Cloning and Identification of Salt Tolerance Function of Soybean Transcription Factor GmMYB069

doi:10.13560/j.cnki.biotech.bull.1985.2025-0348

Abstract

Abstract:

Objective MYB (v-MYB avian myoblastosis viral oncogene homolog) transcription factors play an important regulatory role in plant growth, development, and salt stress response. Soybean GmMYB069 is involved in the salt stress response process. Cloning soybean GmMYB069 and analyzing its salt tolerance function may provide molecular basis and genetic resources for soybean salt tolerance breeding. Method The full-length CDS sequence of GmMYB069 was cloned and its gene and amino acid sequence characteristics were analyzed using bioinformatics software. GmMYB069 overexpression vector was transferred and soybean was transformed using Agrobacterium mediated method. The salt-tolerant phenotype and physiological and biochemical indicators of overexpressed strains were analyzed. Result A 1 059 bp GmMYB069 CDS sequence was successfully cloned, encoding 352 amino acids with a relative molecular weight of 39 309.26 Da and a predicted isoelectric point of 6.76. Its protein was an unstable hydrophilic protein, with the largest proportion of irregular curls in the polypeptide chain. It lacked transmembrane structures and signal peptides and has the closest genetic relationship with Sophora alopecuroides and the farthest genetic relationship with Crassulaceae. GmMYB069 had a higher expression in the roots, followed by the stems and leaves. Under salt stress conditions, the expression of GmMYB069 gene showed an upward trend in the early stage of stress, reaching a peak. With the prolongation of stress time, its expression decreased, indicating that it can respond to salt stress in the early stage. The GmMYB069 overexpression vector was successfully constructed and transformed it into soybean root hairs, and an overexpressed chimeric strain was obtained. Under salt stress, compared with the empty load plants, the relative expression of overexpressing lines significantly increased. The antioxidant enzymes (SOD, POD) in the leaves of overexpressed chimeric plants increased, MDA decreased, and the Na⁺ and K⁺ content did not show significant differences in the roots of overexpressed chimeric plants. The Na⁺ content significantly decreased in the leaves of overexpressed chimeric plants, while the opposite was true for K⁺. Conclusion The overexpression of soybean GmMYB069 may enhance antioxidant enzyme activity, prevent Na⁺ transport from roots to leaves, reduce plant salt damage, and improve the tolerance of transgenic plants to salt.

Key words: soybean, GmMYB069, gene cloning, salt stress, bioinformatics analysis, functional analysis

YANG Yue, LI Chang-ning, SUN Xiao-lu, LI Ya-qian, DU Wei-jun, WANG Li-xiang, WANG Min, JI Yue-mei. Cloning and Identification of Salt Tolerance Function of Soybean Transcription Factor GmMYB069[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0348.

Figures/Tables 11

Table 1 GmMYB069 primer sequence

引物名称 Primer name	引物序列 Primer sequence（5′-3′）
GmMYB069-F	ATGGGAAGGTCACCGTGTTG
GmMYB069-R	CCACAAAATTGGTGACGCAGAA
qRT-GmCYP2-F	CGGGACCAGTGTGCTTCTTCA
qRT-GmCYP2-R	CCCCTCCACTACAAAGGCTCG
qRT-GmMYB069-F	GCCTCATGTGGTGTTGAGTG
qRT-GmMYB069-R	TACAGCCACTGTCCATTTGGT
GmMYB069-pUBi-F	TTGATGTGATTACAGTCTAGAATGGGAAGGTCACCGTGTTG
GmMYB069-pUBi-R	GTTAATTAACCCGCTGGTACCCTACCACAAAATTGGTGACGCA

Fig. 1 Expression analysis of MYB family members in two materials

Fig. 2 Quantitative analysis of soybean (Glycine max) GmMYB069 gene under salt stress

Fig. 3 GmMYB069 gene cloning

Fig. 4 GmMYB069 gene structure

Fig. 5 Characterization of GmMYB069 proteinA. GmMYB069 protein hydrophobicity prediction map. B. GmMYB069 protein transmembrane domain prediction diagram. C. Signal peptide analysis of GmMYB069 protein （The horizontal coordinate indicates the position of the amino acid， and the vertical coordinate indicates the probability of the signal peptide）. D. Conservative domains of GmMYB069 protein. E. Tertiary structure of GmMYB069 protein

Fig. 6 Multiple sequence alignment of GmMYB069 with MYB proteins from other species (A) and phylogenetic tree (B)

Fig. 7 Temporal and spatial expression pattern diagram of soybeanA. Expressions of GmMYB069 gene in different tissues of soybean. B. The expressions of GmMYB069 gene in soybean W82 under salt stress at different time periods. C. The expressions of GmMYB069 gene at different time periods in material 150 and 535. The data shown is mean±variance, and significant differences were analyzed using t-test. ns indicates no significant difference, *P<0.05, **P<0.01, and ***P<0.001, the same below

Fig. 8 Expression analysis of GmMYB069-OE and control EV under salt stress and water treatment

Fig. 9 Phenotypic analysis of GmMYB069 OverexpressionA. Phenotypes of GmMYB069-OE and control EV under water treatment (OE-Control: Overexpressed chimeric plants under water treatment). B. Phenotypes of GmMYB069-OE and control EV under salt treatment (OE-Salt: Overexpressed chimeric plants under salt treatment). C. Subterranean phenotype of GmMYB069-OE and control EV under salt stress (OE-Salt: Overexpressed chimeric plants under salt treatment)

Fig. 10 Physiological indicators and Na+, K+ contents of GmMYB069 overexpressing chimeric plants under salt stressA: SOD activity in GmMYB069-OE leaves. B: POD activity in GmMYB069-OE leaves. C: MDA content in GmMYB069-OE leaves. D: SPAD value of GmMYB069-OE leaves. E: Na+ content in the roots of GmMYB069-OE plants. F: Na+ content in the leaves of GmMYB069-OE plants. G: K+ content in the roots of GmMYB069-OE plants. H: K+ content in the leaves of GmMYB069-OE plants

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