MdWRKY33和MdWRKY127调控苹果果实糖酸代谢提高糖酸比

doi:10.16420/j.issn.0513-353x.2026-0120

摘要/Abstract

摘要：

基于‘绿袖’苹果（Malus × domestica Borkh.）果实不同发育阶段的转录组分析，对两个在成熟期特异高表达的WRKY转录因子编码基因MdWRKY33与MdWRKY127进行功能及生物信息分析。系统发育分析表明MdWRKY33蛋白与苹果野生近缘种山荆子（Malus baccata var. baccata）以及梨（Pyrus × bretschneideri）中的同源蛋白亲缘关系较近，MdWRKY127蛋白与苹果中1个未完全注释的蛋白亲缘关系较近。亚细胞定位分析表明，MdWRKY33与MdWRKY127均定位在细胞核。为验证其功能，以苹果愈伤组织为受体构建转基因材料，结果显示，过表达MdWRKY33或MdWRKY127均能显著降低愈伤组织中苹果酸与柠檬酸含量，同时提高果糖的积累水平，使得转基因愈伤组织中糖酸比升高。进一步对苹果果实进行瞬时转化，发现在果实中过表达MdWRKY33或MdWRKY127所产生的效应与在愈伤组织中的效应一致，而分别沉默任一基因均会导致糖酸比下降。这些结果表明MdWRKY33和MdWRKY127均可以调控苹果果实糖酸代谢，改善苹果果实的风味。

关键词: 苹果, WRKY, 果实风味, 可溶性糖, 有机酸, 糖酸比, 过表达, 愈伤组织

Abstract:

Transcriptome analysis of‘Greensleeves’apple（Malus × domestica Borkh.）fruit during developmental stages identified two WRKY transcription factors，MdWRKY33 and MdWRKY127，which were specifically highly expressed at the ripening stage. To clarify the biological functions of these two WRKY transcription factors，they were cloned and subjected to bioinformatics analysis. Phylogenetic

analysis revealed that the MdWRKY33 protein shares close phylogenetic relationships with its homologous proteins from wild apple relative Siberian crabapple（Malus baccatavar. baccata）and pear（Pyrus× bretschneideri），while the MdWRKY127 protein is closely related to an incompletely annotated protein in apple. Subcellular localization analysis indicated that both MdWRKY33 and MdWRKY127 proteins are located in the nucleus，exhibiting the typical subcellular localization characteristics of WRKY transcription factors. To verify their functions，overexpression experiments were carried out using apple calli as the material. The results showed that overexpressing either MdWRKY33or MdWRKY127gene significantly reduced the content of malic acid and citric acid in the calli，while increasing the accumulation levels of fructose and sucrose，resulting in a higher sugar-acid ratio in the calli. Further transient injection experiments in apple fruits showed that the overexpression results were consistent with those in the calli，whereas silencing either gene led to a decrease in the sugar-acid ratio in the fruit. These results indicate that both MdWRKY33 and MdWRKY127 are involved in regulating sugar and acid metabolism in apple fruit，thereby effectively improving the flavor of apple fruit.

Key words: apple, WRKY, fruit flavor, soluble sugars, organic acids, sugar-acid ratio, overexpression, calli

苏安琪, 庄英钰, 杜连达, 裴翡翡, 陈泽, 赵婷婷, 胡大刚. MdWRKY33和MdWRKY127调控苹果果实糖酸代谢提高糖酸比[J]. 园艺学报, 2026, 53(7): 1991-2003.

SU Anqi, ZHUANG Yingyu, DU Lianda, PEI Feifei, CHEN Ze, ZHAO Tingting, HU Dagang. MdWRKY33 and MdWRKY127 Regulate Sugar and Organic Acid Metabolism to Improve the Sugar-Acid Ratio in Apple Fruit[J]. Acta Horticulturae Sinica, 2026, 53(7): 1991-2003.

