Genome-Wide Identification and Characterization of Melon bHLH Transcription Factors in Regulation of Fruit Development

Abstract

The basic helix-loop-helix (bHLH) transcription factor family is one of the largest transcription factor families in plants and plays crucial roles in plant development. Melon is an important horticultural plant as well as an attractive model plant for studying fruit ripening. However, the bHLH gene family of melon has not yet been identified, and its functions in fruit growth and ripening are seldom researched. In this study, 118 bHLH genes were identified in the melon genome. These CmbHLH genes were unevenly distributed on chromosomes 1 to 12, and five CmbHLHs were tandem repeat on chromosomes 4 and 8. There were 13 intron distribution patterns among the CmbHLH genes. Phylogenetic analysis illustrated that these CmbHLHs could be classified into 16 subfamilies. Expression patterns of the CmbHLH genes were studied using transcriptome data. Tissue specific expression of the CmbHLH32 gene was analysed by quantitative RT-PCR. The results showed that the CmbHLH32 gene was highly expressed in female flower and early developmental stage fruit. Transgenic melon lines overexpressing CmbHLH32 were generated, and overexpression of CmbHLH32 resulted in early fruit ripening compared to wild type. The CmbHLH transcription factor family was identified and analysed for the first time in melon, and overexpression of CmbHLH32 affected the ripening time of melon fruit. These findings laid a foundation for further study on the role of bHLH family members in the growth and development of melon.

Keywords: CmbHLH32; bHLH; fruit ripening; melon.

Figure 1

Sequence logo of bHLH domain of CmbHLH. The high letter represents the conservation of the sequence at that site. Stars represent the conservation sites of bHLH in Arabidopsis. Bold letters below represent the conservation sites of the bHLH domain that have a consensus ratio higher than 50%.

Figure 2

The domain distribution and types of CmbHLH.

Figure 3

ML phylogenetic tree of CmbHLHs with predicted DNA-binding activities. The tree shows the 16 phylogenetic subfamilies marked with different colored backgrounds. The different colored dots indicate four bHLH DNA binding activties groups, A (yellow), B (red), C (blue) and D (green), according to Atchley (2003).

Figure 4

Sequence logo and arrangement of conserved motifs, gene structure and intron pattern of CmbHLH. (A) Sequence logo of 10 conserved motifs of CmbHLH. (B) Arrengement of conserved motifs. Motifs labeled with 1 to 10 in different colores. (C) Intron-exon structure of CmbHLH. The exon, intron and UTR are represented by green rectangle, lines and yellow rectangle, respectively; the numbers (0–3) represent the intron phase. (D) Intron distribution pattern of bHLH of CmbHLH. The bars (labeled from I to XIII) illustrate the intron distribution pattern of the coding sequence of the bHLH domain within CmbHLHs. The arrows indicate the intron positions, and the numbers (0–3) above the arrows represent the intron phase. The count and percentage of genes in each pattern are shown on the right.

Figure 5

Chromosomal distribution and tandem repeat gene of CmbHLH. Blue characters indicate tandem repeat genes.

Figure 6

Collinearity analysis of CmbHLHs between melon and four Cucurbitaceae plants. The grey lines indicate the collinear blocks within melon and other plant genomes, and the numbers represent the chromosome numbers of plant genomes.

Figure 7

The differential expression profile of CmbHLH, different tissues and CmbHLH32-OE transgenic lines expression of CmbHLH32. (A) Heatmap of CmbHLH differential expression genes. (B) Quantitative PCR analysis of transcription levels of the CmbHLH32 gene in different tissues. The root was normalized to 1. (C) Fruit ripening of CmbHLH32-OE transgenic lines were earlier than that of the WT. (D) The relative expression level of CmbHLH32-OE transgenic lines and WT fruit. Quantitative PCR expression level was calculated by 2^−ΔΔCT method, and the results are represented as the means ± standard deviations, ** p ˂ 0.01, N > 3.

Figure 8

Transcriptional activation activity of CmbHLH32 and GO annotation of correlation expression genes of CmbHLH32. (A) Transcriptional activation activity of CmbHLH32. Full-length of CmbHLH32 was fused to the GAL4 domain in the pGADT7-T vector and cotransformed with the pGBKT7-53 vector for monomer transcriptional activation activity of CmbHLH32. For homodimerization transcriptional activation activity tests, Full-length of CmbHLH32 fused to pGADT7 and pGBKT7 was cotransformed into the yeast AH109 strain. The growth performances of the transformed yeast cells were exhibited in different dilutions on SD/-Trp/-His/Ade/3-AT solid medium. (B) GO annotation of correlation expression genes of CmbHLH32 genes.