Comparison of the Efficiency of Different Transformation Methods for Mediating EjHSFB-2a Overexpression in Loquat Fruit under High-temperature Stress

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

Abstract

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

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.

Figures/Tables 6

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

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

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

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

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

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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