三种酶对采后‘海沃德’和‘华特’猕猴桃 AsA的氧化作用

doi:10.3864/j.issn.0578-1752.2020.04.013

摘要/Abstract

摘要：

【目的】探究‘海沃德’‘华特’猕猴桃果实采后影响抗坏血酸（ascorbic acid,AsA）氧化的相关酶活性及基因表达差异,为猕猴桃采后AsA氧化机制的系统研究,调控果实成熟和衰老进程,并有效保持果实采后品质和延长贮藏时间等研究提供理论依据。【方法】以‘海沃德’‘华特’猕猴桃作为试验材料,测定两个品种的果实在采后25℃贮藏条件下AsA、总抗坏血酸（total ascorbic acid,T-AsA）、脱氢抗坏血酸（dehydroascorbic acid,DHA）、AsA/DHA、与AsA氧化相关的抗坏血酸氧化酶（ascorbic acd oxidase,AO）、漆酶（Laccase）、抗坏血酸过氧化物酶（ascorbate peroxidase,APX）的活性及相关酶基因的表达,研究两个品种猕猴桃果实AsA含量变化与AO、漆酶、APX活性及相关酶基因的相关性。【结果】‘海沃德’猕猴桃采后初期AsA含量为86.9 mg/100 g FW,到贮藏末期损失约45%,而‘华特’猕猴桃在采后初期AsA含量较高,为610 mg/100 g FW,中期上升至峰值886 mg/100 g FW,末期下降到778 mg/100 g FW,高于采后第1天AsA含量,整体呈上升趋势;两个品种DHA含量在整个贮藏期整体呈下降趋势,但‘海沃德’猕猴桃中DHA含量始终低于‘华特’;T-AsA含量与其AsA含量的变化趋势接近;整个贮藏后期,‘海沃德’猕猴桃的AsA/DHA比值远低于‘华特’。AO活性与两个品种猕猴桃AsA含量呈显著负相关性,漆酶活性与两个品种猕猴桃AsA含量呈负相关性;从采后第8天开始,‘华特’的AO活性低于‘海沃德’,在整个贮藏期,‘华特’猕猴桃中漆酶活性都低于‘海沃德’;且在采后第11天,‘华特’中AO和漆酶活性均达到最低值,‘海沃德’中漆酶活性在采后第16天达到最高值;APX对AsA含量的影响较小,其活性与AsA变化无显著相关性。AO基因家族中的3个基因中,Achn020161是AsA氧化分解的关键基因,而Achn191341和Achn316521与AsA的氧化无显著相关性;漆酶基因家族中的3个基因中,Achn007661、Achn191341对AsA含量变化有一定影响,而Achn163871与AsA的氧化无显著相关性;APX基因家族中的Achn123021、Achn082241、Achn187071对AsA含量的变化有一定影响,但均不是氧化AsA的关键基因。【结论】AsA/DHA的高比值对AsA的积累起重要作用,AO是氧化AsA的关键酶,漆酶对AsA氧化有一定作用,APX不是主要氧化AsA的酶,推测AO基因家族中的Achn020161是氧化AsA的关键基因,而漆酶基因家族中的Achn007661与Achn19134对氧化AsA有一定作用。

关键词: 猕猴桃, ‘华特’, ‘海沃德’, 抗坏血酸, 酶活性, 基因表达

Abstract:

