激素和逆境胁迫对大豆维生素E和γ-TMT表达的影响

doi:10.13560/j.cnki.biotech.bull.1985.2023-0183

摘要/Abstract

摘要：

明确大豆维生素组分含量及合成相关基因γ-TMT对激素和逆境胁迫的响应，为维生素E参与逆境胁迫研究开辟新思路。以山西省主栽品种晋大88为试验材料，在激素、逆境胁迫条件下对不同组织的维生素E组分含量、生理指标、γ-TMT表达模式进行分析。结果表明，维生素E主要存在于叶中，茎中有少量，根中不存在。在激素及盐等胁迫条件下，叶和茎的总维生素E含量小于对照，但γ-生育酚含量在NaCl、H₂O₂、ABA胁迫下含量高于对照。在激素和逆境胁迫环境下，大豆植株的生长被显著抑制，株高、鲜重、干重、SPAD值以及含水量皆小于对照。基因组数据挖掘到3个γ-TMT，启动子中均存在许多胁迫应答和激素响应元件，在激素和逆境胁迫下，γ-TMT1-γ-TMT3在根、叶中的表达均发生上调或下调。在NaCl胁迫条件下，大豆叶中γ-生育酚含量增加，而γ-TMT2、γ-TMT3的表达明显下调，推测γ-TMT2和γ-TMT3可能影响盐胁迫条件下维生素组分含量。激素和逆境胁迫显著抑制大豆茎和叶中维生素E的含量，进而抑制植株的生长，影响了大豆γ-TMT表达和维生素E组分。

关键词: 大豆, 维生素E, γ-TMT, 激素, 逆境

Abstract:

The response of vitamin content and synthesis-related gene γ-TMT to hormonal and stress stress was clarified, which opened up new ideas for vitamin E to participate in the study of adversity stress. Taking Jinda 88, the main cultivars of Shanxi province, as the experimental materials, the vitamin E component content, physiological indexes and γ-TMT expression patterns in different tissues were analyzed under hormonal and adversity stress. The results showed that vitamin E was mainly present in the leaves, a small amount in stems, and absent in roots, and the total vitamin E content in leaves and stems was smaller than that in control under stress conditions such as hormones and salts, but the content of γ-tocopherols under NaCl, H₂O₂ and ABA stress was higher than that of the control. Under hormonal and adversity stress, the growth of soybean plants was significantly inhibited, and the plant height, fresh weight, dry weight, SPAD value and water content were all lower than those of the control. Genomic data were mined to 3 γ-TMTs, and there were many stresses response and hormone response elements in the promoters, and the expression of γ-TMT1-γ-TMT3 in roots and leaves was upregulated or down-regulated under hormonal and adversity stress. Under NaCl stress, the content of γ-tocopherol in soybean leaves increased, while the expression of γ-TMT2 and γ-TMT3 was significantly down-regulated, and it was speculated that γ-TMT2 and γ-TMT3 may affect the content of vitamin components under salt stress. Hormonal and adverse stress significantly inhibited the contents of vitamin E in soybean stems and leaves, which in turn inhibited plant growth, i.e.,affected soybean γ-TMT expression and vitamin E components.

Key words: soybean, vitamin E, γ-TMT, hormone, adversity

白苗, 田雯青, 武帅, 王敏, 王利祥, 岳爱琴, 牛景萍, 张永坡, 高春艳, 张武霞, 郭数进, 杜维俊, 赵晋忠. 激素和逆境胁迫对大豆维生素E和γ-TMT表达的影响[J]. 生物技术通报, 2023, 39(10): 148-162.

BAI Miao, TIAN Wen-qing, WU Shuai, WANG Min, WANG Li-xiang, YUE Ai-qin, NIU Jing-ping, ZHANG Yong-po, GAO Chun-yan, ZHANG Wu-xia, GUO Shu-jin, DU Wei-jun, ZHAO Jin-zhong. Effects of Hormonal and Adversely Stress on Vitamin E and γ-TMT Gene Expression in Soybeans[J]. Biotechnology Bulletin, 2023, 39(10): 148-162.

图/表 11

表1 γ-TMT的RT-qPCR扩增引物

Table 1 Primers for RT-qPCR amplification of γ-TMT gene

基因名称 Gene name	引物 Primer	序列 Sequence（5'-3'）
γ-TMT1	F	GTGTGGTCCATGGAGAGTGG
γ-TMT1	R	CCCTGTGGCACCATGTTACT
γ-TMT2	F	CTGGCCGTCAGAGAATGGTT
γ-TMT2	R	GGAGCAACCAGTGTAGGCAT
γ-TMT3	F	TGACGCCCTAAAGCAACCAT
γ-TMT3	R	CCCTGTGGCACCATGTTACT
GmCYP2	F	CGGGACCAGTGTGCTTCTTCA
GmCYP2	R	GTGCTGCTACCCGAGCTAAT

