Genome-wide identification and characterization of bHLH family genes from Ginkgo biloba

Abstract

Basic helix-loop-helix (bHLH) proteins, one of the most important and largest transcription factor family in plants, play important roles in regulating growth and development, stress response. In recent years, many bHLH family genes have been identified and characterized in woody plants. However, a systematic analysis of the bHLH gene family has not been reported in Ginkgo biloba, the oldest relic plant species. In this study, we identifed a total of 85 GbbHLH genes from the genomic and transcriptomic databases of G. biloba, which were classified into 17 subfamilies based on the phylogenetic analysis. Gene structures analysis indicated that the number of exon-intron range in GbbHLHs from 0 to 12. The MEME analysis showed that two conserved motifs, motif 1 and motif 2, distributed in most GbbHLH protein. Subcellular localization analysis exhibited that most GbbHLHs located in nucleus and a few GbbHLHs were distributed in chloroplast, plasma membrane and peroxisome. Promoter cis-element analysis revealed that most of the GbbHLH genes contained abundant cis-elements that involved in plant growth and development, secondary metabolism biosynthesis, various abiotic stresses response. In addition, correlation analysis between gene expression and flavonoid content screened seven candidate GbbHLH genes involved in flavonoid biosynthesis, providing the targeted gene encoding transcript factor for increase the flavonoid production through genetic engineering in G. biloba.

Figure 1

Multiple alignment of conserved domain amino acid sequences of multiple bHLH proteins from G. biloba. (A) Multiple sequence alignment of convserved bHLH domain of bHLH proteins from G. biloba. Alignment was carried out using Clustal W and represented by Adobe ExtendScript Toolkit CS6. (B) Analysis of bHLH domain motif by TBtools. Highly conserved amino acid residues in the bHLH domain across all GbbHLHs. The conservation of the sequence at that position was represents height of each stack.

Figure 2

Phylogenetic tree constructed using the sequences of bHLH domain proteins from Arabidopsis thaliana, Manus domestica and G. biloba. The tree was generated using Clustal X2 and MEGA 6 by using neighbor-joining method with 1,000 bootstrap replicates. All bHLH genes are clustered into subclades based on the priority classification rule of Arabidopsis bHLH genes.

Figure 3

Phylogenetic relationship and gene structure analysis of bHLH genes in G. biloba. (A) phylogenetic tree was constructed from the alignment of amino acid sequencing of selected bHLH proteins from G. biloba. (B) Gene structure analysis of selected bHLH genes of G. biloba, showing locations and lengths of the exons and introns. Exons and introns are presented as filled yellow round-corner rectangle and thin single lines, respectively.

Figure 4

Motif composition and distribution of 85 bHLH proteins in G. biloba. The motifs of the GbbHLH proteins were analyzed using the MEME web server. The length of the black line indicates the length of a sequence relative to all the other sequences. The position of each block indicates the location of a motif with a matching sequence.

Figure 5

The Go annotation of bHLH genes in G. biloba. All annotated GO terms including biological process, cellular component and molecular function of 85 GbbHLHs.

Figure 6

Cis-element analysis of 74 bHLH gene promoters in G. biloba. The potential cis-regulatory elements in the promoter regions 2,000 bp upstream of the G. biloba were predicted by PlantCARE software. Different colors indicated the elements related to growth and development (circadian control), plant hormones (abscisic acid, auxin, methyl jasmonate, gibberellic acid, and salicylic acid) and stress responsiveness (anaerobic induction, light, low temperature, and drought inducibility).

Figure 7

Functional regulatory network of 85 G. biloba bHLH proteins. The protein–protein interaction of bHLH proteins was predicted using STRING software. Cyan line presents data from curated databases, purple line experimentally determined, green line gene neighborhood, red line gene fusions, blue line gene co-occurrence; yellow line presents text mining, black line co-expression and gray line protein homology.

Figure 8

Expression of GbbHLH genes and correlation analysis between expression level of GbbHLH genes and the content of flavonoids in different organs of G. biloba. (A) A heatmap shows expression level of 80 GbbHLH genes with different subfamilies (left column) in different organs (bottom row) of G. biloba. Expression differences are observed in different colors. The R, S, IL, ML, M, OS, IF and MF represent root, stem, immature leaf, mature leaf, microstrobilus, ovulate strobilus, immature fruit and mature fruit, respectively. Changes in expression level are indicated by a change in color; green indicates a lower expression level, whereas red indicates a higher expression level. All data shown reflect the average mean of three biological replicates (n = 3). (B): Correlation analysis between the expression level of selected 7 GbbHLH genes and the content of flavonoids and in different organs of G. biloba.

Figure 9

Chromosomal distribution and regional duplication of 82 bHLH genes of G. biloba. The scale bar on the left indicated the length (Mb) of ginkgo chromosomes.