Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (2): 312-326.doi: 10.3864/j.issn.0578-1752.2019.02.010

• HORTICULTURE • Previous Articles     Next Articles

Cloning of Adβgal-1 and Adβgal-2 Genes and Their Roles During Fruit Softening of Kiwifruit

FENG Xin,LAI RuiLian,GAO MinXia,CHEN WenGuang,WU RuJian,CHEN YiTing()   

  1. Fruit Research Institute, Fujian Academy of Agricultural Sciences/Research Centre for Engineering Technology of Fujian Deciduous Fruits, Fuzhou 350013
  • Received:2018-07-01 Accepted:2018-11-12 Online:2019-01-16 Published:2019-01-21
  • Contact: YiTing CHEN


【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

Fig. 1

The softening stages of kiwifruit S1-S7: Ripe fruits with high hardness (control), start stage of softening, stage I of softening, stage II of softening, stage III of softening, late stage of softening, stage of soft and decay, bar: 1 cm. The same as bellow"

Fig. 2

The physiological and biochemical indexes of kiwifruit during fruit softening ** indicate statistically significant differences at P<0.01. The same as below"

Fig. 3

Sequence alignment of the ORF regions from Adβgal-1 and Ach04g123061"

Fig. 4

Sequence alignment of the ORF regions from Adβgal-2 and Ach09g294421"

Fig. 5

Multiple sequence alignments of the Adβgal and other plant beta-galactosidases Identical conserved residues are shaded in black, while similar residues are shaded in gray. The conserved motif of glycoside hydrolase family 35 is boxed. Ccβgal:克萊门柚Citrus clementina,XP006434246;Siβgal:芝麻Sesamum indicum,XP011069386;Muβgal:香蕉Musa acuminata,XP018676452;Ppβgal:碧桃Prunus persica XP007199661;Mnβgal:川桑Morus notabilis,XP010107841;Jrβgal:核桃Juglans regia,XP018840337"

Fig. 6

Phylogenetic tree of the beta-galactosidase proteins from various plant species Numbers at the nodes represent the reliability percent of bootstrap values on 1000 replications. The bar shows branch length"

Fig. 7

Genomic structure of the Adβgal genes A: The extron and intron organization of Adβgal genes. B: The length of extrons and introns of Adβgal genes. The length of introns was in brackets. The unit of length (bp)"

Fig. 8

Relative expression of the Adβgal genes in different tissues"

Fig. 9

Relative expression of the Adβgal genes during fruit softening"

Fig. 10

Relative expression of the Adβgal genes under different storage temperatures indicates statistically significant differences at P<0.05"

Fig. 11

Relative expression of the Adβgal genes after ABA treatment"

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