Functional Study of Osmanthus fragransOfSVB1 in Response to Salt Stress

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

Abstract

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

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.

Figures/Tables 7

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

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

Fig. 2 Subcellular localization of OfSVB1 gene

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

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

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

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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