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

doi:10.16819/j.1001-7216.2023.221113

Abstract

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

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

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.

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.

Figures/Tables 9

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

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.

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

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

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

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).

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

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

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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