Functional Analysis of Soybean Gene GmHMGR Responding to Exogenous Hormones and Abiotic Stresses

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

Abstract

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

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.

Figures/Tables 8

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

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

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

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

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

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

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

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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