不同杨树SOS1基因启动子的克隆及盐胁迫响应分析

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

摘要/Abstract

摘要：

目的 SOS1（salt overly sensitive1）作为定位于质膜上的Na⁺/H⁺反向转运蛋白，在植物盐胁迫响应中扮演着关键角色。比较分析杨属（Populus）不同树种的SOS1基因启动子，为理解和应用SOS1基因及其启动子进行抗逆改良奠定基础。方法以杨属不同树种作为试验材料，通过实时定量PCR分析SOS1基因的表达模式，进一步从新疆杨（P. alba）、胡杨（P. euphratica）和俄罗斯杨（P. russkii）中克隆了SOS1基因的启动子片段，与GUS（β-glucuronidase）报告基因连接并转化毛白杨（P. tomentosa）获得转基因植株，利用转基因毛白杨通过GUS组织化学染色和酶活性定量研究了启动子的组织特异性和对盐胁迫的响应。结果不同树种中SOS1基因的表达模式差异明显，例如，盐胁迫下在新疆杨茎中SOS1基因上调表达，胡杨变化不显著，俄罗斯杨则下调表达。3个启动子片段均可驱动GUS基因在转基因毛白杨叶、茎、根中表达，具有启动子活性，在茎和根中的活性较高。新疆杨的SOS1启动子在茎的表皮和皮层中有活性，而在韧皮部、形成层、木质部中的活性极低；俄罗斯杨的SOS1启动子则在木质部中活性较高，而在形成层和树皮中活性极低；胡杨的SOS1启动子在各个部位均有活性，尤其是在形成层中活性最高。在盐胁迫条件下，3个启动子的活性均上调。结论不同杨树SOS1基因启动子均可响应盐胁迫但具有差异明显的组织特异性。

关键词: 杨树, SOS1, 盐胁迫, 启动子

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

付博晗, 毛华, 赵薪程, 陆虹, 欧庸彬, 姚银安. 不同杨树SOS1基因启动子的克隆及盐胁迫响应分析[J]. 生物技术通报, 2025, 41(7): 205-213.

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.

图/表 4

表1 试验中使用的引物

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

图1 不同杨树中SOS1基因的表达水平及其对盐胁迫的响应A：新疆杨、胡杨、俄罗斯杨、毛果杨和青杨等不同杨树叶片中SOS1的相对表达水平。将SOS1在新疆杨叶片中的表达水平设为1。不同小写字母表示经邓肯氏检验在P<0.05的水平上差异显著。B，C，D：盐胁迫下新疆杨（B）、胡杨（C）和俄罗斯杨（D）植株不同部位中SOS1表达水平的变化。以温室盆栽培养2月（新疆杨和俄罗斯杨扦插苗）或6月（胡杨实生苗）的树苗为试验材料进行盐胁迫处理，在含有5 L土壤的花盆中浇灌1 L 200 mmol/L NaCl溶液，每12 h浇250 mL，在第1次浇灌后48 h取样。以相同方式浇灌1 L去离子水作为对照。将各树种对照组叶片中SOS1的表达水平均设为1。**表示处理与对照之间差异极显著（t-test，P<0.01）

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)

图2 杨树SOS1基因启动子载体的构建和转基因株系鉴定A：杨树SOS1基因启动子扩增片段的电泳检测；B：杨树SOS1基因启动子上的顺式作用元件；C：启动子载体的双酶切鉴定；D：载体结构示意图；E：转基因植株的PCR鉴定；F：转基因试管苗叶片的GUS组织化学染色。M为DNA分子量标准Trans2K Plus （A），TaKaRa DL15000 （C）和Trans15K （E）；Pa：新疆杨，Pe：胡杨，Pr：俄罗斯杨；TSS：转录起始位点，ATG：起始密码子；NPT Ⅱ：新霉素磷酸转移酶Ⅱ，NOS-T：胭脂碱合成酶基因的终止子；+：正对照；-：负对照；CaMV 35Spro：花椰菜花叶病毒35S启动子；Pa-1、Pa-2、Pa-6等为不同转基因株系

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

图3 盐胁迫下转基因杨树的GUS组织化学染色及GUS活性定量分析A：用打孔器从成熟叶片上采集的叶盘进行GUS组织化学染色；B：茎段第5节节间部位横切面的GUS组织化学染色；C：根尖部位GUS组织化学染色；D：不同组织部位的GUS活性。用含100 mmol/L NaCl的1/2霍格兰溶液处理砂培杨树苗，盐胁迫处理7 d时采样。对照为不额外添加NaCl的1/2霍格兰溶液进行培养。每个启动子均采用了3个株系进行试验，变化模式较一致，因此仅展示其中1个株系的染色结果。标尺为1 cm（A）或1 mm（B和C）。*表示处理与对照之间差异显著（t-test， P<0.05），**表示处理与对照之间差异极显著（t-test， P<0.01）

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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