Cloning and Salt-drought Tolerance Analysis of MnDREB6E Gene in Mulberry

doi:10.13560/j.cnki.biotech.bull.1985.2025-0705

Abstract

Abstract:

Objective To elucidate the biological function of MnDREB6E, a member of the DREB transcription factor family in mulberry, and clarify its mechanism of action in response to abiotic stresses, thereby providing critical genetic resources for molecular breeding of stress-tolerant woody plants. Method MnDREB6E was cloned from salt-stressed mulberry using transcriptomic data. Bioinformatics tools were employed to analyze its sequence characteristics and phylogenetic relationships. Cis-acting elements were validated using yeast one-hybrid assays. Overexpression and RNAi-mediated suppression vectors were constructed and transiently transformed into mulberry via Agrobacterium-mediated transformation. Physiological and biochemical parameters as well as the antioxidant gene expression were measured under salt/drought stress. Result MnDREB6E contains a 1 173 bp ORF encoding 390 amino acids with a conserved AP2/ERF domain, classifying it into the DREB A6 subgroup. Its subcellular localization was predicted to be in the nucleus and specifically bound to GCC-box/DRE cis-elements. Under salt/drought stress, the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione S-transferase (GST) in the overexpressed plants were significantly enhanced. The contents of ascorbic acid (AsA), reduced glutathione (GSH), and proline increased, while the levels of reactive oxygen species (ROS), including superoxide anion (O₂^•-), hydrogen peroxide (H₂O₂), and hydroxyl radical (•OH) decreased. Additionally, the relative electrolyte leakage and malondialdehyde (MDA) content were reduced, and the expressions of antioxidant-related genes was significantly upregulated. Conclusion MnDREB6E coordinately regulates antioxidant metabolism and osmoprotection pathways, enhancing mulberry tolerance to salt/drought stress.

Key words: mulberry, DREB transcription factor, drought stress, salt stress, transient expression

DONG Ya-ru, ZHU Hong, WANG Zhao-hong, ZHAO Dong-xiao, LIU Hui-fen. Cloning and Salt-drought Tolerance Analysis of MnDREB6E Gene in Mulberry[J]. Biotechnology Bulletin, 2026, 42(2): 306-316.

Figures/Tables 12

Table 1 Primers used in gene cloning and vector construction

引物名称 Primer name	序列 Sequence （5′‒3′）	用途 Application
MnDREB6E-F	ATGGAAGATCAGTTTCCCAAGATG	基因克隆 Gene cloning
MnDREB6E-R	TCATGATGTATCAGAGACCAG	基因克隆 Gene cloning
pROKⅡ-MnDREB6E-F	CTCTAGAGGATCCCCATGGAAGATCAGTTTCCCAAG	载体构建 Vector construction
pROKⅡ-MnDREB6E-R	TCGAGCTCGGTACCCTCATGATGTATCAGAGACCAG
pFGC5941-MnDREB6E-cis-F	ACAATTACCATGGGGCGTCATCGGTTTCATCACCGGG
pFGC5941-MnDREB6E-cis-R	AAATCATCGATTGGGCCACTCTTTTCTTGTGGGGTTG
pFGC5941-MnDREB6E-anti-F	AGTTAATTAAGACCCCGTCATCGGTTTCATCACCGGG
pFGC5941-MnDREB6E-anti-R	CTAGGGACTAGTCCCCCACTCTTTTCTTGTGGGGTTG
pGADT7-Rec2-F	ATGAACATGGAGGCCAGTG
pGADT7-Rec2-R	GATGGATCCCGTATCGATG
pHIS2-F	GCCTTCGTTTATCTTGCCTGCTC
pHIS2-R	CGATCGGTGCGGGCCTCTTC

Table 2 Primer sequences for RT-qPCR

基因 Gene	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）
MnDREB6E	CCAAATCAAAAGAACAAGCCT	ATTCTACTCAGCTGAACAGCT
MnRPL15	GGCTATGTGATTTACCGTGTT	TTGGTCCAGTATGAGTTGAGAA
β-actin	AGCAACTGGGATGACATGGAGA	CGACCACTGGCGTAAAGGGA
sodc	GCAGCACCAAAGCCACTCT	CCGTCGTCTTGTTGGGTCA
sod1	GACGCTGATGAGAAAGGT	TTAGACCTTGCCGGTGAC
mpod12	CCCCACGAACATCACGGTTGCCACTA	GCACGTATCTCACCTTGGCTGCCTGT
pod4	TCCCCTCTCGACAGCACC	AGTTTACCACCACGCAAT
cat1	CGTCACGCTGAGAGGTAC	TCAAATGCTTGGCCTCAC
gst10	GCCCTAGGTGACAAAGAT	CTACTCAATGCCATTCAT

Fig. 1 Cloning of MnDREB6E geneM: DNA marker DL 2 000; 1: negative control; 2: MnDREB6E gene band

Fig. 2 Sequence alignment of MnDREB6E with protein sequences from group A6 of Arabidopsis thalianaThe green line indicates the conserved DNA-binding domain (AP2/ERF domain), 3 red boxes, 1 blue box and ▲ respectively indicate 3 β-sheets, 1 α-helix and V14

Fig. 3 Phylogenetic relationships between MnDREB6 and DREB family proteins from A. thaliana, Oryza sativa, Zea mays, Populus euphratica, Gossypium hirsutum, and Glycine max

Fig. 4 Analyses of MnDREB6E binding to GCC-box and DRE motifsThe positive control, negative control, and experimental bacterial cells were diluted 5, 50, 500, and 5 000 fold respectively. The TDO (SD/-His/-Leu/-Trp) solid medium contained 60 mmol/L 3-AT; 1 refers to the positive control, 2 refers to the negative control, 3 and 7 correspond to the GCC-box and DRE core sequences respectively, while 4‒6 and 8‒9 refer to core mutant sequences of GCC-box and DRE motifs

Fig. 5 RT-qPCR detection of mulberry transiently transformed with MnDREB6EA and B are the expressions of the MnDREB6E gene in transiently transformed mulberry plants under salt and drought stress treatments, respectively. * indicates significantly different at the P<0.05 level. The same below

Fig. 6 Analysis of electrolyte permeability and MDA content in Control, OE and RNAi mulberry plants under stress conditionsA: Salt stress treatment. B: Drought stress treatment

Fig. 7 Changes in the contents of O2•-, H2O2, and •OH in Control, OE and RNAi mulberry plants under stress conditionsA: Salt stress treatment. B: Drought stress treatment

Fig. 8 Changes in the activities of SOD, POD, CAT and GST in Control, OE and RNAi mulberry plants under stress conditionsA: Salt stress treatment. B: Drought stress treatment

Fig. 9 Changes in the contents of AsA, GSH and proline in Control, OE and RNAi mulberry plants under stress conditionsA: Salt stress treatment. B: Drought stress treatment

Fig. 10 Analysis of oxidase gene expression in Control, OE and RNAi mulberry plants under stress conditionsA: Salt stress treatment (48 h). B: Drought stress treatment (48 h)

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