; B, Off target sequence analysis; C, Gene editing vector T-DNA segment structure; D, Sequencing results and corresponding amino acid sequence.

图2 淮119中sd1突变株农艺性状分析

Fig. 2. Analysis of agronomic characters of sd1 mutant in Huai 119. ***P<0.001.

表2 野生型淮119及sd1突变体群体产量及其倒伏比例

Table 2. Yield and lodging result of the wild-type Huai 119 and sd1 mutant.

试验小区 Experiment plot	倒伏比例 Lodging percentage/%		Yield 产量/(t·hm⁻²)
试验小区 Experiment plot	野生型 Wild type	sd1	野生型 Wild type	sd1
重复小区1 Repeat 1	60	0	10.52	10.31
重复小区2 Repeat 2	90	0	11.26	11.53
重复小区3 Repeat 3	75	0	11.55	11.46
平均 Mean	75	0	11.11	11.10

图3 淮119中sd1突变株表型及抗倒伏性分析

Fig. 3. Phenotype and lodging-resistance of sd1 mutant in Huai 119.

图4 外源GA处理下野生型与突变体株高比较 A—GA处理7 d; B—GA处理10 d. *表示突变体与野生型间的差异达0.05显著水平(n=4)。

Fig. 4. Plant height of the wild type and the mutant under the exogenous GA treatment. A, GA treatment for 7 days; B, GA treatment for 10 days; *Significant difference at 0.05 level between the wild type and the mutant (n=4).

表3 sd1及淮119对水稻不同时期稻瘟病的抗性鉴定结果

Table 3. Resistance evaluation of sd1 and Huai 119 to rice blast.

品种名称Variety name	苗瘟病级Disease rating of seedling blast		叶瘟病级Disease rating of leaf blast	穗颈瘟Neck blast						抗性 Resistance
品种名称Variety name	苗瘟病级Disease rating of seedling blast		叶瘟病级Disease rating of leaf blast	发病率Incidence rate of blast/%	病级Disease rating	损失率 Loss rate of blast /%	病级 Disease rating	抗性综合指数Comprehensive resistance index	病级Disease rating	抗性 Resistance
sd1		0	1	23	5	6	3	3.00	3	中抗Moderately resistant
淮119 Huai 119		0	2	82	9	33	7	6.25	7	感病Sensitive

图5 sd1突变体与野生型穗颈瘟的抗性比较

Fig. 6. The comparison of spike neck blast resistance between sd1 mutant and the wild type.

图6 野生型淮119和sd1突变体氯酸盐处理下的表型 A―萌发期KClO3处理的幼苗，比例尺=2 cm；B―萌发期KClO3处理的苗高，n=10；C―苗期KClO3处理的幼苗，比例尺=5 cm；D和E―苗期氯酸盐处理后幼苗的总叶绿素(D)和类胡萝卜素(E)含量。*P<0.05; ***P<0.01.

Fig. 6. Phenotype of the wild type Huai 119 and sd1 mutant under the chlorate treatment. A, Seedlings treated with KClO3 during the germination period, Bar= 2 cm; B, Height of seedlings treated with KClO3 during germination, n=10; C, Seedlings treated with KClO3 at seedling stage, scale=5 cm; D and E, Total chlorophyll (D) and carotenoid (E) contents of seedlings after KClO3 treatment at seedling stage.

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定向敲除SD1基因提高水稻的抗倒性和稻瘟病抗性

Directed Knockout of SD1 Gene Improves Lodging Resistance and Blast Resistance of Rice

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