Adβgal-1和Adβgal-2克隆及其在猕猴桃果实软化中的作用

doi:10.3864/j.issn.0578-1752.2019.02.010

摘要/Abstract

摘要：

【目的】 β-半乳糖苷酶是一类参与细胞壁降解的糖苷酶,在植物的生长发育和果实成熟软化过程中发挥重要作用。通过对猕猴桃2个β-半乳糖苷酶基因的鉴定及其表达模式研究,明确它们在猕猴桃果实软化中的作用,丰富猕猴桃的后熟软化机理研究。【方法】以‘米良1号’猕猴桃为试材,采用RT-PCR法扩增果实的β-半乳糖苷酶基因,利用在线数据库分析其编码蛋白的特性、功能结构域、系统进化关系、基因结构和调控miRNA,应用qPCR研究其在不同组织部位、果实的不同软化时期、不同贮藏温度和ABA处理后的表达特征,并结合酶活性变化,阐释这2个β-半乳糖苷酶基因在猕猴桃果实软化中的作用。【结果】克隆得到2个猕猴桃β-半乳糖苷酶基因（Adβgal-1和Adβgal-2）,登录号分别为MH319788和MH319789。Adβgal-1的开放阅读框为2 280 bp,编码759个氨基酸;Adβgal-2的开放阅读框为2 025 bp,编码674个氨基酸。结构域分析显示,Adβgal-1和Adβgal-2均含有植物糖苷水解酶家族35的功能结构域（Glyco_hydro_35）。基因结构分析表明,Adβgal-1由18个外显子和17个内含子组成,而Adβgal-2由17个外显子和16个内含子组成。进化树分析显示它们位于两个不同的进化分支中,且序列差异较大,可能源自不同的基因祖先。qPCR分析结果表明,Adβgal-1和Adβgal-2在猕猴桃的各组织部位（根、茎、叶、花、幼果、成熟果）均有表达,但表达水平不同;它们均在果实软化初期上调表达,随着果实硬度的下降,表达量持续增加;在25℃贮藏过程中,它们均在3 d时上调表达,随着时间的推移,表达量增加;而在4℃贮藏过程中,Adβgal-1的表达受到抑制,Adβgal-2的表达呈波浪式;ABA处理诱导Adβgal-1和Adβgal-2的表达。酶活性测定结果显示,β-半乳糖苷酶活性在果实软化初期略有降低,随着果实硬度的下降逐渐升高。【结论】 Adβgal-1和Adβgal-2均参与猕猴桃果实的软化进程。不同贮藏温度和ABA处理对猕猴桃果实软化的影响可能是通过改变β-半乳糖苷酶基因的表达而实现的。

关键词: 猕猴桃, β-半乳糖苷酶基因;, 果实软化, 酶活性, 基因表达

Abstract:

【Objective】 Beta-galactosidase is one type of glycosidases involving in cell wall degradation, which plays an important role in plant growth and fruit ripening and softening. In this paper, the identification and expression analysis of two β-galactosidase genes in kiwifruit, as well as their functions during fruit softening, were carried out to provide new insights for the mechanism of kiwifruit softening.【Method】 Actinidia chinensis var. deliciosa ‘Miliang-1’ was used as material to amplify the β-galactosidase genes by RT-PCR, then their characteristics, functional domains, phylogenetic relationships, gene structures and miRNAs were analyzed by using online databases. qRCR was applied to analyze their expression patterns in different tissues, different stages of softening, different storage temperatures, and ABA treatment. Enzymes activities were also analyzed to clarify the function of these two β-galactosidase genes during fruit softening.【Results】Two β-galactosidase genes (Adβgal-1 and Adβgal-2) were obtained from kiwifruit. Their accession numbers were MH319788 and MH319789, respectively. Adβgal-1 had an open reading frame (ORF) of 2 280 bp and encoded 759 amino acids, while the ORF of Adβgal-2 was 2 025 bp encoding 674 amino acids. Protein domain analysis showed that both Adβgal-1 and Adβgal-2 harbored a functional domain (Glyco_hydro_35) of plant glycoside hydrolase family 35. Gene organization analysis revealed that Adβgal-1 had 18 exons and 17 introns, while Adβgal-2 harbored 17 exons and 16 introns. Moreover, Adβgal-1 and Adβgal-2 were clustered in different evolutionary branching in the phylogenetic tree, which was due to the great difference in their sequences. This implied that Adβgal-1 and Adβgal-2 might originate from different ancestral genes. qPCR analysis showed that Adβgal-1 and Adβgal-2 were expressed in all tested tissues (root, stem, leaf, flower, young fruit and ripe fruit) although their expression levels were different. The expression levels of both Adβgal-1 and Adβgal-2 were increased at the start stage of fruit softening, and the amount of gene expression continued to increase as the firmness of fruit decreased. When stored at 25℃, both the Adβgal-1 and Adβgal-2 were upregulated at 3 d, and the amount of gene expression continued to increase as time over. When stored at 4℃, the expression of Adβgal-1 was inhabited, while that of Adβgal-2 was like wavy. ABA induced the expression of Adβgal-1 and Adβgal-2.【Conclusion】 Both Adβgal-1 and Adβgal-2 participated in the process of fruit softening of kiwifruit. The effect of storage temperatures and ABA on fruit softening might be achieved by changing the expression of β-galactosidase genes.

Key words: Actinidia chinensis, beta-galactosidase genes, fruit softening, enzyme activity, gene expression

冯新,赖瑞联,高敏霞,陈文光,吴如健,陈义挺. Adβgal-1和Adβgal-2克隆及其在猕猴桃果实软化中的作用[J]. 中国农业科学, 2019, 52(2): 312-326.

FENG Xin,LAI RuiLian,GAO MinXia,CHEN WenGuang,WU RuJian,CHEN YiTing. Cloning of Adβgal-1 and Adβgal-2 Genes and Their Roles During Fruit Softening of Kiwifruit[J]. Scientia Agricultura Sinica, 2019, 52(2): 312-326.

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