苹果肌醇-1-磷酸合成酶基因MdMIPS1参与高温胁迫应答的功能分析

doi:10.16420/j.issn.0513-353x.2024-0997

摘要/Abstract

摘要：

通过改变外源和内源苹果植株中的肌醇含量，并对其进行高温处理，解析肌醇在苹果响应高温胁迫中的功能。结果发现，高温胁迫引起苹果叶片枯黄萎蔫，活性氧、丙二醛和电导率迅速升高，叶绿素含量降低；然而100 µmol · L^-1外源肌醇处理显著缓解了高温对苹果植株造成的伤害。MdMIPS1编码肌醇合成的关键限速酶，其在高温胁迫下大量表达。利用实验室前期获得的MdMIPS1过表达的转基因株系（OE，高肌醇含量）进行高温处理，结果表明，与野生型植株相比，OE株系在高温下叶片失绿干枯症状比较轻微，电导率较低，叶绿素含量较高；高温下OE株系的气孔开度更大，光合系统效率更强；与野生型相比，OE植株在高温下表现出更低的活性氧水平、更高的抗氧化酶活性；并且一些编码热激蛋白的基因也在OE植株中表达水平更高。以上这些结果表明，提高肌醇水平能直接提高苹果植株对高温胁迫的抗性。

关键词: 苹果, 肌醇, MdMIPS1, 高温胁迫

Abstract:

In order to reveal the role of Myo-inositol（MI）in apple plants responding to high-temperature stress，apple plants were first exogenously supplied with MI and treated with high temperature stress. The results showed that high-temperature stress significantly caused leaf yellow and wilting，increased the contents of reactive oxygen species（ROS），malondialdehyde（MDA），and relative electrolyte leakage（REL），decreased the contents of the chlorophyll in apple plants. However，100 µmol · L^-1exogenous MI treatment significantly alleviated the damage caused by high temperature. In addition，MdMIPS1 encoding the key rate-limiting enzyme in the MI biosynthesis，expressed highly under high-temperature stress in apple plants. The transgenic lines overexpressed MdMIPS1（OE，high MI content）were obtained in our previous study. They had higher endogenous content of MI，compared with wild type（WT）plants. And these OE lines were also used in this work and treated with high temperature stress. Compared with WT plants under high temperature stress，the OE lines exhibited slighter chlorisis and wilting in leaves. The contents of REL were lower and the contents of the chlorophyll were higher in OE plants than in WT. In addition，the stomatal aperture were larger in OE leaves，and the photosynthetic efficiency were stronger in OE plants than in WT. Meanwhile，the OE plants possessed less ROS accumulation and higher antioxidant enzyme activity than WT. The expression levels of several genes encoding the heat shock protein were also higher in OE plants than in WT under high temperature stress. In sum，all these results indicated that increasing the MI levels can significantly enhance tolerance to high temperature stress in apple plants.

Key words: apple, Myo-inositol, MdMIPS1, high-temperature stress

孙文静, 李小艳, 张静允, 周辉, 黄小军, 马锋旺, 龚小庆. 苹果肌醇-1-磷酸合成酶基因MdMIPS1参与高温胁迫应答的功能分析[J]. 园艺学报, 2025, 52(12): 3143-3156.

SUN Wenjing, LI Xiaoyan, ZHANG Jingyun, ZHOU Hui, HUANG Xiaojun, MA Fengwang, GONG Xiaoqing. The Inositol-Phosphate Synthase Gene，MdMIPS1，Enhances Heat Stress Tolerance in Transgenic Apple Plants[J]. Acta Horticulturae Sinica, 2025, 52(12): 3143-3156.

