桂花OfSVB1响应盐胁迫的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-0980

生物技术通报 ›› 2026, Vol. 42 ›› Issue (5): 332-339.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0980

• 研究报告 • 上一篇

桂花OfSVB1响应盐胁迫的功能研究

顾恒¹^,²^,³(), 郑栋¹^,², 宰舟颖¹^,², 陈贡伟¹^,², 岳远征¹^,², 王良桂¹^,²^,³(), 杨秀莲¹^,²()

^1.南京林业大学林木遗传育种国家重点实验室，南京 210037
^2.南京林业大学风景园林学院，南京 210037
^3.南京林业大学林草学院、水土保持学院，南京 210037

收稿日期:2025-09-11 出版日期:2026-05-26 发布日期:2026-06-10
通讯作者: 杨秀莲，女，博士，教授，研究方向：园林植物生理和栽培应用；E-mail: xly@njfu.edu.cn
王良桂，男，博士，教授，研究方向：园林植物种质创新与利用；E-mail: wlg@njfu.edu.cn
作者简介:顾恒，男，博士研究生，研究方向：园林植物遗传与育种；E-mail: guheng2021@163.com
基金资助:
国家自然科学基金项目(31870695);中央财政林业科技推广示范资金项目（苏［2024］TG04），江苏高校优势学科建设工程资助项目（PAPD），江苏省研究生科研与实践创新计划项目(KYCX24_1255)

Functional Study of Osmanthus fragransOfSVB1 in Response to Salt Stress

GU Heng¹^,²^,³(), ZHENG Dong¹^,², ZAI Zhou-ying¹^,², CHEN Gong-wei¹^,², YUE Yuan-zheng¹^,², WANG Liang-gui¹^,²^,³(), YANG Xiu-lian¹^,²()

^1.State Key Laboratory of Tree Genetics and Breeding, Nanjing Forestry University, Nanjing 210037
^2.College of Landscape Architecture, Nanjing Forestry University, Nanjing 210037
^3.College of Forestry and Grassland, College of Soil and Water Conservation, Nanjing Forestry University, Nanjing 210037

Received:2025-09-11 Published:2026-05-26 Online:2026-06-10

摘要/Abstract

摘要：

目的 SVB（smaller with variable branches）基因在植物生长发育和响应逆境过程中发挥关键作用。探究OfSVB1基因的功能为解析桂花响应盐胁迫的分子机制奠定基础。方法根据课题组前期桂花盐胁迫转录组数据中FPKM水平的变化筛选出关键候选基因OfSVB1，利用生物信息学工具对其理化性质进行分析，以桂花‘日香桂’叶片的cDNA为模板克隆OfSVB1基因，通过注射烟草叶片进行亚细胞定位分析，对瞬时侵染烟草进行盐胁迫后测定相对电导率、脯氨酸、丙二醛、过氧化物酶（POD）、过氧化氢酶（CAT）和超氧化物歧化酶（SOD）等6个生理指标的变化，对转化后的烟草叶片组织进行氮蓝四唑（NBT）、二氨基联苯胺（DAB）和过碘酸希夫（Schiff）化学染色，同时对5个可能参与响应盐胁迫的功能基因（NbNHX1、NbCAT、NbSOD、NbAPX和NbP5CS）进行实时荧光定量PCR（RT-qPCR）分析。结果 OfSVB1基因在盐处理72 h时的FPKM值最高，与其他6个处理组之间存在显著差异。该基因拥有全长为597 bp的完整阅读框，编码199个氨基酸，属于DUF538家族成员。基因OfSVB1定位于细胞膜上。与转空载体（empty vector, EV）相比，转基因烟草叶片中相对电导率、脯氨酸含量和POD活性显著上升；而丙二醛含量、CAT活性和SOD活性与EV相比无显著差异。此外，叶片化学染色中观察到OfSVB1烟草叶片的颜色明显比EV深且分布范围广。最后，与EV相比，OfSVB1过表达后未引起5个响应盐胁迫相关基因的表达量发生显著变化。结论 OfSVB1基因具有降低桂花耐盐性的功能。

关键词: 桂花, SVB, 理化性质, 瞬时转化, 烟草, 叶片染色, RT-qPCR, 盐胁迫

Abstract:

Objective The SVB (smaller with variable branches) gene plays a crucial role in the growth and development of plants and their response to adverse conditions. Exploring the function of the OfSVB1 gene can lay the foundation for understanding the molecular mechanism by which Osmanthus fragrans responds to salt stress. Method The key candidate gene OfSVB1 was screened based on changes in FPKM levels from the research group’s previous transcriptome data of O. fragrans ‘Rixiang Gui’ under salt stress. Its physicochemical properties were analyzed using bioinformatics tools. The OfSVB1 gene was cloned using cDNA from the leaves of O. fragrans ‘Rixiang Gui’ as a template. Subcellular localization analysis was conducted by injecting tobacco leaves. Transient infection of tobacco was conducted with salt stress treatment, the changes in six physiological indicators were measured: Relative conductivity, proline, malondialdehyde (MDA), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities. Chemically staining with Nitroblue Tetrazolium (NBT), 3,3′-Diaminobenzidine (DAB), and Periodic Acid-Schiff (Schiff) was conducted on the transformed tobacco leaf tissues. Simultaneously, quantitative real-time PCR (RT-qPCR) analysis was performed on five functional genes (NbNHX1, NbCAT, NbSOD, NbAPX, and NbP5CS) potentially involved in the salt stress response. Result The FPKM value of the OfSVB1 gene was the highest at 72 h of salt treatment, showing significant differences compared to the other six treatment groups. This gene has open reading frame of 597 bp in whole length, encoding 199 amino acids and belonging to the DUF538 family. The OfSVB1 gene was localized to the cell membrane. Compared with EV (instantaneous conversion of N. benthamiana into empty vector), the relative conductivity, proline content and POD activity in the leaves of transgenic tobacco significantly increased; while the MDA content, CAT activity and SOD activity showed no significant difference compared with EV. Additionally, leaf chemical staining revealed a more intense and widespread coloration in OfSVB1-overexpressing tobacco compared to the EV. Finally, no significant differences were observed in the transcript levels of the five salt stress-responsive genes between the OfSVB1-overexpressing lines and the EV. Conclusion OfSVB1 gene has the function of reducing the salt tolerance of O. fragrans.

