黑果枸杞 LrCYP78A5 基因在干旱与盐胁迫响应中的功能研究

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

摘要/Abstract

摘要：

目的明确黑果枸杞细胞色素P450基因LrCYP78A5在非生物胁迫响应中的功能，为作物抗逆育种提供基因资源。方法构建LrCYP78A5过表达载体，获得转基因酵母和黑果枸杞过表达植株；通过点板实验和液体培养分析酵母在0.5 mol/L甘露醇和0.8 mol/L NaCl胁迫下的生长状况；测定植株在10% PEG-6000干旱胁迫和300 mmol/L NaCl盐胁迫下的相对含水量、叶绿素含量、丙二醛含量及超氧化物歧化酶和过氧化物酶活性；利用离体叶片实验分析胁迫下表型与活性氧积累；利用RNA-seq鉴定差异表达基因，并进行GO和KEGG富集分析。结果过表达LrCYP78A5显著提高酵母胁迫耐受性，在0.5 mol/L甘露醇和0.8 mol/L NaCl胁迫下菌落形成能力和菌液浓度均优于对照。转基因植株在胁迫条件下相对含水量下降幅度较野生型减少约40%，叶绿素含量保持更稳定，丙二醛含量降低约30%，超氧化物歧化酶和过氧化物酶活性提高15%‒20%。离体叶片实验进一步表明，过表达株系在胁迫下呈现显著紫化并积累更少的活性氧。转录组分析共鉴定595个差异表达基因，显著富集于离子转运、活性氧清除和植物信号转导通路，包括ATP结合盒转运蛋白G家族成员、谷胱甘肽还原酶、谷胱甘肽S-转移酶、丙二烯氧化物合成酶AOS3-like和玉米素-O-葡萄糖基转移酶等关键基因。结论 LrCYP78A5通过调控水分保持、光合稳定性、抗氧化防御和膜脂重塑等多层次生理过程，增强黑果枸杞对干旱和盐胁迫的耐受性。

关键词: 黑果枸杞, LrCYP78A5, 转录组, 干旱胁迫, 盐胁迫, 酵母胁迫, 活性氧, 膜脂过氧化

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

王鸿睿, 赵怡如, 饶书培, 陈金焕. 黑果枸杞 LrCYP78A5 基因在干旱与盐胁迫响应中的功能研究[J]. 生物技术通报, 2026, 42(7): 182-192.

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.

图/表 6

表1 前20个差异表达基因

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

表1 前20个差异表达基因

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

图1 异源表达LrCYP78A5增强酵母的盐和渗透胁迫耐受性A：酵母菌落PCR鉴定；1-5：不同pYES2-LrCYP78A5转化菌落；B：转基因酵母在添加0.5 mol/L甘露醇与0.5 mol/L NaCl的固体培养基上的生长状态；C：酵母在正常液体培养基中培养48 h后OD₆₀₀值；D：酵母在1 mol/L甘露醇液体培养基中培养48 h后OD₆₀₀值；E：酵母在0.8 mol/L NaCl液体培养基中培养72 h后OD₆₀₀值；数据为平均值±标准差（n=3）。** P<0.01，*** P<0.001。下同

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

图2 过表达LrCYP78A5植株的分子鉴定与胁迫表型A：转基因株系的DNA水平PCR鉴定；B：转基因株系中LrCYP78A5转录水平的RT-qPCR验证；C：苗期干旱与盐胁迫表型比较；10% PEG代表营养液中添加了10% PEG-6000模拟的干旱胁迫；同理，300 mmol/L NaCl代表模拟盐胁迫；处理0、4和8 d

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

图3 离体叶片胁迫响应与活性氧积累A：离体叶片在10% PEG-6000与300 mmol/L NaCl培养基上处理0和4 d的表型；B：10% PEG-6000处理0和4 d后的DAB与NBT染色；C：300 mmol/L NaCl处理0和4 d后的DAB与NBT染色

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

图4 干旱和盐胁迫下WT和LrCYP78A5-OE植株生理指标的时间动态变化10% PEG-6000干旱处理下相对含水量（A）和叶绿素含量（B）；300 mmol/L NaCl盐胁迫下相对含水量（C）和叶绿素含量（D）；10% PEG-6000干旱胁迫处理下MDA含量（E）、SOD活性（F）和POD活性（G）；300 mmol/L NaCl盐胁迫处理下MDA含量（H）、SOD活性（I）和POD活性（J）。数据为平均值±标准差（n=3），同一时间点上不同字母表示WT与OE株系间差异显著（P<0.05）

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

图5 WT与LrCYP78A5-OE株系差异表达基因的功能富集分析A：显著富集的GO条目（生物过程、细胞组分和分子功能）；B：显著富集的KEGG通路

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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