中国水稻科学 ›› 2023, Vol. 37 ›› Issue (4): 359-367.DOI: 10.16819/j.1001-7216.2023.221113
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()
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:
LI Gang1, GAO Qingsong2, LI Wei2, ZHANG Wenxia2, WANG Jian1, CHEN Baoshan1, WANG Di1, GAO Hao1, XU Weijun1, CHEN Hongqi3(), JI Jianhui2()
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:
【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。
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.
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 |
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.
试验小区 Experiment plot | 倒伏比例 Lodging percentage/% | Yield 产量/(t·hm−2) | ||
---|---|---|---|---|
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 |
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) | ||
---|---|---|---|---|
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).
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 | ||||||
---|---|---|---|---|---|---|---|---|---|---|
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 | |||||
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 |
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 | ||||||
---|---|---|---|---|---|---|---|---|---|---|
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 | |||||
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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