怀玉山三叶青ThActin基因克隆与功能分析

doi:10.11924/j.issn.1000-6850.casb2025-1023

摘要/Abstract

摘要：

肌动蛋白(Actin)是一类在真核生物中普遍存在且高度保守的结构蛋白，作为细胞骨架的主要构成成分，参与细胞分裂、胞质分裂、胞吞作用等基础生命活动，并在抗逆过程中发挥关键作用。为研究怀玉山三叶青的ThActin基因，通过转录组数据筛选并克隆该基因，构建pCambia2301超表达质粒，进行烟草(Nicotiana tabacum W38)叶片表皮细胞的瞬时转化亚细胞定位实验，然后转入烟草中，获得ThActin转基因烟草，通过低温胁迫处理来验证转基因烟草的功能。结果表明：ThActin基因长度为1131 bp，烟草叶片表皮细胞的瞬时转化亚细胞定位实验结果表明，ThActin蛋白分布于细胞质和细胞核中；烟草的遗传转化实验结果表明，在低温胁迫下，与野生型烟草相比，过表达ThActin烟草的过氧化氢、超氧阴离子和丙二醛含量显著下降，抗氧化酶（SOD、POD和CAT）活性显著增强，渗透调节物质（脯氨酸、可溶性糖和可溶性蛋白）含量显著提高，说明过表达ThActin能增强植株的抗低温能力。该结果可为怀玉山三叶青低温胁迫耐受机制的阐明及其高抗品种的选育提供参考。

关键词: 怀玉山三叶青, ThActin, 基因克隆, 亚细胞定位, 基因过表达, 低温胁迫, 抗氧化酶, 渗透调节物质

Abstract:

Actin is a type of structural protein that is ubiquitous and highly conserved in eukaryotes. As a major component of the cytoskeleton, it participates in fundamental biological processes such as cell division, cytokinesis, and endocytosis, and plays a critical role in stress resistance. To investigate the ThActin gene of Tetrastigma hemsleyanum from Huaiyu Mountain, we screened and cloned this gene based on transcriptome data, constructed the pCambia2301 overexpression vector, and performed a transient transformation subcellular localization assay in leaf epidermal cells of tobacco (Nicotiana tabacum W38). Subsequently, the construct was transformed into tobacco to generate ThActin transgenic tobacco plants, and low-temperature stress treatment was applied to characterize the functions of the transgenic lines. The results showed that the full-length of ThActin gene was 1131 bp. Transient transformation subcellular localization analysis in tobacco leaf epidermal cells revealed that the ThActin protein localized to the cytoplasm and nucleus. Genetic transformation experiments in tobacco demonstrated that under low-temperature stress, compared with wild-type tobacco, ThActin-overexpressing tobacco exhibited significantly reduced contents of hydrogen peroxide, superoxide anion and malondialdehyde, markedly increased activities of antioxidant enzymes (SOD, POD and CAT), and significantly elevated levels of osmotic adjustment substances (proline, soluble sugar and soluble protein). These findings indicated that overexpression of ThActin enhances plant cold tolerance. This study provides a reference for elucidating the low-temperature stress tolerance mechanism of Tetrastigma hemsleyanum from Huaiyu Mountain and breeding its cold-resistant cultivars.

Key words: Tetrastigma hemsleyanum from Huaiyu Mountain, ThActin, gene cloning, subcellular localization, gene overexpression, low temperature stress, antioxidant enzymes, osmotic adjustment substances

中图分类号:

S567.239

洪森荣, 陈丹丹, 查淑丽, 吴可桐, 吴静妍. 怀玉山三叶青ThActin基因克隆与功能分析[J]. 中国农学通报, 2026, 42(21): 188-197.

HONG Senrong, CHEN Dandan, CHA Shuli, WU Ketong, WU Jingyan. Cloning and Functional Analysis of ThActin Gene in Tetrastigma hemsleyanum from Huaiyu Mountain[J]. Chinese Agricultural Science Bulletin, 2026, 42(21): 188-197.

图/表 7

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烟草试管苗	生理指标
烟草试管苗	温度/℃	过氧化氢含量/(nmol/g)	超氧阴离子产生速率/[nmol/(g·min)]	MDA含量/(nmol/g)
ThActin转基因植株	25	0.82±0.12 Cc	0.68±0.11 Cc	236.53±23.54 Cc
ThActin转基因植株	4	2.68±0.23 Bb	3.26±0.36 Bb	452.18±41.09 Bb
野生型植株	25	0.95±0.16 Cc	0.72±0.13 Cc	246.95±19.58 Cc
野生型植株	4	4.31±0.24 Aa	5.18±0.48 Aa	587.48±31.57 Aa

烟草试管苗	生理指标
烟草试管苗	温度/℃	过氧化氢含量/(nmol/g)	超氧阴离子产生速率/[nmol/(g·min)]	MDA含量/(nmol/g)
ThActin转基因植株	25	0.82±0.12 Cc	0.68±0.11 Cc	236.53±23.54 Cc
ThActin转基因植株	4	2.68±0.23 Bb	3.26±0.36 Bb	452.18±41.09 Bb
野生型植株	25	0.95±0.16 Cc	0.72±0.13 Cc	246.95±19.58 Cc
野生型植株	4	4.31±0.24 Aa	5.18±0.48 Aa	587.48±31.57 Aa

烟草试管苗	生理指标
烟草试管苗	温度/℃	SOD活性/(U/g)	POD活性/(U/g)	CAT活性/(U/g)
ThActin转基因植株	25	105.62±23.51 Cc	41.33±5.36 Cc	31.22±5.32 Cc
ThActin转基因植株	4	216.84±31.47 Bb	62.34±6.34 Bb	42.39±3.24 Bb
野生型植株	25	112.34±19.44 Cc	39.62±2.49 Cc	32.49±3.94 Cc
野生型植株	4	334.82±42.05 Aa	82.19±4.71 Aa	61.25±6.98 Aa

烟草试管苗	生理指标
烟草试管苗	温度/℃	SOD活性/(U/g)	POD活性/(U/g)	CAT活性/(U/g)
ThActin转基因植株	25	105.62±23.51 Cc	41.33±5.36 Cc	31.22±5.32 Cc
ThActin转基因植株	4	216.84±31.47 Bb	62.34±6.34 Bb	42.39±3.24 Bb
野生型植株	25	112.34±19.44 Cc	39.62±2.49 Cc	32.49±3.94 Cc
野生型植株	4	334.82±42.05 Aa	82.19±4.71 Aa	61.25±6.98 Aa

烟草试管苗	生理指标
烟草试管苗	温度/℃	Pro含量/(µg/g)	可溶性糖含量/(mg/g)	可溶性蛋白含量/(mg/g)
ThActin转基因植株	25	82.53±6.96 Cc	1.52±0.35 Cc	31.28±2.54 Cc
ThActin转基因植株	4	126.28±13.55 Bb	2.31±0.41 Bb	42.35±5.22 Bb
野生型植株	25	86.22±4.28 Cc	1.36±0.22 Cc	29.86±1.67 Cc
野生型植株	4	236.47±26.72 Aa	3.62±0.47 Aa	56.19±4.28 Aa