Genome Structures and Evolution Analysis of Hsp90 Gene Family in Brassica napus Reveal the Possible Roles of Members in Response to Salt Stress and the Infection of Sclerotinia sclerotiorum

Abstract

Heat shock proteins 90 (Hsp90s) are conserved proteins participating in the responses to heat stress and are found to be involved in different kinds of abiotic and biotic stresses. Brassica napus (B. napus) is an important heteropolyploid crop, producing edible oil. Salt stress is one of the most important hazards to the growth of rape in the world, while Sclerotinia stem rot is one of the most serious diseases, caused by Sclerotinia sclerotiorum (S. sclerotiorum). In this study, the evolution of Hsp90 genes and their responses to these two stresses were elucidated. Bioinformatic analysis through the whole genome of B. napus identified 35 Hsp90 gene family members. Five groups were obtained via phylogenetic analysis with the 35 Hsp genes, Hsps from its two ancestor species Brassica rapa, Brassica oleracea, and AtHsps. Gene structure and conservative motif analysis of these 35 Hsps indicated that the Hsps were relatively conservative in each group. Strong collinearity was also detected between the genomes of Brassica rapa, Brassica oleracea and B. napus, along with identifying syntenic gene pairs of Hsps among the three genomes. In addition, whole genome duplication was discovered as the main reason for the generation of BnHsp gene family. The analysis of cis-acting elements indicated that BnHsp90 might be involved in a variety of abiotic and biotic stress responses. Analysis of the expression pattern indicated that BnHsp90 participates in the responses of B. napus to salt stress and the infection of S. sclerotiorum. Fourteen and nine BnHsp90s were validated to be involved in the defense responses of B. napus against salt stress and S. sclerotiorum, respectively. Our results provide new insights for the roles of BnHsp90s in the responses of B. napus to salt stress and S. sclerotiorum.

Keywords: Brassica napus; Hsp90; Sclerotinia sclerotiorum; heat shock protein; salt stress.

Figure 1

Phylogenetic tree of Hsp90 proteins from A. thaliana, B. rapa, B. oleracea, and B. napus. The Hsp90s were divided into five groups (groups I–V) based on the clustering of the protein sequence. The proteins from A. thaliana, B. rape, B. oleracea and B. napus are presented in light green, blue, dark green, and orange, respectively. The IDs of proteins from Brassica napus, Brassica rapa, Brassica oleracea, and Arabidopsis thaliana start with Bn, Bra, Bol, and AT, respectively. Branch length is indicated on each branch.

Figure 2

Gene structure and motif analysis of BnHSP90s. (A) left, Intron and exon structure of the B. napus BnHsp90 genes according to the phylogenetic relationships. The untranslated region (UTR) and exon are indicated as green and pink boxes, respectively. The gray line represents introns. A, right, Distribution of the BnHsp90 conserved motifs in B. napus. There are five types of conserved motifs, which are represented by light green, purple, orange, red, and yellow. (B) MEME motifs are stacked by letters at each investigated site. The x-axis represents the width of the motif, whereas the y-axis represents the frequency of each letter.

Figure 3

Syntenic relationships of Hsp90s among A. thaliana, B. rapa, B. oleracea, and B. napus. (A) Hsp90 genes are plotted against their predicted counterparts in the four species. The chromosomes of A. thaliana are displayed as number 1–5, and the chromosomes of B. napus are showed in the form that symbol starting with A represents the chromosome originating from B. rapa, and symbol starting with C denotes the chromosome from B. oleracea. The chromosomes of B. rapa and B. oleracea are named using symbols beginning with A and C, respectively. The grey lines indicate the collinear blocks between different species. The yellow lines represent the collinear Hsp90 gene pairs between A. thaliana and B. napus. The blue lines denote the collinear Hsp90 gene pairs between B. napus and B. rapa, and the orange lines display the collinear gene pairs between B. napus and B. oleracea. (B) The collinear gene pairs of BnHsp90s on B. napus chromosomes. Red lines were used to link a pair of collinear genes. The ID of each chromosome was represented using “chr” followed by its ID, and the name of each gene was indicated on the corresponding chromosome.

Figure 4

Predicted stress-related cis-elements in BnHsp90 promoters. Promoter sequences (2,000 bp upstream region) of 35 BnHsp90 genes were analyzed online at PlantCARE web server. Different colors were used for indicating different cis-elements, as shown on the right part.

Figure 5

Expression changes of BnHsp90 genes in Brassica napus after treatment with salt. (A) Differential expression of Hsp90 gene in rapeseed after salt stress from public dataset (SRA datasets, PRJNA561674). The variety used in this research is Zhongshuang No. 11. LC, leaf control group; LN, leaf treatment group; RC, root control group; RN, root treatment group. (B) Expression patterns of BnHsp90s in response to NaCl treatments. Transcript levels of BnHsp90s were analyzed by quantitative real-time PCR using actin gene as an internal control. The unstressed expression level (0 h) was regarded as a standard. Values are the mean ± SE, n = 3.

Figure 6

Expression of BnHsp90 genes in Brassica napus infected by Sclerotinia slcerotiorum. (A) Expression changes of BnHsp90 genes after Sclerotinia sclerotiorum infection from public dataset (GEO RNA-seq datasets, GSE81545). The materials used are Westar and ZY821. The treatments are divided into 0 h and 24 h post inoculation. (B) Expression patterns of BnHsp90s in response to Sclerotinia sclerotiorum. Transcript levels of BnHsp90s were analyzed by quantitative real-time PCR using actin gene as an internal control. The non-inoculated expression level (0 h) was regarded as a standard. Values are the mean ± SE, n = 3.