Cloning and Function Analysis of MfWRKY22 Gene in Medicago falcata

doi:10.13560/j.cnki.biotech.bull.1985.2025-0930

Abstract

Abstract:

Objective To explore the role of MfWRKY22 transcription factor in response to salt, drought and low temperature stresses in Medicago falcata, and to provide a theoretical basis for further investigating the molecular mechanism of MfWRKY22 in M. falcata responding to the abiotic stresses. Method Based on the RNA-Seq data of M. falcata treated with salt and drought, MfWRKY22 gene and its promoter sequence were cloned respectively by RT-PCR and Genome Walker method, and the bioinformatics, gene expression, subcellular localization and transcriptional activation activity analysis were performed. Meanwhile, the OE-MfWRKY22 transgenic Arabidopsis lines were constructed, and the fresh weight, root length and lateral root number of transgenic Arabidopsis seedlings were determined respectively at the day 10 of salt, drought and low temperature stresses treatments. Result The CDS of MfWRKY22 is 1 017 bp, encoding a protein with 338 amino acids, with predicted molecular mass of 37.05 kD and an isoelectric point of 5.65. MfWRKY22, localized in the nucleus and with transcriptional activation activity, is a Group Ⅱ member. A 1 888 bp sequence of MfWRKY22 promoter was obtained, and the cis-acting elements, such as salt stress induction, drought responsive, osmotic stress responsive, ethylene responsive and W-box, were identified. Meanwhile, it was found that the MfWRKY22 gene had the highest expression in the leaves of M. falcata, and the expressions of MfWRKY22 gene in the roots, stems and leaves were up-regulated after salt, drought and low temperature treatment. Under salt, drought and low temperature stresses, the fresh weight, root length and lateral root number of transgenic Arabidopsis seedlings were higher than that of wild seedlings. Conclusion MfWRKY22, whose expression is up-regulated by salt, drought and low temperature stresses,is a Group Ⅱ WRKY transcription factor member, and the overexpressing MfWRKY22 could improve the growth of transgenic plants under abiotic stresses.

Key words: Medicago falcata, MfWRKY22, abiotic stresses, function analysis

ZHAO Jing-wei, CHANG Na, JIN Xiao-wei, ZHANG Li-quan. Cloning and Function Analysis of MfWRKY22 Gene in Medicago falcata[J]. Biotechnology Bulletin, 2026, 42(7): 236-245.

Figures/Tables 10

Table 1 Cis-acting elements in MfWRKY22 promoter

顺式作用元件 Cis-acting element	序列 Sequence	位置 Site	功能 Function
ERE	TATGAAAT	-1 731；-1 299	乙烯应答元件 Ethylene response element
MYB	TAACCA	-1 364；-311；-252	参与干旱响应顺式作用元件 Cis-acting element involved in drought-responsiveness
TGACG-motif	TGACG	-840；-543	参与茉莉酸甲酯反应顺式作用元件 Cis-acting regulatory element involved in the MeJA-responsiveness
STRE	AGGGG	-623；-584	渗透压胁迫应答元件 Osmotic stress-responsive elements
GT1-motif	GGTTAA	-1 626	参与盐胁迫诱导 Involvement in salt-stress induction
W-box	TTGACC	-1 616；-599；-236	WRKY结合位点 Binding site of WRKY

Fig. 1 Analysis of MfWRKY22 protein sequenceA： Sequence alignment and analysis of protein MfWRKY22 with WRKY22s from different plant species. B： Phylogenetic tree analysis of protein. C： Prediction of the secondary structure. D： Prediction of protein conserved domain. E： Prediction of the tertiary structure

Fig. 2 Promoter sequence of MfWRKY22

Fig. 3 Tissue-specific expression pattern analysis of MfWRKY22

Fig. 4 Expression analysis of MfWRKY22A: Relative expression under salt stress. B: Relative expression under drought stress. C: Relative expression under 4 ℃. *, ** and *** indicate significant difference at P<0.05, P<0.01, and P<0.001, respectively

Fig. 5 Subcellular localization analysis of MfWRKY22

Fig. 6 Transcriptional activation analysis of MfWRKY22

Fig. 7 Identification of transgenic lines overexpressing MfWRKY22Different letters indicate significant difference at P<0.01

Fig. 8 Growth situation of overexpressing MfWRKY22 lines under stresses

Fig. 9 Effects of stresses on the growths of overexpressing MfWRKY22 linesDifferent letters indicate significant difference at P<0.01

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