Key words: Osmanthus fragrans, SVB, physicochemical property, instantaneous conversion, tobacco, leaf staining, RT-qPCR, salt stress

顾恒, 郑栋, 宰舟颖, 陈贡伟, 岳远征, 王良桂, 杨秀莲. 桂花OfSVB1响应盐胁迫的功能研究[J]. 生物技术通报, 2026, 42(5): 332-339.

GU Heng, ZHENG Dong, ZAI Zhou-ying, CHEN Gong-wei, YUE Yuan-zheng, WANG Liang-gui, YANG Xiu-lian. Functional Study of Osmanthus fragransOfSVB1 in Response to Salt Stress[J]. Biotechnology Bulletin, 2026, 42(5): 332-339.

图/表 7

表1 基因引物序列

Table 1 Primer sequences of genes

基因名 Gene name	引物名 Primer name	引物序列 Primer sequence （5′‒3′）
OfSVB1	OfSVB1-F	caaatcgactcttagaaagcttATGTACAGAAACTCTTTACTCTCTGTACTCC
OfSVB1	OfSVB1-R	tatttaaatgtcgaccccgggCATCTGATTCAAAGAAGAGAAVACG
pSuper1300	1300-F	AACGCTTTACAGCAAGAACGGAATG
pSuper1300	1300-R	TAGGTCAGGGTGGTCACGAGGGT
Nbactin	Nbactin-F	ATCCTCACAGAGCGTGGTTAC
Nbactin	Nbactin-R	CACTGAGCACTATGTTTCCGT
NbNHX1	NbNHX1-F	AACTGGGCTCTTTGGGTA
NbNHX1	NbNHX1-R	AGGGCGGTAATAGACTCA
NbP5CS	NbP5CS-F	ATCTTGATGGCAAGGCTTGTGCTG
NbP5CS	NbP5CS-R	AAGCTGAGCTGAGGTTACGTCCAA
NbCAT	NbCAT-F	GTATCGTCCGTCAAGTGCCT
NbCAT	NbCAT-R	CGGGAGCTCGAAGGAAATCA
NbSOD	NbSOD-F	AAAGGGCACCACAGAATTAAAGTA
NbSOD	NbSOD-R	GCTCCAGTGCTCCATAGTCGTA
NbAPX	NbAPX-F	CTCCTCCATATCCACAACAGAACTA
NbAPX	NbAPX-R	GCAGAGTGCCATGCTAAACG

图1 基因OfSVB1的FPKM值水平变化CK表示对照组，S表示NaCl处理组，数字表示处理时间。不同小写字母表示差异显著（P<0.05）。下同

Fig. 1 The level changes of FPKM values for the gene OfSVB1CK indicates the control group, S indicates the NaCl treatment group, and numbers indicate treatment time. Different lowercase letters indicate significant differences(P<0.05). The same below

图2 OfSVB1基因亚细胞定位

Fig. 2 Subcellular localization of OfSVB1 gene

图3 转基因烟草盐胁迫后相关生理指标EV表示瞬时转化空载体的本氏烟草，OfSVB1表示瞬时转化桂花OfSVB1基因的本氏烟草

Fig. 3 Related physiological indexes of transgenic tobacco after salt stressEV indicates the instantaneous conversion of N. benthamiana into empty vector, and OfSVB1 indicates the instantaneous transformation OfSVB1 gene of O. fragrans

图4 转基因烟草盐胁迫后6个生理指标的正交偏最小二乘判别分析

Fig. 4 Orthogonal partial least squares discriminant analysis of 6 physiological indexes of transgenic tobacco after salt stress

图5 NBT、DAB和Schiff溶液对盐处理后本氏烟草叶片染色

Fig. 5 NBT, DAB, and Schiff solution staining of N. benthamiana leaves after salt treatment

图6 OfSVB1瞬时转化烟草检测（A）及5个盐胁迫相关基因的RT-qPCR（B）Nbaction为本氏烟草的内参基因，L1‒3和L4‒6分别表示注射了pSuper1300空载（EV）和pSuper1300-OfSVB1的3株烟草

Fig. 6 OfSVB1 transient conversion for tobacco detection (A) and RT-qPCR of five salt stress-related genes (B)The Nbaction is the internal reference gene of N. benthamiana, and the distribution of L1-3 and L4-6 refers to three tobacco plants injected with pSuper1300 empty vector (EV) and pSuper1300-OfSVB1, respectively

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桂花OfSVB1响应盐胁迫的功能研究

Functional Study of Osmanthus fragransOfSVB1 in Response to Salt Stress

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