大豆GmPM31基因启动子响应高温高湿胁迫的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0435

摘要/Abstract

摘要：

目的研究GmPM31启动子响应高温高湿胁迫过程中对种子活力形成的功能，为全面揭示大豆小热激蛋白参与高温高湿胁迫响应的功能奠定基础。方法对纯合T3代转GmPM31启动子拟南芥进行高温高湿胁迫（40℃/相对湿度100%）处理，以野生型拟南芥为对照，检测其耐受性以及气孔开度；通过GUS组织化学染色和实时荧光定量PCR检测分析大豆GmPM31启动子所驱动的GUS基因的表达情况；通过2，3，5-氯化三苯基四氮唑（TTC）染色法检测种子活力变化。结果与对照组相比，经高温高湿胁迫处理后的转大豆GmPM31启动子拟南芥对高温高湿的耐受性提高，GUS活性提高，在叶、根和花等中均有表达，发芽率以及种子活力增加。结论 GmPM31启动子可提高种子对高温高湿胁迫的抗性和抗劣变的能力。

关键词: 大豆, 启动子, 高温高湿, 种子活力

Abstract:

Objective To study the function of GmPM31 promoter on seed vigor formation in response to high temperature and high humidity stress, and to lay a foundation for comprehensively unravelling the function of soybean small heat shock proteins involved in the response to high temperature and high humidity stress. Method Homozygous T3 transgenic Arabidopsis thaliana with GmPM31 promoter was treated with high temperature and high humidity stress (40℃/100% relative humidity), and wild-type A. thaliana was used as control to detect its tolerance and stomatal opening. Analysis of GUS gene expression driven by the soybean GmPM31 promoter by GUS histochemical staining and quantitative real-time PCR. The seed vigor was detected by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Result Compared with the control group, the tolerance of GmPM31-transgenicpromotor A. thaliana to high temperature and high humidity increased. The activity of GUS increased, and they were expressed in the leaves, roots and flowers, and germination rate and vigor increased. Conclusion GmPM31 promoter improve the resistance of seeds to high temperature and high humidity stress and the ability to resist deterioration.

Key words: soybean, promoter, high temperature and high humidity, seed vigor

林紫依, 吴一舟, 叶芳贤, 朱淑颖, 刘燕敏, 刘骕骦. 大豆GmPM31基因启动子响应高温高湿胁迫的功能分析[J]. 生物技术通报, 2025, 41(3): 90-97.

LIN Zi-yi, WU Yi-zhou, YE Fang-xian, ZHU Shu-ying, LIU Yan-min, LIU Su-shuang. Functional Analysis of Soybean GmPM31 Gene Promoter Involvement in Response to High Temperature and Humidity Stress[J]. Biotechnology Bulletin, 2025, 41(3): 90-97.

图/表 5

图1 转GmPM31基因启动子拟南芥植株经高温高湿处理后的表型

Fig. 1 Phenotypes of GmPM31-transgenic Arabidopsis plants under HTH stress

图2 转GmPM31基因启动子拟南芥植株在高温高湿条件下气孔观察统计A：生长28 d的野生型和转GmPM31基因启动子拟南芥植株的气孔形态；B：转GmPM31基因启动子拟南芥植株经高温高湿处理1 d、2 d后的气孔开度；C：转GmPM31基因启动子拟南芥植株经高温高湿处理1 d、2 d后的气孔长度；**代表极显著水平差异（P<0.01），下同

Fig. 2 Stomata assay of GmPM3-transgenic Arabidopsis plants under HTH conditionA: Stomatal morphology of WT and transgenic Arabidopsis grown for 28 d under HTH stress. B: Stomatal opening of WT and transgenic Arabidopsis under the HTH stress for 1 d and 2 d. C: Stomatal length of WT and transgenic Arabidopsis under the HTH stress for 1 d and 2 d. ** indicate that the differences are markly significant at 0.01 level. The same below

图3 大豆GmPM31基因启动子驱动GUS基因在拟南芥各组织或器官中的表达模式A：整株生长25 d的对照组拟南芥；B：整株生长28 d的对照组拟南芥；C：整株生长25 d的实验组拟南芥；D-H：分别为生长28 d的实验组拟南芥的花、叶片、种子、荚、根；I：大豆GmPM31基因启动子驱动GUS基因在拟南芥各组织或器官中的相对表达量。不同字母代表极显著水平差异（P<0.01）

Fig. 3 Expression patterns of GUS genes driven by soybean GmPM31 gene promoter in various tissues and organs of ArabidopsisA: The control group of Arabidopsis grown for 25 d; B: the control group of Arabidopsis grown for 28 d; C: the control group of Arabidopsis grown for 25 d;D-H: the experimental group of Arabidopsis's flowers, blade, seeds, pod and root grown for 28 d, respectively; I: relative expressions of soybean GmPM31 gene promoter driving GUS genes in various tissues and organs of Arabidopsis. Different letters indicate the difference significant (P<0.01)

图4 大豆GmPM31基因启动子驱动GUS基因在拟南芥幼苗中的表达模式A：正常条件下转GmPM31启动子拟南芥幼苗植株GUS染色情况；B：高温高湿处理转GmPM31启动子拟南芥幼苗植株GUS染色情况；C：大豆GmPM31基因启动子驱动GUS基因在拟南芥幼苗中的相对表达量

Fig. 4 Expression pattern of GUS genes driven by soybean GmPM31 gene promoter in Arabidopsis seedlingsA: GUS staining of Arabidopsis seedlings with GmPM31 promoter under normal condition; B: GUS staining of transgenic Arabidopsis seedlings with GmPM31 promoter treated with high temperature and humidity; C: relative expression of GUS genes driven by soybean GmPM31 gene promoter in Arabidopsis seedlings

图5 高温高湿胁迫对GmPM31转启动子拟南芥植株种子萌发的影响A：播种7 d后经高温高湿胁迫处理2 d后收获的野生型和转GmPM31启动子拟南芥幼苗；B：对照条件下的野生型和转GmPM31启动子拟南芥种子发芽率；C：未经高温高湿处理的野生型和转GmPM31启动子拟南芥种子发芽率；D：经高温高湿处理的野生型和转GmPM31启动子拟南芥种子发芽率；E：经高温高湿处理后种子的TTC染色结果

Fig. 5 Effect of HTH stress on seed germination of GmPM31 transgenic ArabidopsisA: WT and GmPM31-transgenic Arabidopsis seedlings harvested after 7 d of sowing and then 2 d of HTH stress treatment; B: germination percentage of WT and GmPM3-transgenic Arabidopsis seeds under control conditions; C: germination percentage of WT and GmPM31-transgenic Arabidopsis seeds without HTH treatment; D: germination percentage of WT and GmPM31-transgenic Arabidopsis seeds after HTH treatment; E: TTC staining results of seeds after control and treatment groups

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