Genome-wide characterization and expression analysis of the heat shock transcription factor family in pumpkin (Cucurbita moschata)

Abstract

Background: Crop quality and yield are affected by abiotic and biotic stresses, and heat shock transcription factors (Hsfs) are considered to play important roles in regulating plant tolerance under various stresses. To investigate the response of Cucurbita moschata to abiotic stress, we analyzed the genome of C. moschata.

Results: In this research, a total of 36 C. moschata Hsf (CmHsf) members were identified and classified into three subfamilies (I, II, and III) according to their amino acid sequence identity. The Hsfs of the same subfamily usually exhibit a similar gene structure (intron-exon distribution) and conserved domains (DNA-binding and other functional domains). Chromosome localization analysis showed that the 36 CmHsfs were unevenly distributed on 18 of the 21 chromosomes (except for Cm_Chr00, Cm_Chr08 and Cm_Chr20), among which 18 genes formed 9 duplicated gene pairs that have undergone segmental duplication events. The Ka/Ks ratio showed that the duplicated CmHsfs have mainly experienced strong purifying selection. High-level synteny was observed between C. moschata and other Cucurbitaceae species.

Conclusions: The expression profile of CmHsfs in the roots, stems, cotyledons and true leaves revealed that the CmHsfs exhibit tissue specificity. The analysis of cis-acting elements and quantitative real-time polymerase chain reaction (qRT-PCR) revealed that some key CmHsfs were activated by cold stress, heat stress, hormones and salicylic acid. This study lays the foundation for revealing the role of CmHsfs in resistance to various stresses, which is of great significance for the selection of stress-tolerant C. moschata.

Keywords: Cis-acting elements; Conserved domain; Cucurbita moschata; Expression pattern; Gene duplication; Heat shock transcription factor.

Fig. 1

Classification and conserved motifs of 36 CmHsfs. a. The unrooted phylogenetic tree of 36 CmHsfs was constructed using the Neighbor-joining (NJ) method with 1000 bootstrap replicates, and a 60% cut-off value was used for the condensed tree. Three different subfamilies (I-III) were highlighted with different colored branch lines. b. Schematic representation of conserved motifs in 36 CmHsfs. Each motif was represented by a numbered colored box on the right. The same number in different proteins referred to the same motif. Motif 1, motif 2 and motif 3 together formed the DBD, and motif 4 formed the HR-A/B domain. The function of other motifs was unknown

Fig. 2

Exon-intron organization of 36 CmHsfs constructed by GSDS (Gene structure display server). The exons and introns were represented by pink boxes and grey lines, respectively. Untranslated regions (UTRs) were indicated by blue boxes. The sizes of the exons and introns can be estimated using the scale at the bottom

Fig. 3

Chromosomal distribution and duplication events of Hsf genes in C. moschata. The chromosomal locations of the CmHsf genes were mapped with visualization tools. The duplicated CmHsf genes were shown in blue boxes and black lines

Fig. 4

Phylogenetic trees of the Hsf gene family in C. moschata, C. sativa and A. thaliana. The 9 clades (Clade Ia-b, Clade II and Clade IIIa-e) were displayed with different background colors. The phylogenetic tree was constructed with MEGA 5.0 software using the Neighbor-joining (NJ) method with 1000 bootstrap replicates. Cm, C. moschata; Cs, C. sativa; At, A. thaliana

Fig. 5

Synteny analysis of the Hsf genes between C. moschata and five other species. The synteny relationship maps were constructed using the Advanced Circos program in TBtools. At, A. thaliana; Ls, L. siceraria; Cs, C. sativus; Cma, C. maxima; Cg, C. lanatus; Cmo, C. moschata. The gray lines in the background indicated the collinear blocks in the genome of C. moschata and other plants, while blue lines in the background highlighted syntenic Hsf gene pairs. All the data for the various species was extracted from Cucurbit genomics database

Fig. 6

Heat map and hierarchical clustering of 36 CmHsf genes in the roots, stems, cotyledons and true leaves. Quantitative real-time PCR was performed in three biological replicates and three technical replicates, and the heat map and hierarchical clustering were constructed by TBtools. The results were calculated via the 2^−ΔΔCt method, and the reference gene (β-Actin) was used to correct the expression level of target genes. All data were standardized by Log₁₀ (2^−ΔΔCt). The bar on the right of the heat map represented the data that has been converted to Log₁₀ (2 ^-ΔΔCt)

Fig. 7

Distribution of cis-acting elements in 36 CmHsfs and the proportions of corresponding genes in 9 stress response elements. a. The cis-acting elements of 36 heat shock transcription factors in C. moschata. They were predicted by PlantCare program (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and visualized by Simple BioSequence Viewer in TBtools. The squares on the right represented cis-acting elements that respond to a total of 9 stresses. Different colors indicated cis-acting elements that participate in different stresses. The coordinates at the bottom of the figure indicated the length of the gene promoter. The promoter sequence was defined as 2 kb before the start codon. b. The distribution of 429 cis-acting elements related to 9 abiotic stresses. c. The proportion of 36 CmHsfs related to 9 abiotic stresses

Fig. 8

Heat map and hierarchical clustering of 36 CmHsf genes in true leaves under cold stress and heat stress. Quantitative real-time PCR and hierarchical clustering were performed in three biological replicates and three technical replicates, and the heat map was constructed by TBtools. The results were calculated via the 2^−ΔΔCt method, and the reference gene (β-Actin) was used to correct the expression level of target genes. All data were standardized by Log₁₀ (2^−ΔΔCt). The bar on the right of the heat map represented the data that has been converted to Log₁₀ (2 ^-ΔΔCt)

Fig. 9

Expression profiles of 36 CmHsf genes in true leaves under MeJA, ABA and SA treatments. The data represented the expression levels of CmHsf genes at 10 h after the MeJA, ABA and SA treatments. CK referred to untreated plants (control plants) under normal conditions. The results were calculated via the 2^−ΔΔCt method, and the reference gene (β-Actin) was used to correct the expression level of target genes. The expression level of CK was set as 1. The data were presented as the means of three biological replicates and three technical replicates, and the error bars represented the standard deviations of the means. According to Welch’s t-test, different letters above the bars indicated significant differences (p < 0.05) between different treatments