图/表 8

表1 qRT-PCR引物序列

Table 1 Primer sequences of qRT-PCR

引物名称Primer name	正向引物序列（5′-3′）Forward primer sequence	反向引物序列Reverse primer sequence
qEF-1α	ATTCAAGTATGCCTGGGTGC	CAGTCAGCCTGTGATGTTCC
qMdMIPS1	AGGCCAAGTTCCACTCATTCCAC	CAAGCCCTCAGTATGTTCTCCAG
qMdHY5	AACTAGCATCCAAAGCTAGCC	CCACAGTCTGCAGTTTCACAAG
qMdDREB2A	CAGAGAACTTCACAACCGAC	CCAGTGCTCATGGCAAGGTTTT
qMdHSP17.3	AAGTTTGTGTTGCCGGAG	ATGTTATAATCCCTGCACGA
qMdHSP70-2	CTCTGTCTCTCGGTTTGGAG	GTCTTGTCCTCGGCAGAAA
qMdHSP70-1	CAAGCTGAAAGAGCTCAAGG	CTGTCCCAGCTGCATTACTT
qMdHSP90-5	ACACTCACTTCACCACTGAG	CATTTGAATCCACCACACCC

图1 高温胁迫下外源肌醇对苹果耐热性的影响 A：表型；B：MDA含量；C：叶绿素含量；D：相对电导率。采用单因素方差分析进行邓肯氏多重比较检验，不同字母表示处理间差异显著（P < 0.05）。下同

Fig. 1 Effect of exogenous MI on heat resistance of apple under high temperature stress A：The performance；B：MDA content；C：Total chlorophyll content；D：Electrolyte leakage. The data were shown as the mean of three replicates，and univariate analysis of variance and duncan multiple comparison were used，with different letters indicating significant differences between the data（P < 0.05）. The same below

图2 高温胁迫下外源肌醇对苹果抗氧化系统的影响

Fig. 2 Effect of exogenous MI on antioxidant capacity of apple under high temperature stress

图3 高温下苹果MdMIPS1表达模式分析 A：组织表达量；B：叶片中的时空表达模式

Fig. 3 Analysis of expression of apple MdMIPS1 A：Relative expression levels in tissues；B：Spatio-temporal expression patterns in leaves

图4 MdMIPS1转基因苹果在高温胁迫下的耐热性 A：植株与叶片表型；B：相对电导率；C：叶绿素含量。WT：GL-3野生型；OE-4、OE-6：GL-3苹果MdMIPS1过表达株系。下同

Fig. 4 Analysis of heat resistance of MdMIPS1 transgenic apple plants under high temperature stress A：The performance of plant and leaf；B：Electrolyte leakage；C：Chlorophyll content. WT：GL-3 wild-type. OE-4/OE-6：Transgenic apple plants with overexpression of MdMIPS1. The same below

图5 MdMIPS1转基因苹果在高温胁迫下的光合作用能力 A：叶绿素荧光；B：最大光化学效率；C：蒸腾速率；D：净光合速率；E：胞间二氧化碳浓度；F：扫描电子显微镜下叶片气孔；G：气孔开度；H：气孔导度。不同字母表示WT、OE-4、OE-6株系分别在对照或者高温处理数据间具有显著差异（P < 0.05）

Fig. 5 Analysis of photosynthetic capacity of MdMIPS1 transgenic apple plants under high temperature stress A：The chlorophyll fluorescence；B：Fv/Fm；C：Tr；D：Pn；E：Ci；F：Leaf stomata；G：Stomatal opening；H：Gs. Different letters indicated WT，OE-4 and OE-6 strains have significant differences in control or heat stress data significant differences between the data（P < 0.05）

图6 MdMIPS1转基因苹果在高温胁迫下的抗氧化能力 A：活性氧组织化学染色；B：超氧阴离子含量；C：过氧化氢含量；D：SOD活性；E：POD活性

Fig. 6 Analysis of antioxidant capacity of MdMIPS1 transgenic apple plants under high temperature stress A：NBT and DAB staining；B： $\mathrm{O}_{2}^{\overline{·}}$ content；C：H2O2 content；D：SOD activity；E：POD activity

图7 高温胁迫下野生型及过表达株系中热激响应基因的相对表达水平按行进行Z-score归一化处理

Fig. 7 Relative expression levels of heat shock proteins genes in WT and OE plants under high temperature stress Row Z-score normalization

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