Function Study of OsST2 in Regulating Salt Tolerance in Rice Seedlings

doi:10.16819/j.1001-7216.2026.250413

Abstract

Abstract:

【Objective】To investigate the function of OsST2, a candidate gene associated with seedling-stage salt tolerance, and to elucidate its molecular mechanism in regulating salt tolerance in rice, an analysis under salt stress conditions was conducted.【Method】The CRISPR-Cas9 gene editing technology was used to generate OsST2 knockout mutants, and the phenotypic and physiological characteristics of these mutants under salt stress were analyzed.【Result】Under salt stress, the OsST2 gene knockout mutants exhibited significant changes in traits such as seedling height, dead leaf rate, root traits, and leaf SPAD value compared to the wild type. Moreover, the OsST2 knockout mutants demonstrated stronger salt tolerance than the wild type. qRT-PCR analysis revealed that the relative expression levels of known genes positively regulating rice salt tolerance, such as SKC1, OsMYB84, OsNAC3, OsMsr9, and ROS-related genes, were significantly higher in the OsST2 knockout mutants compared to the wild type. After salt stress, the levels of malondialdehyde, hydrogen peroxide, and superoxide anion in the OsST2 knockout mutants were significantly lower than those in the wild type. Conversely, the activities of peroxidase and superoxide dismutase, as well as glutathione content, were significantly higher in the mutants compared to the wild type. These findings suggest that the knockout of OsST2 enhances the rice plant’s ability to scavenge reactive oxygen species, thereby improving its salt tolerance.【Conclusion】OsST2 negatively regulates salt tolerance in rice. Loss of OsST2 function enhanced the scavenging capacity of ROS in rice, thereby improving salt tolerance in rice, confirming that OsST2 plays an important role in the regulation of salt tolerance in rice.

Key words: salt stress, rice, OsST2 knockout mutants, physiological mechanism, gene function

摘要：

【目的】探究苗期耐盐相关候选基因OsST2在盐胁迫下的功能，为解析其调控水稻耐盐性的分子机制奠定基础。【方法】利用CRISPR-Cas9基因编辑技术，构建OsST2基因敲除突变体，并分析盐胁迫下突变体表型和生理特性。【结果】盐胁迫下，OsST2基因敲除突变体苗高、死叶率、根系、叶片SPAD值等性状相较于野生型都发生显著的变化，且更耐盐。qRT-PCR分析表明，已知的正向调控水稻耐盐性的基因SKC1、OsMYB84、OsNAC3、OsMsr9及ROS相关基因在OsST2敲除突变体中的相对表达量都显著高于野生型。盐胁迫处理后，OsST2基因敲除突变体的丙二醛含量、过氧化氢含量、超氧阴离子含量均显著低于野生型，而过氧化物酶和超氧化物歧化酶的活性、谷胱甘肽含量等指标均显著高于野生型，说明OsST2敲除增强了水稻清除活性氧的能力，提高了水稻耐盐性。【结论】说明OsST2负向调控水稻耐盐性，且OsST2基因敲除增强了水稻清除活性氧的能力，进而提高了水稻耐盐性，证实OsST2在水稻耐盐调控中发挥重要作用。

关键词: 盐胁迫, 水稻, OsST2敲除突变体, 生理机制, 基因功能

FU Yao, LI Na, XU Jingru, QIN Yiyan, CHENG Xiaoran, SUN Haofeng, ZHANG Qi, CUI Zhibo, YANG Xinyu, ZHAO Minghui. Function Study of OsST2 in Regulating Salt Tolerance in Rice Seedlings[J]. Chinese Journal OF Rice Science, 2026, 40(4): 436-446.

付瑶, 李娜, 徐静茹, 覃奕琰, 程笑然, 孙浩峰, 张琪, 崔志波, 杨新宇, 赵明辉. OsST2调控水稻苗期耐盐性的功能研究[J]. 中国水稻科学, 2026, 40(4): 436-446.

Figures/Tables 10

Table 1. Primers for qRT-PCR of the genes involved in this study

登录号 Gene ID		前引物（5'-3'） Forward primer（5'-3'）	后引物（5'-3'） Reverse primer（5'-3'）
	OsST2	CAAAGAAGCGGGAGGGGA	GAGGATGCGAGAGATGTGGT
	Actin	CCACACCCCTGCTATGTACG	CATCACCAGAGTCCAACACAA
Os01g0307500	SKC1	TTCTGGCTCCAACTGCTGTA	TGGTGAAGTAGAGGCCAAGG
Os03g0771100	OsMYB84	CGAGGCTCAAGAAGAAGCT	AACAGAAGCTTCCGGATCGA
Os07g0225300	OsNAC3	CGCGAGTGGTACTTCTTCAC	TGCATGATCCACTCCGTCTT
Os07g0561300	OsMsr9	GCTCGAATCACTGCCTCAAG	CCGTTGCTTCGCTAACTTGT
Os07g0665200	OssodCc1	CGACGCCGAGGAATTTCTAG	AGGTGGTGTAAGTGTCTCTCATGC
Os03g0351500	OssodCc2	TGTGCGCATGGTTTTTGGTG	CATAGTTCATTGGGCGCCA
Os01g0263300	OsPOX1	CAGCTGCTCCAAGGTGAACTC	AGATTTGCTTCCAGCAACGAA
Os01g0734200	OsrbohA	CAGCACTCATCACCATGCT	TTCGTTGTGCATTTCCCGTT

Fig. 1. Expression analysis of candidate gene OsST2 for salt tolerance in different tissues of WT

Fig. 2. Sequences of target sites of wild-type Nipponbare and osst2

Fig. 3. Salt tolerance analysis of OsST2-knockout mutants at seedling stage A, Observation of osst2 mutant phenotype after salt treatment；B, Dead leaf rate（n=6）; Scale bar=5 cm. * indicates significant difference at the 0.05 probability level; ** indicates extremely significant difference at the 0.01 probability level. The same below.

Fig. 4. Seedling height and root morphology of OsST2 knockout mutant and wild type after 7 days of salt stress（n=15）

Fig. 5. Seedling root morphology of OsST2 knockout mutant and wild type after 14 days of salt stress（n=15） A, Observation of osst2 mutant phenotype of root morphology after salt treatment; B, Total root length; C, Root surface area; D, Total root volume.

Fig. 6. Effects of salt stress on SPAD of leaves of OsST2-knockout mutants and wild type at seedling stage A, Phenotypic observation of osst2 leaves in vitro after salt treatment; B, SPAD values of leaves after salt treatment(n=15)；C, SPAD values of detached leaves after salt treatment(n=15). Scale bar=1 cm.

Fig. 7. Analysis of expression levels of known genes related to salt tolerance（n=3）

Fig. 8. Physiological and antioxidant parameters in leaves of rice seedlings under salt stress(n=3)

Fig. 9. DAB and NBT staining results of leaves of rice seedlings under salt stress（n=3） A, DAB staining results of leaves; B, NBT staining results of leaves.

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