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. 2024 May 11;15(5):613.
doi: 10.3390/genes15050613.

Cloning and Function Analysis of the CsTAU1 in Response to Salt-Alkali Stress

Fan Zhang  1 , Dandan Li  1 , Rina Sa  1 , Ling Wang  1 , Yunyan Sheng  1
Affiliations

Cloning and Function Analysis of the CsTAU1 in Response to Salt-Alkali Stress

Fan Zhang et al. Genes (Basel). .

Abstract

To investigate the role of candidate genes for salt-alkali tolerance in cucumber (Cucumis sativus L.), this study screened CsTAU1 in the glutathione pathway from previous transcriptome data for cloning and functional analysis. Clone cucumber CsTAU1 contains one 675 bp open reading frame, containing one GST-N-Tau domain and one GST-C-Tau domain, and is expressed in cytoplasm. After successfully constructing overexpression vectors of CsTAU1 (+) and CsTAU1 (-), they were transferred into cucumber varieties 'D1909' (high salt alkali resistance) and 'D1604' (low salt alkali resistance) for salt-alkali resistance identification. It was found that under salt-alkali stress, CsTAU1 (+)-overexpressing plants showed strong resistance to salt-alkali stress, while CsTAU1 (-)-overexpressing plants showed the opposite situation. qRT-PCR analysis was performed on other glutathione pathway-related genes in CsTAU1-overexpressing plants. The expression patterns of LOC101219529 and LOC105434443 were the same as CsTAU1, and the introduction of CsTAU1 (+) increased the chlorophyll, α-Naphthylamine oxidation, glutathione S-transferase (GST), and catalase (CAT) content of cucumber. The research results provide a theoretical basis for cultivating salt-alkali-tolerant cucumber varieties.

Keywords: CsTAU1; cucumber; functional analysis; salt–alkali stress.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Cloning of CsTAU1. (A) Gel electrophoresis of CsTAU1; (B) CsTAU1 protein domain; M: DNA marker 2K; a, b, c: PCR product.
Figure 2
Figure 2
Subcellular localization of CsTAU1 protein. Images show protoplasts prepared from 3- to 4-week-old Arabidopsis leaves expressing CsTAU1-pGII-EGFP (bottom row) or pGII-EGFP (upper row). Bright-field illumination, GFP fluorescence, chlorophyll fluorescence, and an overlay of GFP and chlorophyll fluorescence are shown. Scale bars, 10 μm.
Figure 3
Figure 3
Phylogenetic analysis of CsTAU1.
Figure 4
Figure 4
Relative expression of CsTAU1 under salt–alkali stress. (A) The expression level of CsTAU1 in ‘D1909’ roots; (B) the expression level of CsTAU1 in ‘D1604’ roots; (C) the expression level of CsTAU1 in ‘D1909’ stems; (D) the expression level of CsTAU1 in ‘D1604’ stems; (E) the expression level of CsTAU1 in ‘D1909’ leaves; (F) the expression level of CsTAU1 in ‘D1604’ leaves; (G) the expression level of CsTAU1 in ‘D1909’ fruits; (H) the expression level of CsTAU1 in ‘D1604’ fruits. The “*” presents p < 0.05; The “**” presents p < 0.01.
Figure 5
Figure 5
Expression analysis of CsTAU1 under different hormone and stress conditions. (A) Expression analysis of CsTAU1 in ‘D1909’. (B) Expression analysis of CsTAU1 in ‘D1604’. “**” presents p < 0.01; CK is distilled water treatment, SAT is salt–alkali treatment.
Figure 6
Figure 6
Genetic transformation of overexpression cucumber; (A): germination culture of overexpressing cucumber; (B): cotyledon node of overexpressing cucumber; (C): overexpressing cucumber in differentiation culture; (D): differentiated buds grow from overexpressing cucumber; (E): rooting culture of overexpressing cucumber; (F): overexpressing cucumber domestication culture.
Figure 7
Figure 7
Molecular biological verification of overexpressing cucumbers. (A) PCR detection of ‘D1909’ CsTAU1 (+) and ‘D1909’ CsTAU1 (−)-overexpressing plants; (B) PCR detection of ‘D1604’ CsTAU1 (+)- and ‘D1909’ CsTAU1 (−)-overexpressing plants; a, b, c, d, e, f, g, h: PCR product. (C) qRT-PCR detection of ‘D1909’ CsTAU1 (+)- and ‘D1909’ CsTAU1 (−)-overexpressing plants; (D) qRT-PCR detection of ‘D1604’ CsTAU1 (+)- and ‘D1604’ CsTAU1 (−)-overexpressing plants. “**” presents p < 0.01.
Figure 8
Figure 8
CsTAU1 (+)-overexpression cucumbers growth status under salt–alkali stress.
Figure 9
Figure 9
Metabolic pattern diagram of CsTAU1 in reducing salt–alkali stress. (A) Heatmap of SAT and NSAT; (B) metabolic pathway diagram.
Figure 10
Figure 10
The expression of key genes in the glutathione pathway in CsTAU1-overexpressing cucumbers. (A) Expression of LOC101219529 under salt−alkali stress. (B) Expression of LOC101219529 under non−salt–alkali stress. (C) Expression of LOC105434443 under salt−alkali stress. (D) Expression of LOC105434443 under non−salt−alkali stress. (E) Expression of LOC101203032 under salt−alkali stress. (F) Expression of LOC101203032 under non−salt−alkali stress. (G) Expression of LOC101212771 under salt−alkali stress. (H) Expression of LOC101212771 under non−salt−alkali stress; “*” presents p < 0.05; “**” presents p < 0.01.
Figure 11
Figure 11
Physiological and biochemical indexes under salt−alkali treatment. (A) Chlorophyll content of ‘D1909’. (B) chlorophyll content of ‘D1604’. (C) α-Naphthylamine oxidation of ‘D1909’. (D) α-Naphthylamine oxidation of ‘D1604’. (E) GST content of ‘D1909’. (F) GST content of ‘D1604’. (G) CAT content of ‘D1909’. (H) CAT content of ‘D1604’. “*” presents p < 0.05; “**” presents p < 0.01.
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