Cloning and Expression Analysis of Two G6PDH Genes from Safflower

doi:10.13560/j.cnki.biotech.bull.1985.2025-1123

Abstract

Abstract:

Objective Glucose-6-phosphate dehydrogenase (G6PDH) plays a crucial role in plant growth, development, and stress responses. This study aimed to clone the G6PDH genes from safflower (Carthamus tinctorius L.), analyze their sequences and expression patterns, and investigate their involvement in abiotic stress responses. These findings will provide valuable candidate genes for stress-resistant safflower breeding. Method Based on the genome data of safflower, G6PDH genes were identified and cloned, followed by bioinformatic analysis. Using RT-qPCR, the tissue-specific expression patterns of CtG6PDH genes and their responses to hormones (MeJA, ABA, and SA) and drought, salt and alkali stress were analyzed. Additionally, the activities of glucose-6-phosphate dehydrogenase (G6PDH) under these abiotic stresses were determined by ultraviolet spectrophotometry. Result Two G6PDH family members (CtG6PDH1 and CtG6PDH2) were identified and cloned from the safflower genome, located on chromosomes 1 and 3, respectively. Subcellular localization and phylogenetic analysis revealed that CtG6PDH1 is a cytosolic isoform localized in the cytoplasm, while CtG6PDH2 belongs to the plastidial P1-type and is located in chloroplasts. Their promoter regions contain multiple cis-acting elements associated with light response, phytohormone signaling, and stress responses. Tissue expression analysis showed that CtG6PDH1 exhibits higher expression in roots, while CtG6PDH2 demonstrated the highest expression in leaves. Under drought and saline-alkaline stress, the cytosolic CtG6PDH1 gene was markedly upregulated, while CtG6PDH2 expression exhibited induction by multiple phytohormones. Further enzymatic assays confirmed that cytosolic G6PDH activity was significantly enhanced under both drought and saline-alkali stress conditions. Conclusion Two G6PDH genes were identified and cloned from safflower, exhibiting distinct subcellular localizations (cytosol and chloroplast). Promoter analysis revealed putative cis-regulatory elements associated with growth, hormone responses, and stress adaptation. RT-qPCR and enzyme activity analyses demonstrated that the cytosolic CtG6PDH1 plays a major role in safflower’s response to abiotic stresses, including drought, salt, and alkali.

Key words: glucose-6-phosphate dehydrogenase (G6PDH), clone, subcellular localization, expression analysis, G6PDH activity, safflower

QIN Juan, LU Wei-Wen, LI Jia-Hui, LI Chen, LI Bin, WANG Zhi-ye. Cloning and Expression Analysis of Two G6PDH Genes from Safflower[J]. Biotechnology Bulletin, 2026, 42(8): 217-228.

Figures/Tables 9

Fig. 1 Cloning and chromosome localization of G6PDHs gene in safflower (Carthamus tinctorius L.)

Fig. 2 Prediction of the phosphorylated site, secondary structure and tertiary structures of CtG6PDH protein

Fig. 3 Phylogenetic tree of G6PDH proteins in different plants

Fig. 4 Analysis of conserved domains and multiple sequence alignment of CtG6PDH

Fig. 5 Analysis of promoter cis-acting elements of the CtG6PDHs gene

Fig. 6 Subcellular localization of CtG6PDH1 and CtG6PDH2

Fig. 7 Expression patterns of CtG6PDHs genes in different tissues

Fig. 8 CtG6PDHs expression patterns under different stress treatments and hormone inductionDifferent lowercase letters indicate the significant difference at P<0.05 level

Fig. 9 Determination of G6PDH enzyme activity under different stress treatments

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