Research on the Function of LrCYP78A5 Gene from Lycium ruthenicum in Response to Drought and Salt Stress

doi:10.13560/j.cnki.biotech.bull.1985.2025-1127

Abstract

Abstract:

Objective This study characterized the cytochrome P450 gene LrCYP78A5 from Lycium ruthenicum to understand its role in abiotic stress tolerance. Method LrCYP78A5 was expressed in transgenic yeast and L. ruthenicum plants. Stress responses were evaluated under 0.5 mol/L mannitol and 0.8 mol/L NaCl (yeast) and 10% PEG-6000/300 mmol/L NaCl (plants). Physiological parameters including relative water content, chlorophyll content, malondialdehyde (MDA) levels, and antioxidant enzyme activities were measured. Detached leaf assays were conducted to analyze phenotypic changes and ROS accumulation. RNA-seq identified differentially expressed genes (DEGs) followed by GO and KEGG enrichment analysis. Result Heterologous expression of LrCYP78A5 significantly enhanced stress tolerance in yeast, with transgenic strains showing improved growth under osmotic and ionic stress. In L. ruthenicum, overexpression lines maintained approximately 40% higher relative water content, more stable chlorophyll levels (approximately 25% less reduction), 30% lower MDA accumulation, and 15%‒20% higher SOD and POD activities under stress compared to wild-type. Detached leaves of overexpression lines exhibited enhanced anthocyanin accumulation and significantly less ROS production under stress conditions. RNA-seq identified 595 differentially expressed genes (DEGs) enriched in ion transport (ABCG transporters), ROS scavenging (glutathione reductase and GST genes), and plant signal transduction (AOS3-like and zeatin O-glucosyltransferase). These molecular changes correlated with improved membrane stability and reduced oxidative damage in transgenic plants. Conclusion LrCYP78A5 functions as a positive regulator of abiotic stress tolerance in L. ruthenicum through coordinated regulation of multiple defense mechanisms. The gene enhances water retention capacity, maintains photosynthetic pigment stability, strengthens antioxidant systems, and promotes membrane lipid remodeling under drought and salt stress conditions.

Key words: Lycium ruthenicum, LrCYP78A5, transcriptome, drought stress, salt stress, yeast stress, reactive oxygen species, lipid peroxidation

WANG Hong-rui, ZHAO Yi-ru, RAO Shu-pei, CHEN Jin-huan. Research on the Function of LrCYP78A5 Gene from Lycium ruthenicum in Response to Drought and Salt Stress[J]. Biotechnology Bulletin, 2026, 42(7): 182-192.

Figures/Tables 6

Table 1 Top 20 differentially expressed genes (DEGs)

基因ID Gene ID	基因名称 Gene name	log₂FC	q-value	KO_description	功能描述 Functional description
LruCao09G0374300	---	-6.37	3.95E-96	---	未知功能 Unknown function
LruCao07G0318870	SAMS2	-7.70	3.45E-57	S-腺苷甲硫氨酸合成酶 S-adenosylmethionine synthetase	S-腺苷甲硫氨酸合成酶 S-adenosylmethionine synthase 2
LruCao01G0002990	---	5.53	2.52E-38	海藻糖-6-磷酸合成酶/磷酸酶 Trehalose 6-phosphate synthase/phosphatase	未知功能 Unknown function
LruCao10G0446870	FST-like	-2.90	1.03E-34	---	类黄酮醇磺基转移酶 Flavonol sulfotransferase-like
LruCao07G0318290	TMV-N-like	5.43	1.87E-34	---	类烟草花叶病毒抗性蛋白N TMV resistance protein N-like
LruCao06G0270540	LOC132598731	-4.60	1.53E-32	---	未知功能 Unknown function
LruCao02G0051350	AP1B	2.81	1.57E-32	---	β-衔接蛋白样蛋白B Beta-adaptin-like protein B
LruCao07G0280600	R1A-10-like	-5.71	1.61E-31	---	类晚疫病抗性蛋白 R1A-10 Late blight resistance protein R1A-10-like
LruCao09G0368190	LOC132626143	4.36	8.78E-31	---	未知功能 Unknown function
LruCao06G0269340	KH-like	-3.18	1.86E-28	---	含KH结构域蛋白 KH domain-containing protein
LruCao11G0483750	LOC132617431	4.12	1.17E-27	---	未知功能 Unknown function
LruCao09G0397570	BEACH-B	-2.00	1.03E-25	---	含BEACH结构域蛋白B BEACH domain-containing protein B
LruCao09G0364230	RLK902	2.41	1.57E-25	---	类受体激酶RLK902 Receptor-like kinase RLK902
LruCao03G0096480	LOC132629991	-3.91	3.65E-25	E3泛素-蛋白连接酶 RFWD3 E3 ubiquitin-protein ligase RFWD3	未知功能 Unknown function
LruCao08G0322360	RPP13-like	2.18	5.03E-24	---	类霜霉病抗性蛋白 RPP13 Downy mildew resistance protein RPP13-like
LruCao07G0281170	R-protein	2.43	4.72E-23	---	抗病蛋白同源物 Disease resistance protein homolog
LruCao02G0050030	LOC132634780	-1.96	1.01E-22	---	未知功能 Unknown function
LruCao08G0324650	ABCB11-like	2.53	1.45E-22	ABC转运蛋白B家族成员 ABC transporter subfamily B member	ABC转运蛋白，B亚家族成员 ABC transporter， subfamily B member
LruCao01G0024590	LOC132632404	6.10	3.53E-22	---	未知功能 Unknown function
LruCao03G0098210	LOC132057225	-4.90	5.13E-19	---	未知功能 Unknown function

