不同转化方法介导高温胁迫下枇杷果实EjHSFB-2a过表达效率比较

doi:10.13560/j.cnki.biotech.bull.1985.2025-1341

摘要/Abstract

摘要：

目的探究不同转化方法介导的EjHSFB-2a瞬时过表达对转色期枇杷果实耐热性的影响，为枇杷抗逆功能基因组学研究及育种提供技术依据。方法分别采用果柄注射法、果面注射法及真空渗透法3种转化方法导入pC1300-EjHSFB-2a过表达载体，45 ℃高温胁迫7 h后，通过测定基因表达水平、丙二醛含量、过氧化氢含量、超氧阴离子产生速率及抗氧化酶活性，比较3种转化方法的差异。结果 3种处理方法均不同程度地降低了果实热害指数与热害发生率。RT-qPCR分析证实，所有方法均能在果肉组织中成功过表达EjHSFB-2a，而仅果面注射法在果皮中有效转化。在果肉组织中，3种方法均显著降低了MDA、H₂O₂和O₂^-的积累，其中真空渗透法对MDA和H₂O₂的抑制效果最佳，降幅分别为43.33%和7.82%；果面注射法对O₂^-的清除能力最强，降幅达29.70%。此外，3种处理均提升了果肉中SOD、POD和CAT的活性。结论 3种转化方法均可通过EjHSFB-2a过表达增强枇杷果肉的耐热性。不同转化方法对抗高温胁迫的调控效果具有组织特异性，转化效率由高到低依次为果面注射法＞真空渗透法＞果柄注射法，其中果面注射法是鉴定果皮及果肉组织基因功能的最适方法。

关键词: 枇杷, 高温胁迫, EjHSFB-2a, 瞬时转化, 果面注射法, 真空渗透法, 果柄注射法, 生理效应

Abstract:

Objective To explore the effects of transient overexpression of EjHSFB-2a on enhancing the heat tolerance of loquat fruit at the turning-color stage, thereby providing a technical basis for stress-resistant functional genomics and breeding in loquat. Method The pC1300-EjHSFB-2a overexpression vector was introduced using three transformation methods: fruit stalk injection, fruit surface injection, and vacuum infiltration. After exposure to high-temperature stress at 45 ℃ for 7 h, the differences among the three methods were compared by measuring gene expression levels, malondialdehyde content, hydrogen peroxide content, superoxide anion production rate, and antioxidant enzyme activities. Result All three treatment methods reduced the heat injury index and incidence to varying degrees. RT-qPCR analysis confirmed that all methods successfully overexpressed EjHSFB-2a in the flesh tissue, while only fruit surface injection achieved effective transformation in the peel. In the flesh tissue, all three methods significantly reduced the accumulation of MDA, H₂O₂ and O₂^-. Among them, vacuum infiltration was most effective in suppressing MDA and H₂O₂, with reductions of 43.33% and 7.82%, respectively. Fruit surface injection showed the strongest scavenging capacity for O₂^-, with a reduction of 29.70%. Furthermore, all three treatments enhanced the activities of SOD, POD and CAT in the flesh tissue. Conclusion All three transformation methods enhanced the thermotolerance of loquat flesh via EjHSFB-2a overexpression. The regulatory effects of different transformation methods on resistance to high-temperature stress exhibited tissue specificity, with transformation efficiency ranking from high to low as fruit surface injection > vacuum infiltration > fruit stalk injection. Among these, fruit surface injection is the most suitable method for functional gene identification in both peel and flesh tissues.

Key words: loquat, high temperature stress, EjHSFB-2a, transient transformation, fruit surface injection, vacuum infiltration, fruit stalk injection, physiological effects

牛洁, 汪红霞, 任艺华, 易鹏翰, 周佳, 孟鹏蕊, 邓群仙, 张慧芬. 不同转化方法介导高温胁迫下枇杷果实EjHSFB-2a过表达效率比较[J]. 生物技术通报, 2026, 42(10): 177-187.

NIU Jie, WANG Hong-xia, REN Yi-hua, YI Peng-han, ZHOU Jia, MENG Peng-rui, DENG Qun-xian, ZHANG Hui-fen. Comparison of the Efficiency of Different Transformation Methods for Mediating EjHSFB-2a Overexpression in Loquat Fruit under High-temperature Stress[J]. Biotechnology Bulletin, 2026, 42(10): 177-187.

