马铃薯StTrihelix30的基因克隆及在低温胁迫下的功能分析

doi:10.16420/j.issn.0513-353x.2025-0363

摘要/Abstract

摘要：

为探究马铃薯（Solanum tuberosum L.）Trihelix基因在低温响应中的功能，通过在拟南芥中过表达前期已鉴定马铃薯低温响应基因StTrihelix30，并测定过表达株系的抗低温生理指标。结果表明： StTrihelix30不具备毒性，具有自激活活性；过表达StTrihelix30的转基因拟南芥株系在低温胁迫下表现出更高的抗寒性，相对电导率较野生型降低27.3%，MDA含量减少34.5%，抗氧化酶活性显著提升，SOD、POD和CAT活性分别增加至野生型的1.8、2.1和1.5倍，清除活性氧（ROS）的能力明显增强。综上所述，StTrihelix30能够通过提高植物的抗氧化能力来增强其在低温胁迫下的适应性。

关键词: 马铃薯, Trihelix转录因子家族, 低温胁迫, 基因克隆

Abstract:

In order to explore the functional divergence of the Trihelix gene from potato（Solanum tuberosum L.）in low temperature response，the previously identified potato low-temperature-responsive gene StTrihelix30 was overexpressed in Arabidopsis thaliana，and the low-temperature tolerance physiological indices of the overexpression lines were determined. The results showed that StTrihelix30 is non-toxic and exhibits self-activation activity. In cold tolerance assays，transgenic Arabidopsis lines overexpressing StTrihelix30 displayed enhanced cold resistance under low-temperature stress，as evidenced by a 27.3% reduction in relative electrical conductivity and a 34.5% decrease in malondialdehyde（MDA）content compared with the wild type. Furthermore，the activities of antioxidant enzymes were significantly increased，with superoxide dismutase（SOD），peroxidase（POD），and catalase（CAT）activities rising to 1.8-fold，2.1-fold，and 1.5-fold those of the wild type，respectively，indicating a markedly enhanced capacity to scavenge reactive oxygen species（ROS）. In conclusion，StTrihelix30 enhances plant adaptability to low-temperature stress by improving its antioxidant capacity.

Key words: Solanum tuberosum L., Trihelix transcription factor family, low-temperature stress, gene cloning

杨宏羽, 王言, 刘涛涛, 姚文霞, 范相军, 余斌, 师桂英. 马铃薯StTrihelix30的基因克隆及在低温胁迫下的功能分析[J]. 园艺学报, 2026, 53(8): 2306-2318.

YANG Hongyu, WANG Yan, LIU Taotao, YAO Wenxia, FAN Xiangjun, YU Bin, SHI Guiying. Cloning of the StTrihelix30 Gene in Potatoes and Functional Analysis Under Low-temperature Stress[J]. Acta Horticulturae Sinica, 2026, 53(8): 2306-2318.

图/表 12

表1 StTrihelix30基因克隆及载体构建所用引物序列

Table 1 Primer sequences used for the cloning of the StTrihelix30 and vector construction

用途Purpose	引物名称 Primer name	引物序列（5′-3′）Primers sequence
瞬时表达/过表达载体 Transient expression / overexpression vector	StTrihelix30-F	TTTGGAGAGGACACGCTCGAGATGCTGGAAAGTTCTGTTTTGTTG
瞬时表达/过表达载体 Transient expression / overexpression vector	StTrihelix30-R	GCCCTTGCTCACCATGAATTCACATTCTGTTCTTTCTGATGCTTGTT
BD载体 BD carrier	StTrihelix30-F	AGGCCGAATTCCCGGGGATCCTTATGCTGGAAAGTTCTGTTTTGTTG
BD载体 BD carrier	StTrihelix30-R	CCGCTGCAGGTCGACGGATCCTCAACATTCTGTTCTTTCTGATGCT

图1 马铃薯StTrihelix30基因的克隆及重组载体菌落PCR鉴定 M：DL2000 marker。A：StTrihelix30基因克隆；B：大肠杆菌菌液PCR；C：农杆菌菌液PCR

Fig. 1 Cloning of the StTrihelix30 of potato and PCR identification of recombinant vector colonies M：DL2000 marker. A：StTrihelix30 gene clone；B：PCR of Escherichia coli bacteriophage；C：PCR of Agrobacterium bacteriophage

图2 马铃薯StTrihelix30和同源基因的物种进化关系（A）和基因结构分析（B）

Fig. 2 Phylogenetic relationships（A）and gene structure analysis（B）of StTrihelix30 and its homologous genes

表2 马铃薯StTrihelix30同源蛋白理化性质分析

Table 2 Analysis of physicochemical properties of the homologous protein StTrihelix30 in potato