图/表 10

表1 本研究中所用的引物序列

Table 1 Primers used in this study

引物用途Primer application	引物名称 Primer name	引物序列（5′-3′）Primer sequence
基因克隆 Gene cloning	MdWRKY33-F	ATGACGGTGGAGCTGATGGAAT
	MdWRKY33-R	CGTTGACTCGTACACCAACCCC
	MdWRKY127-F	ATGGCTATAGATCTAGTTGGCTTCT
	MdWRKY127-R	GGACGACTGGAAAACAAGGCC
亚细胞定位及愈伤过表达 Subcellular localization and callus overexpression	MdWRKY33-3HA-F	GGTACCCGGGGATCCATGACGGTGGAGCTGATGGAAT
	MdWRKY33-3HA-R	CACCATGGTGTCGACCGTTGACTCGTACACCAACCCC
	MdWRKY127-3HA-F	GGTACCCGGGGATCCATGGCTATAGATCTAGTTGGCTTCT
	MdWRKY127-3HA-R	CACCATGGTGTCGACGGACGACTGGAAAACAAGGCC
实时荧光定量PCR qRT-PCR	qMdWRKY33-F	GAGATGGCAGCGTTTCGAAC
	qMdWRKY33-R	CCGTTGAGAGAGGAGGCTTC
	qMdWRKY127-F	ATCGATTTCTGTGCCTACGC
	qMdWRKY127-R	ACTCTGATCGTCCTCTTAACCT
内参基因 Reference gene	18S-F	ACACGGGGAGGTAGTGACAA
内参基因 Reference gene	18S-R	CCTCCAATGGATCCTCGTTA
基因过表达 Gene overexpression	MdWRKY33-pIR-F	TCTGAATTCGTCGACAAGCTTATGACGGTGGAGCTGATGGAAT
	MdWRKY33-pIR-R	CCCCCACACGTGTGGTCTAGACGTTGACTCGTACACCAACCCC
	MdWRKY127-pIR-F	TCTGAATTCGTCGACAAGCTTATGGCTATAGATCTAGTTGGCTTCT
	MdWRKY127-pIR-R	CCCCCACACGTGTGGTCTAGAGGACGACTGGAAAACAAGGCC
基因沉默 Gene silencing	MdWRKY33-TRV-F	AGAAGGCCTCCATGGGGATCCATGACGGTGGAGCTGATGGAATT
	MdWRKY33-TRV-R	TGTCTTCGGGACATGCCCGGGGGAGACGGTGTGATCGGTGATG
	MdWRKY127-TRV-F	AGAAGGCCTCCATGGGGATCCATGGCTATAGATCTAGTTGGCTTCT
	MdWRKY127-TRV-R	TGTCTTCGGGACATGCCCGGGGTGCTTGAACTTGGTGACGGTGA

图1 ‘绿袖’苹果花后16、41、70、94和128 d果实MdWRKY33与MdWRKY127基因表达量热图

Fig. 1 Heat map of MdWRKY33 and MdWRKY127 gene expression in‘Greensleeves’apple fruits at 16，41，70，94，and 128 days after flowering n = 3

图2 苹果MdWRKY33及MdWRKY127基因结构分析黑色部分表示外显子，黑线（intron）表示内含子，白色部分代表非翻译区

Fig. 2 Gene structural analysis of MdWRKY33 and MdWRKY127 in apple The black part represents the exon，the black line（intron）represents the intron，and the white part represents the untranslated region

图3 苹果MdWRKY33及MdWRKY127蛋白系统发育进化树

Fig. 3 Phylogenetic tree of MdWRKY33 and MdWRKY127 proteins in apple

图4 苹果MdWRKY33、MdWRKY127蛋白及其他物种同源蛋白多序列比对红色框线表示WRKY结构域

Fig. 4 Multiple sequence alignment of apple MdWRKY33，MdWRKY127 proteins and their homologous proteins from other species The red box shows the WRKY domain

图5 苹果MdWRKY33及MdWRKY127蛋白的亚细胞定位

Fig. 5 Subcellular localization of apple MdWRKY33 and MdWRKY127 proteins

图6 苹果MdWRKY33及MdWRKY127转基因愈伤的表型（A）、表达量（B）及鉴定（C） WT为野生型对照。用Duncan’s 进行显著性分析，不同小写字母表示差异显著（P < 0.05）。下同

Fig. 6 Phenotypes（A），gene expression levels（B）and molecular identification（C）of apple MdWRKY33 and MdWRKY127 transgenic calli WT represents wild-type control. Duncan’s multiple range test was used for significance analysis，and different lowercase letters indicate significant differences at P < 0.05.The same below

图7 苹果MdWRKY33（A）及MdWRKY127（B）转基因愈伤的糖酸含量

Fig. 7 Sugar and organic acid contents in apple transgenic calli overexpressing MdWRKY33（A）and MdWRKY127（B）

图8 MdWRKY33（A）及MdWRKY127（B）瞬时注射苹果的表型及表达量 1：pIR + TRV（对照1）；2：MdWRKY33-pIR + TRV；3：pIR + MdWRKY33-TRV；4：pIR + TRV（对照2）；5：MdWRKY127-pIR + TRV；6：pIR + MdWRKY127-TRV. 下同

Fig. 8 Phenotype and identification of transiently injected MdWRKY33（A）and MdWRKY127（B）in apples 1：pIR + TRV（Control 1）；2：MdWRKY33-pIR + TRV；3：pIR + MdWRKY33-TRV；4：pIR + TRV（Control 2）；5：MdWRKY127-pIR + TRV；6：pIR + MdWRKY127-TRV. The same below

图9 MdWRKY33（A）及MdWRKY127（B）瞬时注射苹果的糖酸含量

Fig. 9 Sugar and acid content of transiently injected MdWRKY33（A）and MdWRKY127（B）in apples

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