水稻OsSULTR2;2基因功能及调控苗期耐盐性分析

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

摘要/Abstract

摘要：

目的探究OsSULTR2;2基因功能及其调控水稻苗期耐盐性的机制。方法以籼稻品种晶4155S为材料，从盐处理前后根部转录组数据中筛选出响应基因OsSULTR2;2；通过RT-qPCR研究该基因表达模式；构建启动子GUS报告载体和CRISPR/Cas9敲除载体；使用RiceXPro和RGAP在线数据库预测基因及启动子表达模式，通过GUS化学染色分析基因启动子活性；在140 mmol/L NaCl条件下，统计敲除株系与野生型的存活率和株高；并通过NBT和DAB染色比较盐胁迫下敲除株系与野生型叶片超氧阴离子（O₂^-）和过氧化氢（H₂O₂）的积累情况。结果 OsSULTR2;2在盐处理后转录水平发生显著上调，并在水稻的各个组织中都能观察到GUS信号；盐胁迫下，OsSULTR2;2敲除株系的存活率和株高均显著高于野生型，且其活性氧（ROS）积累量显著低于野生型。结论在水稻中敲除OsSULTR2;2基因可增强水稻的活性氧清除能力，提高转基因水稻的耐盐性，为创制耐盐水稻新种质提供参考。

关键词: 水稻, 转录组, 耐盐性, OsSULTR2, 2, 活性氧

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)

陈义焰, 张冬儿, 张涛, 刘育灏, 唐杰, 盛夏冰, 胡远艺, 艾治勇, 李应将, 刘小林. 水稻OsSULTR2;2基因功能及调控苗期耐盐性分析[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1144.

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

图/表 7

图1 水稻硫酸盐转运蛋白基因家族响应及OsSULTR2;2表达分析A：盐胁迫下晶4155S根部硫酸盐转运蛋白基因家族的转录组分析；B：14 d苗期ZH11经140 mmol/L NaCl处理0-48 h后叶中OsSULTR2;2的表达水平；C：14 d苗期ZH11经140 mmol/L NaCl处理0-48 h后根中OsSULTR2;2的表达水平。***表示处理与对照之间差异极显著（t-test，P<0.001）

Fig. 1 Response of rice (Oryza sativa L.) 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)

图2 水稻OsSULTR2;2基因的启动子顺式作用元件示意图

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

图3 OsSULTR2;2Promoter::GUS载体构建示意图及转基因植株的分子检测A：OsSULTR2;2Promoter::GUS载体构建示意图；B：转OsSULTR2;2Promoter::GUS T0代转基因水稻PCR鉴定；M：DL2000 DNA Marker；H2O：超纯水；ZH11：阴性对照；1：阳性对照；2-7：OsSULTR2;2Promoter::GUS转基因阳性植株

Fig. 3 Schematic diagram of OsSULTR2;2Promoter::GUS vector construction 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

图4 OsSULTR2;2器官水平表达谱及转基因水稻OsSULTR2;2Promoter::GUS不同器官与组织GUS染色A：OsSULTR2;2器官水平表达谱；B：转基因水稻OsSULTR2;2Promoter::GUS不同器官与组织的GUS染色；ZH11：阴性对照。图中标尺为1 mm

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. Bars are 1 mm

图5 OsSULTR2;2基因的转基因材料分析A：CRISPR/Cas9敲除载体sgRNA-OsSULTR2;2-cas9示意图；B：CRISPR/Cas9敲除材料KO-OsSULTR2;2-1测序结果显示在第一靶点位置缺失CTCA碱基，第二靶点位置缺失G碱基。KO-OsSULTR2;2-2测序结果显示在第一靶点位置缺失ACCCTCAGTATGG碱基，第二靶点位置缺失G碱基。导致OsSULTR2;2在突变体中ORF提前终止。其中米色方块表示外显子，卡其色方块表示内含子

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

图6 OsSULTR2;2敲除纯合突变体水稻苗期耐盐性分析A：各株系在NaCl处理前后的生长状况；B：水稻幼苗的存活率；C：水稻幼苗的株高；差异显著性分析通过t检验完成，*P < 0.05，***P < 0.001， n = 24。图中标尺为10 cm

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, with *: P < 0.05 and ***: P < 0.001, n = 24. Bars are 10 cm

图7 OsSULTR2;2敲除株系盐胁迫下ROS积累检测A：NBT染色结果；B：DAB染色结果。标尺长度为1 mm

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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