【Objective】This study explored the differences in activities of enzymes and their gene expressions related with oxidation of ascorbic acid (AsA) in postharvest Hayward and Huate kiwifruit to provide a theoretical basis for the oxidation mechanism of AsA in postharvest kiwifruit to regulate fruit ripening and senescence, maintain postharvest quality and extend storage life.【Method】 Hayward and Huate kiwifruit were harvested, and total ascorbic acid (T-AsA), dehydroascorbic acid (DHA), AsA/DHA, and AsA content were measured under the storage conditions of 25℃. Enzyme activity of ascorbic acid oxidase (AO), laccase, ascorbate peroxidase (APX) and gene expression related with AsA oxidation were measured. The relationships between AsA content and the enzyme activity of AO, laccase, APX and gene expression were investigated in the two varieties of kiwifruit.【Result】The content of AsA in Hayward was 86.9 mg/100 g FW at the beginning of harvest, and the loss was about 45% at the end of storage. The content of AsA in Huate was higher at the beginning of harvest (610 mg/100 g FW), and it rose to 886 mg/100 g FW, reached its peak value during the storage, and dropped to 778 mg/100 g FW at the end, which was higher than that on the first day after harvest. The DHA content of the two varieties showed a downward trend throughout the whole storage period, but the DHA content of Hayward was lower than that of Huate. The T-AsA content and AsA content change trend was similar. In the later stage of storage, the AsA/DHA ratio of Hayward was much lower than that of Huate. AO activity was significantly negatively correlated with AsA content of two varieties of kiwifruit, and laccase activity was negatively correlated with AsA content. From the 8th day after harvest, the AO activity of Huate was lower than Hayward, and the laccase activity was lower than Hayward during the whole storage period of Huate kiwifruit. The activity of AO and laccase got the lowest value on the 11th day after harvest; laccase activity reached its highest value in Hayward on the 16th day after harvest. APX had little effect on AsA content, and its activity had no significant correlation with AsA content. Among the three genes in the AO gene family, Achn020161 was a key gene for the oxidative decomposition of AsA, while Achn191341 and Achn316521 had no significant correlation with the oxidation of AsA. Among the three genes in the laccase gene family, Achn007661 and Achn191341 had certain effect on the change of AsA content, while Achn163871 had no significant correlation with the oxidation of AsA. Achn123021, Achn082241 and Achn187071 in the APX gene family had certain effect on the change of AsA content, but they were not key genes for oxidative degradation of AsA.【Conclusion】The high ratio of AsA/DHA played an important role in the accumulation of AsA. AO was the key enzyme on the oxidation of AsA, laccase had certain effect, and APX was not a main enzyme in oxidation of AsA. It’s speculated that Achn020161 in the AO gene family was the key gene for oxidizing AsA, and Achn007661 and Achn19134 in the laccase gene family had certain effects on the oxidation of AsA.

Key words: kiwifruit, Huate, Hayward, ascorbic acid, enzyme activity, gene expression

封琦,李朝政,高贵田,吴悠,肖妍,赵武奇,雷玉山. 三种酶对采后‘海沃德’和‘华特’猕猴桃 AsA的氧化作用[J]. 中国农业科学, 2020, 53(4): 811-822.

Qi FENG,ChaoZheng LI,GuiTian GAO,You WU,Yan XIAO,WuQi ZHAO,YuShan LEI. Influence of Three Enzymes on Oxidation of Ascorbic Acid in Postharvest 'Hayward' and 'Huate' Kiwifruit[J]. Scientia Agricultura Sinica, 2020, 53(4): 811-822.

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酶名称 Enzyme name	基因名称 Gene name	上游引物 Forward primer	下游引物 Reverse primer
抗坏血酸氧化酶 AO	Achn020161	CGGGTCCTGAACTCTACTGC	GCAAGGTAGTCCTGTTTGGA
	Achn230551	CCTGTGTGGAACGATGTGGA	GCGAGGTAAAGGACAGCGTA
	Achn316521	CCAAGTACTCACGGCCACAT	AGTGTCTGCTGGGATGAACG
漆酶Laccase	Achn007661	CAGTTGTTGCTGTGGATGCT	TGAGAAGTGTCGGCTGGTTA
	Achn191341	GGTGAAATCTGGGGAGACAA	GACTACGACGGATGTGGTTC
	Achn163871	CCCAACAAGTGGACAAACATT	CCACTTTGGTTGGAGTAAGC
抗坏血酸过氧化物酶 APX	Achn123021	CAACTGAAGAGTGCCCGAGA	GAATCACCAGCAGCATTGGC
	Achn082241	TCCCGGTACTTTCAGAGGTG	ACCACAACGACCTCAAGTTC
	Achn187071	GGTGGAATGAATGGCTCAAT	CACACGCCTCCAGGTTTATA

	总抗坏血酸 T-AsA	抗坏血酸/脱氢抗坏血酸 AsA/DHA	抗坏血酸氧化酶 AO	漆酶 Laccase	抗坏血酸过氧化物酶 APX
海沃德 Hayward	0.999^**	0.839	-0.910^*	-0.875	0.253
华特 Huate	0.993^**	0.834	-0.958^*	-0.803	-0.500

	海沃德 Hayward	华特 Huate
Achn020161	-0.922*	-0.920*
Achn191341	0.769	0.494
Achn316521	0.02	0.494
Achn007661	-0.947*	-0.950*
Achn191341	-0.852	-0.423
Achn163871	-0.325	0.599
Achn123021	-0.84	0.536
Achn082241	-0.390	0.246
Achn187071	-0.803	-0.517