图/表 6

表1 三种转化方法生理指标平均隶属函数值

Table 1 Average membership function values of physiological indicators for the three transformation methods

测定指标Measured parameters	隶属函数值 Membership function value
		果皮 Peel			果肉 Flesh
	果柄注射法 Fruit stalk injection		果面注射法 Fruit surface injection	真空渗透法 Vacuum infiltration	果柄注射法 Fruit stalk injection	果面注射法 Fruit surface injection	真空渗透法 Vacuum infiltration
转化成功率 Transformation success rate	0		1.000 0	0	0	1.000 0	1.000 0
基因表达水平 Gene expression level	0		1.000 0	0.065 4	0	0.785 7	1.000 0
热害发生率 Heat injury incidence	0.666 7		0	1	0.666 7	0	1.000 0
热害指数 Heat injury index	0.166 7		0	1	0.166 7	0	1.000 0
MDA	-3.664 2		1.000 0	0.214 3	0	0.578 7	1.000 0
H₂O₂	0		1.000 0	0.097 9	1.000 0	0.792 1	0
O₂^-	0.324 2		1.000 0	0	0.899 6	1.000 0	0
SOD	0.002 8		1.000 0	0	0.859 1	1.000 0	0
POD	0		1.000 0	0.059 6	0	0.577 7	1.000 0
CAT	0.386 3		1.000 0	0	0	1.000 0	0.507 3
平均值 Average	-0.211 7		0.800 0	0.243 7	0.359 2	0.674 3	0.650 7

图1 不同转化方法对高温胁迫下枇杷果实表型的影响A：不同转化方法过表达EjHSFB-2a后表型变化；B：45 ℃高温处理4 h果皮中EjHSFB-2a相对表达量变化；C：45 ℃高温处理4 h果肉中EjHSFB-2a相对表达量变化；D‒F：果柄注射法（D）、果面注射法（E）、真空渗透法（F）过表达EjHSFB-2a热害发生率与热害指数； * P＜0.05，** P＜0.01。下同

Fig. 1 Effects of different transformation methods on the phenotype of loquat fruits under high temperature stressA: Phenotypic changes after overexpression of EjHSFB-2a via different transformation methods. B: Relative expression level of EjHSFB-2a in peel after 4 h of high-temperature stress at 45 ℃. C: Relative expression level of EjHSFB-2a in flesh after 4 h of high-temperature stress at 45 ℃. D-F: Heat injury incidence and index after overexpression of EjHSFB-2a via fruit stalk injection (D), fruit surface injection (E), and vacuum infiltration (F). * P < 0.05, ** P < 0.01. The same below

图2 不同转化方法转化后枇杷果实中EjHSFB-2a相对表达量A‒C：果柄注射法过表达EjHSFB-2a后3份样本RT-qPCR检测结果；D‒F：果面注射法过表达EjHSFB-2a后3份样本RT-qPCR检测结果；G‒I：真空渗透法过表达EjHSFB-2a后3份样本RT-qPCR检测结果；J‒K：不同转化方式在果皮（J）和果肉（K）中转化成功率

Fig. 2 Relative expression of EjHSFB-2a in loquat fruit after different transformation methodsA-C: RT-qPCR results of three replicate samples after overexpression of EjHSFB-2a via fruit stalk injection. D‒F: RT-qPCR results of three replicate samples after overexpression of EjHSFB-2a via fruit surface injection. G‒I: RT-qPCR results of three replicate samples after overexpression of EjHSFB-2a via vacuum infiltration. J‒K: Transformation success rates in peel (J) and flesh (K) for different transformation methods

图3 不同转化方法对高温胁迫下枇杷果实细胞膜损伤程度的影响

Fig. 3 Effects of different transformation methods on cell membrane damage of loquat fruit under high temperature stress

图4 不同转化方法对高温胁迫下枇杷果实抗氧化酶活性的影响

Fig. 4 Effects of different transformation methods on antioxidant enzyme activities in loquat fruit under high temperature stress

表2 三种转化方法综合评价

Table 2 Comprehensive evaluation of the three transformation methods

部位 Tissue	方法 Methods	综合隶属函数值 Comprehensive membership function value	排名 Ranking
果皮 Peel	果柄注射法 Fruit stalk injection	-0.211 7	3
	果面注射法 Fruit surface injection	0.800 0	1
	真空渗透法 Vacuum infiltration	0.243 7	2
果肉 Flesh	果柄注射法 Fruit stalk injection	0.359 2	3
	果面注射法 Fruit surface injection	0.673 4	1
	真空渗透法 Vacuum infiltration	0.650 7	2

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