Cloning,Analysis and Functional Verification of ZmRAV1 Gene

doi:10.7668/hbnxb.20195345

Abstract

Abstract:

Preliminary transcriptomic analysis identified ZmRAV1 as a candidate gene involved in maize's response to drought stress. To further investigate its function, this study cloned the ZmRAV1 gene, conducted bioinformatics analysis of its coding sequence, and overexpressed this gene in Arabidopsis thaliana. The function of ZmRAV1 was validated by assessing the phenotypes and physiological and biochemical indices of the transgenic Arabidopsis lines under drought conditions. The results showed that the ZmRAV1 gene had a total length of 1 176 bp and encoded 389 amino acids.It had the highest proportion of irregular coils in its secondary structure and was a hydrophilic protein that did not contain signal peptides and was non transmembrane.Subcellular localization indicated that the protein was located in the nucleus.ZmRAV1 exhibited high conservation across different species.Phylogenetic analysis indicated that ZmRAV1 shares the closest evolutionary relationship with its homolog in Miscanthus sinensis, showing a high degree of homology. After drought stress treatment,the root length of Arabidopsis thaliana lines overexpressing ZmRAV1 during germination was significantly higher than that of wild-type (WT)lines.In the seedling stage,WT showed withering or even death after drought stress,while the survival rate was lower than that of overexpressing lines.Moreover,the POD and SOD activities of ZmRAV1 overexpressing lines were higher than those of WT after drought treatment,indicating that overexpression of ZmRAV1 gene could enhance Arabidopsis thaliana's resistance to drought stress.

Key words: Zea mays L., Drought treatment, ZmRAV1, Subcellular localization, Functional verification

ZHAI Xiaoting, WANG Xinwei, HAO Xiying, SHEN Junrui, GAO Xuhong, LIU Guixue, LIU Songtao. Cloning,Analysis and Functional Verification of ZmRAV1 Gene[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 18-26. doi: 10.7668/hbnxb.20195345.

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Figures/Tables 8

Fig.1 Results of full-length amplification of ZmRAV1 gene M.DL2000 Marker;1,2.Target fragment.

Fig.2 The secondary structure(A) and tertiary structure(B) of ZmRAV1 protein

Fig.3 Protein sequence analysis of ZmRAV1 A.Protein transmembrane domain prediction; B.Protein signal peptide prediction; C.Protein hydrophilicity/hydrophpbicity prediction; D.Phosphorylation site prediction.

Fig.4 Sequence alignment of RAV1 proteins in different plants

Fig.5 Phylogenetic tree for RAV1 proteins in different plants

Fig.6 Observation of GFP fluorescence localization in maize protoplasts A.Observation on GFP fluorescence of maize protoplasts infected with pBWA (V) HS-1-GLosgfp-ZmRAV1;B.GFP fluorescence of protoplasts in the control group was observed.a,e.GFP green fluorescence;b,f.Chloroplast autofluorescence;c,g.Bright;d,h.Overlapping.

Fig.7 Sensitivity of ZmRAV1 overrxpressing seedlings to mannitol stress A.Growth of wild-type and overexpressing seedlings L1,L2,L4 lines at different concentrations of mannitol; B.Statistical graph of root length of wild-type and overexpressing seedlings.Different lowercase letters indicated significant difference(P<0.05).The same as Fig.8.

Fig.8 Identification of drought resistance during seedling stage of transgenic materials A.Phenotypic changes in transgenic Arabidopsis and WT before and after drought treatment; B.The change of survival rate before and after drought treatment; C.POD activity before and after drought treatment; D.SOD activity before and after drought treatment.

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