Cloning of SOS1 Gene Promoters from Poplar and Analysis of Its Response to Salt Stress

doi:10.13560/j.cnki.biotech.bull.1985.2025-0069

Abstract

Abstract:

Objective SOS1 (salt overly sensitive 1), a Na⁺/H⁺ antiporter located on the plasma membrane, plays a crucial role in plant responses to salt stress. Comparative analysis of SOS1 gene promoters from different Populus species would provide an important foundation for understanding and applying SOS1 gene and its promoters for stress resistance improvement. Method The study used different tree species of the genus Populus as experimental materials and the expression patterns of the SOS1 gene was analyzed by real-time quantitative PCR. The promoter fragments of the SOS1 gene were cloned from P. alba, P. euphratica, and P. russkii, and were ligated with the GUS (β-glucuronidase) reporter gene and transformed into Populus tomentosa to obtain transgenic plants. The tissue specificity and response to salt stress of the promoter were studied by GUS histochemical staining and enzyme activity quantification using transgenic P. tomentosa. Result The expression pattern of SOS1 genes varies significantly among different tree species, e.g. under salt stress, the expression of SOS1 gene in the stem of P. alba is upregulated, while there is no significant change in P. euphratica, and is downregulated in P. russkii. All three cloned promoter fragments can drive the expression of the GUS gene in the leaves, stems, and roots of transgenic P. tomentosa, presenting promoter activity, with higher activity in the stems and roots. The SOS1 promoter from P. alba is active in the epidermis and cortex of the stem, but has very low activity in the phloem, cambium, and xylem; the SOS1 promoter from P. russkii shows higher activity in the xylem, but very low activity in the cambium and bark; the SOS1 promoter from P. euphratica is active in all parts, with the highest activity in the cambium. Under salt stress conditions, the activity of all three promoters increases. Conclusion Different poplar SOS1 gene promoters may respond to salt stress but have different tissue specificity.

Key words: poplar, SOS1, salt stress, promoter

FU Bo-han, MAO Hua, ZHAO Xin-cheng, LU Hong, OU Yong-bin, YAO Yin-an. Cloning of SOS1 Gene Promoters from Poplar and Analysis of Its Response to Salt Stress[J]. Biotechnology Bulletin, 2025, 41(7): 205-213.

Figures/Tables 4

Table 1 Primers used in this study

引物Primer

序列Sequence （5′-3′）

功能Function

qRT-PopSOS1-F

qRT-PopSOS1-R

ACTTGGCTTCCCATGAACCT

AGCAATACCACTGTCACCAA

检测SOS1基因表达水平

Detection of SOS1 gene expression level

qRT-PopUBQ-F

qRT-PopUBQ-R

CCAAGCCCAAGAAGATCAAGC

GCACCGCACTCAGCATTAGG

内参，校正上样量的差异

Internal reference， correcting for loading variance

pro-PopSOS1Pro-F

pro-PopSOS1Pro-R

GGAAGCTTGGTGCAmkArAGGAAArATyATyAATTAGCCAT

CCGGATCCCTTGCTCCTCCACTGAAACTGGT

克隆杨树SOS1基因启动子

Cloning of the promoters of poplar SOS1 genes

NPT Ⅱ-F

NPT Ⅱ-R

GCTATGACTGGGCACAACAG

ATACCGTAAAGCACGAGGAA

鉴定转基因阳性植株

Checking transgenic positive plants

Fig. 1 Relative expressions of SOS1 genes in different poplar species and their responses to salt stressA: The relative expressions of SOS1 in the leaves of P. alba (Pa), P. euphratica (Pe), P. russkii (Pr), P. trichocarpa (Ptr) and P. cathayana (Pc). The relative expression of SOS1 in the leaves of Pa was set to 1. Different normal letters indicate statistical significance among different tree species at P<0.05 level by Duncan's test. B, C, D: The response to salt stress of SOS1 genes in different parts of Pa (B), Pe (C) and Pr (D). Two-month-old greenhouse potted cuttings of Pa or Pr, or six-month-old seedlings of Pe were used as experimental materials for salt stress treatment. In pots with 5 L soils, 200 mmol/L NaCl solution was irrigated every 12 h, 250 mL each time, for a total of 4 times. Samples were collected 48 h after the first irrigation. The same way, 1 L of deionized water was irrigated as the mock. The expressions of SOS1 genes in the leaves of the mock group of each tree species was set to 1. ** indicates statistical significance between the treatment and the mock (t-test, P<0.01)

Fig. 2 Construction of vectors for poplar SOS1 promoters and the identification of transgenic linesA: Electrophoresis detection of the amplified fragment of the poplar SOS1 promoters. B: Cis-acting elements of the PopulusSOS1 gene promoters. C: Double digestion of the promoter vectors. D: Schematic diagrams of the vectors. E: Identification of transgenic plants by PCR. F: GUS histochemical staining of transgenic plantlet leaves. M indicates Trans2K Plus DNA marker (A), TaKaRa DL15000 (C), and Trans15K (E); Pa: P. alba, Pe: P. euphratica, Pr: P. russkii; TSS: transcription start site; ATG: start codon. NPT Ⅱ: Neomycin phosphotransferase Ⅱ; NOS-T: nopaline synthase gene terminator. +: Positive control; -: negative control; CaMV35Spro: Cauliflower mosaic virus 35S promoter; Pa-1, Pa-2, Pa-6, etc. are different transgenic lines

Fig. 3 GUS histochemical staining and quantitative analysis of GUS activity in transgenic poplar under salt stressGUS histochemical staining of leaf discs (A) collected from mature leaves using a punch, transverse section of the 5th internode of the stem (B), and root tips (C). D: GUS activity of different plant parts. Sand-cultured poplars were treated with 1/2 Hoagland solution containing 100 mmol/L NaCl for 7 d before sampling. The mock was cultured with 1/2 Hoagland solution without additional NaCl. Three lines were used for each promoter, and the change pattern was consistent, thus the staining results of only one of them were shown. Scale bars: 1 cm (A) or 1 mm (B and C). * and ** indicate statistical significance by t-test between the treatment and the mock at P<0.05 or P<0.01, respectively

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