大豆GmHMGR基因响应外源激素及非生物胁迫功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-0015

摘要/Abstract

摘要：

GmHMGR基因是大豆甲羟戊酸途径的关键酶基因之一，在大豆逆境胁迫调控方面具有重要的作用。探究GmHMGR基因在逆境胁迫中的功能，为大豆抗氧化、抗盐机制解析和分子育种提供依据。采用实时荧光定量PCR、酵母转化、拟南芥遗传转化等技术，分析GmHMGR基因在响应外源激素和非生物胁迫中发挥的功能。结果显示，GmHMGR基因对PEG6000、NaCl、H₂O₂胁迫以及MeJA、ABA外源激素处理均有响应；过量表达GmHMGR1、GmHMGR3、GmHMGR5、GmHMGR6和GmHMGR7可以提高酵母的抗盐、抗氧化能力；过量表达GmHMGR4与GmHMGR6的拟南芥上调了萜类代谢相关酶基因（AtDXR、AtSQS1、AtSQS2与AtCAS）的表达水平，改善了拟南芥在氧化及盐胁迫下的表型。GmHMGR基因可能参与大豆抗氧化、抗盐胁迫的应答。

关键词: 大豆, GmHMGR, 异源表达, 氧化胁迫, 盐胁迫, 外源激素

Abstract:

GmHMGR gene is one of the key enzyme genes in the mevalonate pathway of soybean（Glycine max L.）, and plays an important role in the stress regulation. Exploring the function of the GmHMGR gene in soybean stress resistance will serve as a foundation for the analysis of soybean antioxidant, salt resistance mechanisms, and molecular breeding. The function of the GmHMGR gene under abiotic stress and hormone treatments was analyzed using real-time quantitative PCR, yeast transformation, and A. thaliana genetic transformation. The results showed that the GmHMGR gene responded to PEG6000, NaCl, H₂O₂, MeJA and ABA treatments. Overexpressing GmHMGR1, GmHMGR3, GmHMGR5, GmHMGR6 and GmHMGR7 genes improved yeast's salt resistance and antioxidant capacity. Overexpressing GmHMGR4 and GmHMGR6 genes in A. thaliana upregulated the expression levels of terpenoids metabolism-related enzymes genes（including AtDXR, AtSQS1, AtSQS2 and AtCAS）, and improved its resistance capacity under oxidative and salt stress. GmHMGR gene may be involved in response to oxidative and salt stresses in soybean.

Key words: soybean, GmHMGR, heterologous expression, oxidative stress, salt stress, exogenous hormones

王帅, 冯宇梅, 白苗, 杜维俊, 岳爱琴. 大豆GmHMGR基因响应外源激素及非生物胁迫功能研究[J]. 生物技术通报, 2023, 39(7): 131-142.

WANG Shuai, FENG Yu-mei, BAI Miao, DU Wei-jun, YUE Ai-qin. Functional Analysis of Soybean Gene GmHMGR Responding to Exogenous Hormones and Abiotic Stresses[J]. Biotechnology Bulletin, 2023, 39(7): 131-142.

