Cloning SmERF B3-45 from Salix matsudana and Functional Analysis on Its Tolerance to Salt

doi:10.13560/j.cnki.biotech.bull.1985.2024-0385

Abstract

Abstract:

【Objective】To validate whether SmERF B3-45 plays a positive regulatory role in plant response to salt stress may lay the foundation for revealing the role of AP2/ERF transcription factors in regulating the salt tolerance of Salix matsudana.【Method】The cis-acting elements in the promoter region of SmERF B3-45 from the AP2/ERF superfamily of Populus euphratica were analyzed. The full-length CDS sequence of SmERF B3-45 was cloned using specific primers, and bioinformatics and subcellular localization analysis were conducted. The function was elucidated by constructing overexpression vectors and transforming Arabidopsis thaliana mutants ERF-OE1 and ERF-OE2, and by using virus-induced gene silencing（VIGS）.【Result】Cis-acting element analysis suggested that SmERF B3-45 may be involved in the expression regulation pathways responding to stress. RT-qPCR results showed that the expression of SmERF B3-45 was induced by NaCl treatment and was widely expressed in different tissues of Populus euphratica. Subcellular localization indicated that the SmERF B3-45 protein was localized in the nucleus. In transgenic Arabidopsis, the expression of SmERF B3-45 significantly increased. Under salt stress, compared with the wild type, the root length of overexpressing SmERF B3-45 Arabidopsis significantly increased, while the total protein content, Na⁺ content, MDA content, and Na⁺/K⁺ ratio significantly decreased, and the CAT content and K⁺content significantly increased. Gene-silenced plants showed a significant downregulation of SmERF B3-45 expressions. Compared with the control, the total protein content of the gene-silenced plants significantly reduced, while MDA and proline content were significantly higher than that of the negative control plants. Additionally, the silenced plants demonstrated wilting leaves, indicating that the silencing of SmERF B3-45 reduced the tolerance of Salix matsudana to salt. 【Conclusion】SmERF B3-45 is confirmed as a positive regulatory transcription factor in the plant response to salt stress.

Key words: Salix matsudana, gene cloning, salt stress, ERF transformation factor, genetic transformation, virus-induced gene silencing（VIGS）

HUA Xuan, TIAN Bo-wen, ZHOU Xin-tong, JIANG Zi-han, WANG Shi-qi, HUANG Qian-hui, ZHANG Jian, CHEN Yan-hong. Cloning SmERF B3-45 from Salix matsudana and Functional Analysis on Its Tolerance to Salt[J]. Biotechnology Bulletin, 2024, 40(12): 124-135.

Figures/Tables 11

Fig. 1 AP2/ERF family classification The red star indicates that members of the Soloist subfamily contain AP2 domains with a lower degree of conservatism

Table 1 Information of primers used in this study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
SmERF B3-45F	ATGTCGCAAGGAGGGAACA
SmERF B3-45R	TCATTTTCTTCTCTTGGGC
pWM101-SmERF B3-45F	TCGAGCTTTCGCGAGCTCGGTACCATGTCGCAAGGAGGGAACA
pWM101-SmERF B3-45R	GCATGCCTGCAGGTCGACTCTAGATCATTTTCTTCTCTTGGGC
P2300-SmERF B3-45F	GACGAGCTGTACAAGGGATCCATGTCGCAAGGAGGGAACA
P2300-SmERF B3-45R	CTGCAGGTCGACTCTAGATCATCATTTTCTTCTCTTGGGC
qPCR-SmERF B3-45F	AGCAAGGATGTGGCTTGGAA
qPCR- SmERF B3-45R	CTTCTCTTGGGCGAGCCATC
TRV2- SmERF B3-45F	TAAGGTTACCGAATTCTCGCAAGGAGGGAACAACAG
TRV2- SmERF B3-45R	CGAGACGCGTGAGCTCTCTTACACGCCACTTCCACC

Fig. 2 Cis-acting elements of SmERF B3-45 promoter in S. matsudana Koidz

Table 2 Predicted results of cis-acting elements in the SmERF B3-45 promoter region

种类Type	功能 Function	元件 Element	数目 Amount
光响应元件Light responsive elements	光响应Light responsiveness	G-box	5
		TCT-motif	2
		Box 4	4
		ATCT-motif	1
		AT1-motif	1
		I-box	1
激素响应元件Phytohormone responsive elements	脱落酸响应Abscisic acid responsiveness	ABRE	6
激素响应元件Phytohormone responsive elements	赤霉素响应Gibberellin-responsiveness	TATC-box	1
逆境响应元件Stress responsive elements	抗氧Antioxidant	O2-site	1
逆境响应元件Stress responsive elements	干旱、盐和低温响应 Drought, salt and low temperature responsive	MYB	1

Fig. 3 Cloning of SmERF B3-45 and construction of plant expression vector construction A: PCR amplification product detection of SmERF B3-45. B: Overexpression vector pWM101-SmERF B3-45 single enzyme digestion（BamH I）verification. C: VIGS vector pYL1561-SmERF B3-45 double enzyme digestion（EcoR I+Sal I）verification. D: Subcellular localization vector p2300-SmERF B3-45 double enzyme digestion（Kpn I+Sal I）verification

Fig. 4 Phylogenetic tree construction and protein alignment on SmERF B3-45 protein and its homologs A: Phylogenetic tree. B: Homologous protein alignment. Sabra14G0034100: S. alba; Potri.014G047000, Potri.003G150700, Potri.003G150800: Populus alba; Gohir.D08G067700: G. herbaceum; BraA04t18813Z, BraA05t19475Z: B. rapa; Manes.05G054400: M. esculenta; AT2G44840_ERF13, AT2G31230_ERF15, AT4G17490_ERF6: Arabidopsis thaliana. The SmERF B3-45 protein shares an AP2 domain with its orthologous proteins, which has been marked with a red line

Fig. 5 Expression pattern analysis of SmERF B3-45 A: Expressions of SmERF B3-45 in different tissues of Salix matsudana. B: Expressions of SmERF B3-45 after treatment with 200 mmol/L NaCl for 0, 4, 8 and 12 h. Different small letters indicate significant differences（P<0.05）. The same below

Fig. 6 Subcellular localization analysis of SmERF B3-45 protein A: Laser confocal microscopy to display the stomatal structure of leaf cells.B: GFP signal channel displays the localization signal of SmERF B3-45 protein. C: mCherry signal channel displays histone H2B nuclear localization signals.D: Overlay of channels of A, B, and C, display co-localization of SmERF B3-45 protein localization signal and histone H2B nuclear localization signal

Fig. 7 Phenotypic analysis of SmERF B3-45 overexpressing transgenic Arabidopsis thaliana lines A: Growth conditions of various lines after treatment with 0 and 100 mmol/L NaCl for 7 d. B: Statistical analysis of root length of various lines after treatment with 0 and 100 mmol/L NaCl for 7 d

Fig. 8 Overexpression of SmERF B3-45 in transgenic Arabidopsis enhancing its tolerance to salt

Fig. 9 Silencing SmERF B3-45 through VIGS compromised the tolerance in willow to salt A: Expression of SmERF B3-45 in transgenic plants. B: Growth condition of plants after treatment with 100 mmol/L NaCl, where the red arrows indicate severe wilting, withering, and leaf drop phenomena. C: Total protein content. D: MDA content. E: Proline content. pTRV2: Negative control plants. pTRV2-SmERF B3-45-1, pTRV2-SmERF B3-45-2, pTRV2-SmERF B3-45-3: Gene-silenced plants

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