Analysis on Function of Rice OsSULTR2;2 and Regulation of Seedling Tolerance to Salt

doi:10.13560/j.cnki.biotech.bull.1985.2025-1144

Abstract

Abstract:

Objective To elucidate the function of gene OsSULTR2;2 and its role in regulating tolerance to salt stress during rice (Oryza sativa L.) seedling development. Method Using the indica rice variety Jing 4155S as material, the response gene OsSULTR2;2 was screened from the transcriptome data of the root before and after salt treatment. Its expression patterns were characterized using quantitative real-time PCR. A promoter-GUS fusion vector for histological localization and CRISPR/Cas9-mediated knockout mutants was constructed. The gene and promoter expression patterns were predicted using the RiceXPro and RGAP online databases. Promoter activity was validated through GUS histochemical staining. Phenotypic assessments under 140 mmol/L NaCl stress included survival rate and plant height measurements. Additionally, superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂) accumulation in the leaves were detected by nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) staining, respectively. Result OsSULTR2;2 transcription was significantly upregulated after salt treatment, with GUS staining demonstrating constitutive promoter activity across multiple tissues. Compared to wild-type plants, OsSULTR2;2 knockout mutants showed markedly improved survival rates and plant height under salt stress. Furthermore, these mutants showed substantially reduced accumulation of reactive oxygen species (ROS) in the leaves. Conclusion Targeted disruption of OsSULTR2;2 enhances salt tolerance in rice by augmenting cellular ROS scavenging capacity. This study provides molecular evidence for OsSULTR2;2 function in stress physiology and offers potential strategies for developing salt-resilient rice varieties.

Key words: rice, transcriptome, tolerance to salt, OsSULTR2, 2, reactive oxygen species (ROS)

CHEN Yi-yan, ZHANG Dong-er, ZHANG Tao, LIU Yu-hao, TANG Jie, SHENG Xia-bing, HU Yuan-yi, AI Zhi-yong, LI Ying-jiang, LIU Xiao-lin. Analysis on Function of Rice OsSULTR2;2 and Regulation of Seedling Tolerance to Salt[J]. Biotechnology Bulletin, 2026, 42(7): 116-125.

Figures/Tables 7

Fig. 1 Response to salt stress of rice sulfate transporter gene family and expression analysis of OsSULTR2;2A: Transcriptome analysis of the sulfate transporter gene family in the roots of ‘Jing 4155S’ under salt stress. B: Expression of OsSULTR2;2 in the leaves of 14-day-old ZH11 after treatment with 140 mmol/L NaCl for 0-48 h. C: Expression of OsSULTR2;2 in the roots of 14-day-old ZH11 after treatment with 140 mmol/L NaCl for 0-48 h. *** indicates statistical significance between the treatment and the mock (t-test, P<0.001)

Fig. 2 Analysis of cis-acting elements in the promoter of rice OsSULTR2;2 gene

Fig. 3 Schematic diagram of OsSULTR2;2Promoter::GUS vector and molecular detection of transgenic plantsA: Schematic diagram of OsSULTR2;2Promoter::GUS vector construction. B:PCR identification of T0 generation transgenic rice harboring OsSULTR2;2Promoter::GUS. M: DL2000 DNA Marker; H₂O: ultrapure water; ZH11: negative control; 1: positive control; 2-7: OsSULTR2;2Promoter::GUS positive transgenic plants

Fig. 4 Organ-level expression profile of OsSULTR2;2 and GUS staining of different organs and tissues in transgenic rice carrying OsSULTR2;2Promoter::GUSA: Organ-level expression profile of OsSULTR2;2. B: GUS staining of different organs and tissues in transgenic rice with OsSULTR2;2Promoter::GUS. ZH11: Negative control

Fig. 5 Analysis of transgenic materials for gene OsSULTR2;2A: Schematic diagram of the CRISPR/Cas9 knockout vector sgRNA-OsSULTR2;2-cas9. B: Sequencing results of the CRISPR/Cas9 knockout material KO-OsSULTR2;2-1 showed a deletion of the CTCA base at the first target site and a deletion of the G base at the second target site; sequencing results of KO-OsSULTR2;2-2 showed a deletion of the ACCCTCAGTATGG base at the first target site and a deletion of the G base at the second target site. These deletions led to the premature termination of the open reading frame (ORF) of OsSULTR2;2 in the mutants. Among them, the light brown squares indicate exons, and the light khaki squares indicate introns

Fig. 6 Analysis of salt tolerance at the seedling stage of OsSULTR2;2 knockout homozygous mutant riceA: Growth status of each line before and after NaCl treatment. B: Survival rate of rice seedlings. C: Plant height of rice seedlings. Significant difference analysis was performed by t-test, * P < 0.05 and *** P < 0.001, n = 24. Bars are 10 cm

Fig. 7 Detection of ROS accumulation in OsSULTR2;2 knockout lines under salt stressA: NBT staining results. B: DAB staining results. The scale bar is 1 mm

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