Table 1 Top 20 differentially expressed genes (DEGs)

基因ID Gene ID	基因名称 Gene name	log₂FC	q-value	KO_description	功能描述 Functional description
LruCao09G0374300	---	-6.37	3.95E-96	---	未知功能 Unknown function
LruCao07G0318870	SAMS2	-7.70	3.45E-57	S-腺苷甲硫氨酸合成酶 S-adenosylmethionine synthetase	S-腺苷甲硫氨酸合成酶 S-adenosylmethionine synthase 2
LruCao01G0002990	---	5.53	2.52E-38	海藻糖-6-磷酸合成酶/磷酸酶 Trehalose 6-phosphate synthase/phosphatase	未知功能 Unknown function
LruCao10G0446870	FST-like	-2.90	1.03E-34	---	类黄酮醇磺基转移酶 Flavonol sulfotransferase-like
LruCao07G0318290	TMV-N-like	5.43	1.87E-34	---	类烟草花叶病毒抗性蛋白N TMV resistance protein N-like
LruCao06G0270540	LOC132598731	-4.60	1.53E-32	---	未知功能 Unknown function
LruCao02G0051350	AP1B	2.81	1.57E-32	---	β-衔接蛋白样蛋白B Beta-adaptin-like protein B
LruCao07G0280600	R1A-10-like	-5.71	1.61E-31	---	类晚疫病抗性蛋白 R1A-10 Late blight resistance protein R1A-10-like
LruCao09G0368190	LOC132626143	4.36	8.78E-31	---	未知功能 Unknown function
LruCao06G0269340	KH-like	-3.18	1.86E-28	---	含KH结构域蛋白 KH domain-containing protein
LruCao11G0483750	LOC132617431	4.12	1.17E-27	---	未知功能 Unknown function
LruCao09G0397570	BEACH-B	-2.00	1.03E-25	---	含BEACH结构域蛋白B BEACH domain-containing protein B
LruCao09G0364230	RLK902	2.41	1.57E-25	---	类受体激酶RLK902 Receptor-like kinase RLK902
LruCao03G0096480	LOC132629991	-3.91	3.65E-25	E3泛素-蛋白连接酶 RFWD3 E3 ubiquitin-protein ligase RFWD3	未知功能 Unknown function
LruCao08G0322360	RPP13-like	2.18	5.03E-24	---	类霜霉病抗性蛋白 RPP13 Downy mildew resistance protein RPP13-like
LruCao07G0281170	R-protein	2.43	4.72E-23	---	抗病蛋白同源物 Disease resistance protein homolog
LruCao02G0050030	LOC132634780	-1.96	1.01E-22	---	未知功能 Unknown function
LruCao08G0324650	ABCB11-like	2.53	1.45E-22	ABC转运蛋白B家族成员 ABC transporter subfamily B member	ABC转运蛋白，B亚家族成员 ABC transporter， subfamily B member
LruCao01G0024590	LOC132632404	6.10	3.53E-22	---	未知功能 Unknown function
LruCao03G0098210	LOC132057225	-4.90	5.13E-19	---	未知功能 Unknown function

Fig. 1 Heterologous expression of LrCYP78A5 enhances the tolerances of yeast to salt and osmotic stressA. PCR verification of yeast colonies. 1-5: Different yeast colonies transformed with pYES2-LrCYP78A5. B: Growth phenotype of transgenic yeast on solid media supplemented with 0.5 mol/L mannitol or 0.5 mol/L NaCl. C: OD₆₀₀ values of yeast cultured in non-stress liquid medium for 48 h. D: OD₆₀₀ values of yeast cultured in liquid medium with 1 mol/L mannitol for 48 h. E: OD₆₀₀ values of yeast cultured in liquid medium with 0.8 mol/L NaCl for 72 h. Values are mean ± SD (n = 3). ** indicates P<0.01; *** indicates P<0.001. The same below

Fig. 2 Molecular identification and stress phenotype of LrCYP78A5-overexpressing plantsA: PCR identification of transgenic lines at the DNA level. B: Validation of LrCYP78A5 transcription level in transgenic lines by RT-qPCR. C: Phenotypic comparison under drought and salt stress at the seedling stage. 10% PEG treatment indicates the addition of 10% PEG-6000 to the nutrient solution for simulating drought stress; similarly, 300 mmol/L NaCl treatment represents the simulation of salt stress. The treatments were conducted for 0, 4, and 8 d

Fig. 3 Detached leaf response to stress and ROS accumulationA: Phenotype of detached leaves cultured on media containing 10% PEG-6000 or 300 mmol/L NaCl treatment for 0 and 4 d. B: DAB and NBT staining of leaves treated with 10% PEG-6000 for 0 and 4 d. C: DAB and NBT staining of leaves treated with 300 mmol/L NaCl for 0 and 4 d

Fig. 4 Temporal dynamics changes in physiological indices of WT and LrCYP78A5-OE plants under drought and salt stressRelative water content (A) and chlorophyll content (B) under 10% PEG-6000 drought stress. Relative water content (C) and chlorophyll content (D) under 300 mmol/L NaCl salt stress. MDA content (E), SOD activity (F), and POD activity (G) under 10% PEG-6000 induced drought stress. MDA content (H), SOD activity (I), and POD activity (J) under 300 mmol/L NaCl stress. Values are mean ± SD (n = 3). Different letters above bars indicate significant differences (P<0.05) between WT and OE lines at the same time point.stress

Fig. 5 Functional enrichment analysis of differentially expressed genes between WT and LrCYP78A5-OE linesA: Significantly enriched GO terms (biological process, cellular component, and molecular function). B: Significantly enriched KEGG pathways

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