Functional characterization of AsGRAS24 in Avena sativa reveals its role in enhancing salt tolerance via antioxidant and hormonal regulation in transgenic Arabidopsis thaliana

Abstract

Background: GRAS transcription factors are a type of transcription factor found only in plants. Among these, PAT1 subfamily proteins are known to help plants resist abiotic stress.

Results: To study the GRAS family genes in oat and check if GRAS transcription factors are linked to salt tolerance, this research cloned the AsGRAS24 gene using oat leaf cDNA. The gene was analyzed using bioinformatics tools, and its cellular localization was determined. Through the Agrobacterium-mediated genetic transformation method, the target gene was introduced into the model plant Arabidopsis thaliana, and then the functional mechanism and regulatory network of this gene in plant salt stress tolerance were systematically studied. The results showed that the AsGRAS24 gene produces a protein with 291 amino acids, weighing 32.44 kDa, and has a pI value of 7.1. The protein was found in the nucleus. The AsGRAS24 protein shows the highest sequence similarity to a specific gene in OsGRAS24 (Oryza sativa). The study also compared wild-type A. thaliana seeds (WT) and transgenic AsGRAS24 A. thaliana plants under salt stress. It was found that the AsGRAS24 gene made the transgenic plants more salt-tolerant. As the salt stress continued, the transgenic plants exhibited higher levels of antioxidant enzymes, osmotic adjustment substances, hormones, and gene expression compared to the wild-type (WT) plants (P < 0.05). The results suggest that AsGRAS24 is involved in helping transgenic A. thaliana tolerate salt. Therefore, the AsGRAS24 gene is essential in enhancing salt tolerance in transgenic A. thaliana.

Conclusions: This research helps in understanding how oats resist stress at the molecular level, providing a reference for further study of its function in the development and stress resistance of Gramineae.

Keywords: Avena sativa; Bioinformatics analysis; Phenotype; Physiological index; Salt tolerance; Transcription factors.

Fig. 1

AsGRAS24 gene cloning. M: DL2000 Marker; 1–5: PCR amplification product of AsGRAS24 gene. The same below

Fig. 2

Nucleotide sequence and amino acid sequence of AsGRAS24 gene. Gray marked as amino acid sequence

Fig. 3

Subcellular localization of AsGRAS24 protein

Fig. 4

Protein evolution analysis of AsGRAS24 in Avena sativa

Fig. 5

The acquisition of AsGRAS24 transgenic A. thaliana. (a) PCR identification of positive strain of AsGRAS24 gene. (b) Screening of A. thaliana. WT: WT A. thaliana; OE-1, OE-2, OE-5: AsGRAS24 transgenic A. thaliana

Fig. 6

:The effect of overexpression of the AsGRAS24 gene under salt stress on the root configuration of transgenic A. thaliana. (a) Germination of A. thaliana seedings under salt stress. (b) Growth of A. thaliana under salt stress. (c) The change of fresh weight of Arabidopsis roots under salt stress. (d) The changes in root length of Arabidopsis under salt stress

Fig. 7

The changes in antioxidant enzyme activities of A. thaliana under salt stress. (a) The changes of peroxidase activity in A. thaliana under salt stress. (b) The changes of superoxide dismutase activity in A. thaliana under salt stress. (c) The changes of malondialdehyde content in A. thaliana under salt stress

Fig. 8

The changes in the content of osmotic regulating substances in A. thaliana under salt stress. (a) The changes in proline content of A. thaliana under salt stress. (b) The changes of soluble sugar content in A. thaliana under salt stress

Fig. 9

Changes in the activity of reactive oxygen metabolic enzymes in A. thaliana under salt stress. (a) The changes of ascorbic acid peroxidase activity in A. thaliana under salt stress. (b) The changes of glutathione peroxidase activity in A. thaliana under salt stress

Fig. 10

The changes in endogenous hormone content of A. thaliana under salt stress. (a) The changes in auxin content of A. thaliana under salt stress. (b) The changes in abscisic acid content of A. thaliana under salt stress. (c) The changes in jasmonic acid content of A. thaliana under salt stress. (d) The changes in salicylic acid content of A. thaliana under salt stress

Fig. 11

The changes in the expression level of AsGRAS24 gene in A. thaliana under salt stress