Genome-Wide Identification and Expression Analysis of the Strawberry FvbZIP Gene Family and the Role of Key Gene FabZIP46 in Fruit Resistance to Gray Mold

Abstract

A total of 54 FvbZIP genes were identified from the strawberry genome. These genes were found to be unevenly distributed on seven different chromosomes, and two of the genes had no matching chromosomal localization. FvbZIP genes were divided into 10 subfamilies according to protein sequence, and the structures of these genes were found to be highly conserved. Based on the bioinformatics analysis of FvbZIP genes, the expression of FabZIP genes changed during different stages of its growth and of its infection with gray mold disease. FabZIP46 was substantially upregulated, and its expression remained relatively high. FabZIP46 was cloned from cultivated strawberries by homologous cloning. The results of a transient transgenic assay revealed that the damage to the fruit tissue was markedly alleviated in strawberries overexpressing FabZIP46, with the incidence rate being substantially lower than that in the control group. By contrast, a brief silencing of FabZIP46 had the opposite effect. The results revealed that FabZIP46 played a positive role in the resistance of strawberries to Botrytis cinerea. The study findings provide valuable insights into the role of bZIP transcription factors as well as a theoretical reference for the regulation of resistance to gray mold disease in strawberry fruit.

Keywords: FabZIP46; bZIP transcription factors; bioinformatics; gray mold disease; strawberry.

Figure 1

Phylogenetic analysis of bZIP genes. At represented A. thaliana, Os represented O. sativa. The phylogenetic tree was generated using the amino acid sequences of selected bZIP genes via NJ methods. All strawberry bZIP genes, together with their A. thaliana and O. sativa homologues, were classified into 10 groups.

Figure 2

Chromosome distribution of FvbZIP genes. The chromosome numbers are demonstrated at the top of each chromosome and the scale is in megabases (Mb).

Figure 3

Multiple alignments of conserved domains of all identified FvbZIP proteins. Asterisks show the conserved amino acids of the bZIP domain.

Figure 4

(a) Phylogenetic analysis of strawberry bZIP genes. The phylogenetic tree was generated using the amino acid sequences of bZIP proteins via NJ methods. All strawberry bZIP genes were classified into 10 groups. (b) Gene structure. Through the website Gene Structure Display Server, the whole genome sequence and CDS sequence of FvbZIP genes were formulated into an information map containing the structure of each FvbZIP gene. (c) Motif patterns of strawberry bZIP proteins. The protein sequence of all the strawberry bZIP proteins were entered into the MEME, with the highest motif number set to 20 and the other parameters set to the default.

Figure 5

(a) Photographs showing the different developmental stages of strawberries. The whole process of strawberry development was divided into SG, MG, BG, Wh, Tu, HF, and Re stages. Phenotypes of red fruit after Botrytis cinerea inoculation. (b) The expression of FabZIP genes in different strawberry growth stages and in different stages after Botrytis cinerea inoculation of red fruit.

Figure 6

(a) Manipulated FabZIP46 gene expression levels in strawberries, obtained using transient overexpression and gene silencing techniques. Green fluorescence emitted by transformed strawberries was detected at four days after injection, indicating that the FabZIP46 gene was overexpressed or silenced in strawberries. (b) Phenotypes of FabZIP46-OE and FabZIP46-RNAi strawberries after Agrobacterium tumefaciens injection and Botrytis cinerea inoculation. Strawberries at the red stage of development were injected with A. tumefaciens, and B. cinerea inoculation was performed four days after A. tumefaciens injection. To avoid tissue damage that might affect the experimental results, approximately 1 mL (depending on the fruit size) of A. tumefaciens suspension was evenly injected into the fruits through the pedicel with a sterile hypodermic syringe. The injection depth was approximately half of the longitudinal diameter of the fruit to ensure that the A. tumefaciens suspension was fully released in the pith of the fruit. The first vertical line, second vertical line, fourth vertical line, and fifth vertical line represent the phenotypes of non-transgenic control fruits and transgenic fruits with empty vectors. The third vertical line and sixth vertical line show the phenotypes of transgenic fruits with FabZIP46-OE and FabZIP46-RNAi, respectively. (c) Changes in relative FabZIP46 gene expression levels in FabZIP46-OE and FabZIP46-RNAi fruits. Values are means ± SD (n ≥ 3). Asterisks above the columns denote a significant difference at ** p < 0.05 levels according to the Student’s t-test. (d) Comparison of the incidence of FabZIP46-OE and FabZIP46-RNAi in strawberries after Botrytis cinerea inoculation.

Figure 7

Effects of FabZIP46-OE and FabZIP46-RNAi on the transcription of resistance-related genes. EV-OE and EV-RNAi denote controls for the overexpressed or silenced fruits transformed with the corresponding empty vector. Values are means ± SD (n ≥ 3). Asterisks above the columns denote significant difference at ** p < 0.05 levels according to the Student’s t-test.