图1 γ-TMT氨基酸序列的比对 A：丙氨酸；R：精氨酸；N：天冬酰胺；D：天冬氨酸；C：半胱氨酸；Q：谷氨酰胺；E：谷氨酸；G.甘氨酸；H：组氨酸；I：异亮氨酸；L：亮氨酸；K：赖氨酸；M：蛋氨酸；F：苯丙氨酸；P：脯氨酸；S：丝氨酸；T：苏氨酸；W：色氨酸；Y：酪氨酸；V：缬氨酸

Fig. 1 Amino acid sequence alignment of γ-TMT A: Alanine. R: Arginine. N: Asparagine. D: Aspartic acid. C: Cystine. Q: Glutarnine. E: Lutamic acid. G: Glycine. H: Histidine. I: Isoleucine. L: Leucine. K: Lysine. M: Methionine. F: Phenylalanine. P: Proline. S: Serine. T: Threonine. W: Tryptophan. Y: Tyrosine. V: Valine

图2 γ-TMT启动子顺式作用元件预测热图

Fig. 2 Prediction heat map of γ-TMT gene promoter cis-acting element

图3 激素和逆境胁迫环境下SPAD值不同小写字母表示与对照相比差异显著（P<0.05）。下同

Fig. 3 SPAD values in hormonal and adversely stress environment Different lowercase letters indicate significant differences compared with the control（P<0.05）. The same below

图4 激素和逆境胁迫环境下植物各生理指标测定

Fig. 4 Determination of physiological indexes of plants under hormonal and adversely stress environment

表2 生育酚标准品的回归方程与检出限

Table 2 Regression equation and detection limit of tocopherol standard

标准品 Standard	回归方程 Regression equation	R²	检出限 Detection limit/（μg·g^-1）
α-生育酚 α-Tocopherol	Y=5.281 3X+0.104 2	1	0.17
γ-生育酚 γ-Tocopherol	Y=22.477X+0.258 3	0.999 7	0.03
δ-生育酚 δ-Tocopherol	Y=25.108X-1.6	0.999 8	0.03

表3 激素和逆境胁迫环境下大豆叶中维生素E含量分析

Table 3 Analysis of vitamin E content in soybean leaves under hormonal and adversely stress environment

不同处理 Different treatment	α-生育酚 α-tocopherol/（μg·g^-1）	γ-生育酚 γ-tocopherol/（μg·g^-1）	δ-生育酚 δ-tocopherol/（μg·g^-1）	总维生素E Total vitamin E/（μg·g^-1）
CK	21.35±9.41a	93.04±42.07a	17.36±7.52a	131.75±55.16a
PEG	12.28±4.81b	89.35±34.07a	9.77±5.26b	111.40±43.08b
NaCl	15.26±0.29b	109.37±15.00b	10.32±0.44b	124.25±14.47a
H₂O₂	8.93±0.61b	105.83±7.39b	3.86±0.38b	118.62±8.26b
ABA	11.95±2.14b	106.33±7.45b	8.93±0.63b	127.21±8.77a
SA	11.96±3.21b	81.33±25.48b	9.22±1.11b	102.50±25.49b

表4 激素和逆境胁迫环境下大豆茎中维生素E含量分析

Table 4 Analysis of vitamin E content in soybean stems under hormonal and adversely stress environment

不同处理 Different treatment	α-生育酚 α-tocopherol/（μg·g^-1）	γ-生育酚 γ-tocopherol/（μg·g^-1）	δ-生育酚 δ-tocopherol/（μg·g^-1）	总维生素E Total vitamin E/（μg·g^-1）
CK	0.00±0.00	8.63±0.69a	0.57±0.10a	9.20±0.64a
PEG	0.00±0.00	3.21±0.47b	0.68±0.22b	3.89±0.27b
NaCl	0.00±0.00	1.00±0.27b	0.69±0.11b	1.68±0.29b

图5 激素及逆境胁迫环境下γ-TMT1的表达分析 A、D：SA处理下叶、根中相对表达水平；B、E：ABA处理下叶、根中相对表达水平；C、F：H2O2处理下叶、根中相对表达水平；G、I：PEG处理下叶、根中相对表达水平；H、J：NaCl处理下叶、根中相对表达水平。下同

Fig. 5 Analysis of γ-TMT1 gene expressions in hormonal and advsrsely stress environment A, D: Relative expression in the leaf and root under SA treated. B, E: Relative expression in the leaf and root under ABA treated. C, F: Relative expression in the leaf and root under H2O2 treated. G, I: Relative expression in the leaf and root under PEG treated. H, J: Relative expression in the leaf and root under NaCl treated. The same below

图6 激素及逆境胁迫环境下γ-TMT2基因表达分析

Fig. 6 Analysis of γ-TMT2 gene expressions in hormonal and adversely stress environment

图7 激素及逆境胁迫环境下γ-TMT3基因表达分析

Fig. 7 Analysis of γ-TMT3 gene expressions in hormonal and adversely stress environment

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