图/表 8

表 1 实时荧光定量引物序列

Table 1 Primers sequences by real-time quantitative PCR

基因Gene	登录号Gene ID	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
GmActin	XM_003552652	GGTGGTTCTATCTTGGCATC	CTTTCGCTTCAATAACCCTA
qPCR-GmHMGR1	XM_003517069.4	GAAAAATCTTACTGGGTCTGCTATG	CTGATTGAGACGCCAGTTGTG
qPCR-GmHMGR2	XM_014775260.2	TGCTGGTTCTGCTGTCGC	CAAGTAAATTCAAGCAAGCAGATTA
qPCR-GmHMGR3	XM_003537651.3	TTTCTCCAGAGTGATTTTCC	CAGACCCAGTAAGGTTTTTC
qPCR-GmHMGR4	XM_003534178.4	TTCTTCAGAGTGACTTCC	GTCTTCAACACCTTCTTC
qPCR-GmHMGR5	XM_006605513.3	TTCTTCGGCATCCACTTCGTCCAG	GCTGTGCGTCTCCTTCTATGATGG
qPCR-GmHMGR6	XM_003547838.4	AAGCCGCAGCCGTGAATTGG	CCACCAAGAGCACCAGCCATG
qPCR-GmHMGR7	XM_003519426.4	TTCGGACAAGAAACCTGC	AAACCCACCAAGAGCACC
qPCR-GmHMGR8	XM_003545508.4	GCCGTTGTTGTTGGATGG	GCAATCCCTCAAAACCACA
AtActin	XM_021028231.1	GGTAACATTGTGCTCAGTGGTGG	AACGACCTTAATCTTCATGCTGC
qPCR-AtDXS	NM_117647.3	GAAGTCGCAAAGGGTATGACAAAGCA	CTGGATCAAATTTCACAACACCATGGTAT
qPCR-AtDXR	NM_125674.3	TTCTGCCATATTTCAGTGTATTCAAGGTT	GCACAGATGAATCCTGTGTTTCAATC
qPCR-AtSQS1	NM_119630.4	GGAGAAGCAGATCCCTCCTG	AGTAGAACACACACGGCG
qPCR-AtSQS2	NM_119631.3	ATCTCAAAGCAAACCAATGTAAG	AGAACAACTGAAAAGGAAGAGAG
qPCR-AtCAS	NM_126681.3	GACGGAGGTTGGGGTTTA	TGATTTAGTATCCAGTCTCGTCC
qPCR-AtIPI	NM_121649.6	TGGGATCATGTTGAGAAAGGAAC	GTTGCCCAGTTTTGTCTGTAATCA

图1 非生物胁迫下大豆GmHMGR基因的表达模式分析

Fig. 1 Expression patterns of GmHMGR gene in response to abiotic stress in soybean

图2 激素处理后大豆GmHMGR基因的表达模式分析 A：MeJA处理；B：ABA处理；数据为3个生物学重复的平均值 ± SE

Fig. 2 Expression patterns of GmHMGR gene in response to hormone treatment in soybean A: MeJA treatment; B: ABA treatment.The data represent the mean ± SE of three biological replicates

图3 不同胁迫下过量表达酵母的生长情况 A：H2O2处理；B：NaCl处理

Fig. 3 Growth of overexpressed yeast under different stress A: H2O2 treatment; B: NaCl treatment

图4 T3代转基因拟南芥的鉴定 A：T3转基因拟南芥的PCR鉴定；M：DNA分子量标准；1-5：GmHMGR4转基因株系；6-10：GmHMGR6转基因株系；W：野生型对照；N：阴性对照；P：阳性对照；B：过量表达GmHMGR4转基因拟南芥的qPCR检测；C：过量表达GmHMGR6转基因拟南芥的qPCR检测。*表示0.05水平上显著差异，**表示0.01水平上显著差异。下同

Fig. 4 Identification of T3 transgenic A. thaliana A: Identification of T3 generation A. thaliana by PCR; M: DNA molecular weight marker. Lane 1-5: GmHMGR4 transgenic lines; lane 6-10: GmHMGR6 transgenic lines; W: wild-type control; N: negative control; P: positive control; B: qPCR detection of overexpressed GmHMGR4 transgenic A. thaliana; C: qPCR detection of overexpressed GmHMGR6 transgenic A. thaliana. * indicates significant difference at 0.05 level, and ** indicates significant difference at 0.01 level. The same below

图5 萜类化合物合成相关基因在转基因拟南芥中的表达分析

Fig. 5 Expression analysis of terpenoid synthesis-related genes in transgenic A. thaliana

图6 氧化和盐胁迫后转基因拟南芥的表型分析 A：野生型与转基因拟南芥在氧化和盐胁迫处理后的生长表型；B：拟南芥莲座直径统计；C、D：相对电导率与叶片损伤度的分析；E：MDA含量的测定

Fig. 6 Phenotypic analysis of transgenic A. thaliana after oxidative treatment and salt stress A: Growth phenotypes of wild-type and transgenic A. thaliana after oxidative and salt stress treatments. B: Size statistics of A. thaliana rosette diameter. C, D: Analysis of relative conductivity and leaf damage. E: Determination of MDA content

图7 氧化和盐胁迫处理后拟南芥的抗逆能力分析 A：拟南芥叶片在氧化和盐胁迫处理后的NBT染色；B：染色面积百分比的统计；C、D：CAT与POD活性的测定

Fig. 7 Stress-resistant ability analysis of transgenic A. thaliana after oxidative treatment and salt stress A: NBT staining of A. thaliana leaves after oxidative and salt stress treatments. B: Statistics of stained area percentage. C, D: Determination of CAT and POD activities

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