Comparative analysis of B-BOX genes and their expression pattern analysis under various treatments in Dendrobium officinale

Abstract

Background: Studies have demonstrated that BBX (B-BOX) genes play crucial roles in regulatory networks controlling plant growth, developmental processes and stress response. Nevertheless, comprehensive study of BBX genes in orchids (Orchidaceae) is not well studied. The newly released genome sequences of Dendrobium officinale and Phalaenopsis equestris have allowed a systematic analysis of these important BBX genes in orchids.

Results: Here we identified 19 (DoBBX01-19) and 16 (PeBBX01-16) BBX genes from D. officinale and P. equestris, respectively, and clustered into five clades (I-V) according to phylogenetic analysis. Thirteen orthologous, two DoBBXs paralogous and two PeBBXs paralogous gene pairs were validated. This gene family mainly underwent purifying selection, but five domains experienced positive selection during evolution. Noteworthy, the expression patterns of root, root_tips, stem, leaf, speal, column, lip, and flower_buds revealed that they might contribution to the formation of these tissues. According to the cis-regulatory elements analysis of BBX genes, qRT-PCR experiments were carried out using D. officinale PLBs (protocorm-like bodies) and displayed that these BBX genes were differentially regulated under AgNO₃, MeJA (Methyl Jasmonate), ABA (abscisic acid) and SA (salicylic acid) treatments.

Conclusions: Our analysis exposed that DoBBX genes play significant roles in plant growth and development, and response to different environmental stress conditions of D. officinale, which provide aid in the selection of appropriate candidate genes for further functional characterization of BBX genes in plants.

Keywords: Abiotic stress; BBX; D. officinale; Evolution; qRT-PCR.

Fig. 1

Domain composition of DoBBX and PeBBX proteins. a, b and c indicate the amino acid sequence alignment of the B-BOX 1, B-BOX 2 and CCT domain, respectively. The y-axis and x-axis indicated the conservation rate of each amino acid and the conserved sequences of the domain, respectively

Fig. 2

Phylogenetic relationships and domain structures of the BBX genes. a. Phylogenetic analysis of BBX genes in Populus trichocarpa, A. thaliana, O. sativa, D. officinale and P. equestris, (b). Domain structures of the BBX proteins in D. officinale and P. equestris. CCT and BBX indicated CCT and B-BOX domains, respectively. c. A hypothetic model for the BBX evolutionary trajectory in D. officinale and P. equestris. B1, B2, CCT indicated B-Box 1, B-Box 2, and CCT domains, respectively

Fig. 3

Species used in this study (a) and Maximum-likelihood gene tree (b) for the BBX gene family. The HMM (hidden Markov model) profile of BBX domain (Pfam00643) was used to identify for all of BBXs in these plant genomes with HMMER 3.0 software. Each putative BBX protein was further examined the presence of B-BOX domain by submitting them to SMART database, Pfam and InterProScan, respectively. The Maximum likelihood (ML) tree was generated by FastTree software with JTT model. A total of 1340 BBX genes were divided into four clades and were distinguished by different colors and legends

Fig. 4

The analysis of homologous gene pairs in the BBX gene family. a Identification of paralogs and orthologs in D. officinale and P. equestris. Green, bule and red lines indicated orthologs gene pairs (Do-Pe), paralogs gene pairs (Pe-Pe), and paralogs gene pairs (Do-Do), respectively. b and c The distribution of Ka/Ks, Ks, Ka values of homologous gene pairs. The slopes of Ka/Ks =1 and 0.5 were represented by black and gray lines, respectively

Fig. 5

Investigation of cis-acting element numbers in all BBX genes of D. officinale and P. equestris. a The different colors and numbers of the grid indicated the numbers of different promoter elements in these BBX genes. b The different colored histogram represented the sum of the cis-acting elements in each category. c Pie charts of different sizes indicated the ratio of each promoter element in each category, respectively

Fig. 6

Expression pattern of DoBBX genes. a Organ-specific expression pattern of DoBBX genes in eight tissues: root, root_tips, stem, leaf, speal, column, lip and flower_buds. Blue and red indicated lower and higher transcript abundance, respectively. b Identification of highly expressed BBX genes in D. officinale. According to the previous studies [22], yellow, green, blue, red indicated low (1–6.8 FPKM), mid-low (6.8–17.5 FPKM), mid-high (17.5–44.7 FPKM), and high (44.7–17,092 FPKM) expression, respectively. c Venn diagram of DoBBX genes in different tissues

Fig. 7

Expression patterns of DoBBX genes in D. officinale under ABA treatment stresses as determined by qRT-PCR experiment. The x-axis indicated the time course of each stress treatment, and the y-axis represented the relative expression level. Mean values and standard deviations (SDs) indicated by error bars. ** significant difference (P < 0.01), * significant difference at P < 0.05

Fig. 8

Expression patterns of DoBBX genes in D. officinale under SA treatment stresses as determined by qRT-PCR experiment. The x-axis indicated the time course of each stress treatment, and the y-axis represented the relative expression level. Mean values and standard deviations (SDs) indicated by error bars. ** significant difference (P < 0.01), * significant difference at P < 0.05

Fig. 9

Expression patterns of DoBBX genes in D. officinale under AgNO₃ treatment stresses as determined by qRT-PCR experiment. The x-axis indicated the time course of each stress treatment, and the y-axis represented the relative expression level. Mean values and standard deviations (SDs) indicated by error bars. ** significant difference (P < 0.01), * significant difference at P < 0.05

Fig. 10

Expression patterns of DoBBX genes in D. officinale under MeJA treatment stresses as determined by qRT-PCR experiment. The x-axis indicated the time course of each stress treatment, and the y-axis represented the relative expression level. Mean values and standard deviations (SDs) indicated by error bars. ** significant difference (P < 0.01), * significant difference at P < 0.05