物种 Species	基因 Gene	氨基酸数量 No. of amino acid	分子量/Da Molecular weight	等电点 pI	脂溶性系数 Aliphatic index	亲水性 Hydropathicity
马铃薯Solanum tuberosum	StTrihelix30	503	57 413.52	7.99	59.96	-1.001
多疣茄Solanum verrucosum	XM_049514945.1	497	56 894.05	7.98	61.09	-0.998
欧白英Solanum dulcamara	XM_055976455.1	502	57 445.28	7.58	58.92	-1.061
辣椒Capsicum annuum	XM_016695686.2	506	57 402.60	8.74	60.20	-1.001
枸杞Lycium barbarum	XM_060327770.1	480	55 030.00	8.31	61.60	-1.008
黄瓜Cucumis sativus	XM_004147307.3	499	57 254.69	5.55	61.04	-1.097
甜瓜Cucumis melo	XM_008462691.3	500	57 063.60	5.62	61.72	-1.061
牵牛Ipomoea nil	XM_019321946.1	480	54 300.42	6.00	58.42	-1.037

表3 马铃薯StTrihelix30及其同源序列二级结构和亚细胞预测

Table 3 Secondary structure and subcellular prediction of potato StTrihelix30 and its homologous sequences

物种 Species	基因 Gene	a-螺旋/% Alpha helix	β-折叠/% Beta turn	不规则卷曲/% Random coil	延伸链/% Extended strand	亚细胞定位 Subcellular localization
马铃薯Solanum tuberosum	XM_006350958.2	43.94	4.57	48.51	2.98	细胞核Nuclear
多疣茄Solanum verrucosum	XM_049514945.1	43.66	4.63	48.89	2.82	细胞核、叶绿体Nuclear、Chloroplast
欧白英Solanum dulcamara	XM_055976455.1	45.22	4.98	48.41	1.39	细胞核Nuclear
辣椒Capsicum annuum	XM_016695686.2	43.87	4.15	50.00	1.98	细胞核Nuclear
枸杞Lycium barbarum	XM_060327770.1	46.04	3.75	48.54	1.67	细胞核Nuclear
黄瓜Cucumis sativus	XM_004147307.3	47.90	3.61	45.29	3.21	细胞核Nuclear
甜瓜Cucumis melo	XM_008462691.3	47.00	4.60	45.60	2.80	细胞核Nuclear
牵牛Ipomoea nil	XM_019321946.1	46.66	3.75	47.71	2.08	细胞核Nuclear
马铃薯Solanum tuberosum	StTrihelix30	43.94	4.57	48.51	2.98	细胞核Nuclear

图3 马铃薯StTrihelix30-EGFP融合蛋白在本氏烟草叶片细胞中的亚细胞定位

Fig. 3 Subcellular localization of potato StTrihelix30-EGFP fusion protein in Nicotiana benthamiana leaf cells

图4 马铃薯StTrihelix30-pGBKT7重组质粒的PCR验证 M：DL2000 marker。A：StTrihelix30基因克隆；B：大肠杆菌菌液PCR

Fig. 4 Potato StTrihelix30- pGBKT7 PCR amplification map M：DL2000 marker. A：Gene clone of StTrihelix30；B：PCR detection of Escherichia coli

图5 马铃薯StTrihelix30在酵母中的自激活性及毒性检测 M：DNA marker。A：酵母菌落PCR检测；B：StTrihelix30的自激活性及毒性检测

Fig. 5 Self-activation and toxicity assays for potato StTrihelix30 M：DNA marker. A：Yeast colony PCR assay；B：Self-activation and toxicity assays for StTrihelix30

图6 StTrihelix30转基因拟南芥的筛选与PCR鉴定 M：DNA marker。A：StTrihelix30转基因拟南芥筛选；B：StTrihelix30转基因拟南芥检测条带。N：WT阴性对照，P：pART-CAM-StTrihelix30-EGFP大肠重组质粒阳性对照

Fig. 6 Screening and PCR identification of StTrihelix30 transgenic Arabidopsis thaliana plants M：DNA marker. A：StTrihelix30 transgenic Arabidopsis thaliana；B：StTrihelix30 transgenic Arabidopsis detection band. N：WT negative control，P：the pART-CAM-StTrihelix30-EGFP large intestine recombinant plasmid positive control

图7 低温胁迫下StTrihelix30转基因拟南芥表型 WT：野生型株系；OE：转基因株系。下同

Fig. 7 StTrihelix30 transgenic Arabidopsis thaliana phenotype under low temperature stress WT：Wild-type strain；OE：Transgenic strain. The same below

图8 低温胁迫下StTrihelix30在转基因拟南芥中的相对表达量不同小写字母表示差异显著（P < 0.05）。下同

Fig. 8 Expression analysis of StTrihelix30 in transgenic Arabidopsis thaliana after low-temperature stress Different lowercase letters indicate significant differences（P < 0.05）. The same below

图9 低温胁迫下StTrihelix30过表达拟南芥的生理指标

Fig. 9 Physiological indicators of Arabidopsis thaliana overexpressing StTrihelix30 under low-temperature stress

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