Genome-wide identi cation and characterization of melon bHLH transcription factors in regulation of fruit development

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.

illustrated that these CmbHLHs could be classi ed into 16 subfamilies. Intron distribution pattern analysis of bHLH domain found 13 intron distribution patterns in CmbHLHs. CmbHLH genes were unevenly distributed on chromosomes 1 to 12 of the melon genome, and ve CmbHLH s were tandem repeat on chromosomes 4 and 8. Expression characters of CmbHLH genes were studied using the transcriptome data. Tissue analysis of indicated CmbHLH32 high expressed in female owers and early fruit growth stage. Transgenic plant lines of overexpression of CmbHLH32 were constructed, and overexpression of CmbHLH32 result in early fruit ripening compared to the wild type fruit.
Conclusions: The bHLH transcription factor family was identi ed and analyzed for the rst time in the melon, overexpression of CmbHLH32 will affect the ripening time of melon fruit, these ndings laid a theoretical foundation for further study on the role of bHLH family members in the growth and development of melon .

Background
Transcription factors (TFs) play important roles in regulating plant growth, development, stress response and signaling transduction [1][2][3][4]. Basic helix-loop-helix (bHLH) TFs is one of the largest TF superfamily in plant [5]. Studies on the bHLH gene family in various species will increase our understanding of their evolution and functions. So far, comprehensive identi cation of the bHLH gene family has performed in range of plant species. Such as, Arabidopsis, Brachypodium distachyon, Solanum lycopersicum, Arachis hypogae,Malus pumila [6][7][8][9][10]. Plant bHLH is characterized by a basic helix-loop-helix domain and the domain is highly conserved in evolution [11]. The bHLH domain contains 50-60 amino acids and can be separated into two regions: the region at N-terminal end is a DNA binding domain, comprises about 13-17 amino acid [5]; the C-terminal end of the region is HLH domain, containing two amphipathic α-helices connected by a loop region of variable length, which helps to form a dimerization domain, and allows the formation of homo-or hetero-dimeric complexes [12]. With regard to the bHLH proteins domain, 19 amino acids were conserved and functional for DNA binding or dimerization formation. A highly conserved HERmotif (His 5-Glu 9-Arg 13) was considered important for binding to speci c DNA sequences, however some atypical bHLH lack of the N-terminal binding region [13].
Phylogenic analysis of 544 plant bHLH shows that plant bHLH proteins formed 26 distinct subfamilies, and these subfamilies are highly conserved throughout plant evolution; among them, 20 subfamilies were present in early land plants 443 Ma [14]. However, the plant bHLH subfamilies lack of discernible phylogenetic relationships and possibly plant bHLH proteins are monophyletic [5].
Plant bHLH proteins involved in a wide arrange of biological processes, and participate in the regulation of plant growth and development, abiotic/biotic stress response, hormone signaling, iron homeostasis and secondary metabolism [15][16][17][18][19][20][21]. Nevertheless, researches on bHLH genes regulation in fruit growth and development were limited. Fruit growth is triggered by the fertilization of ovule, and changed drastically, these processes including cell division and expansion, secondary metabolites accumulation, carbohydrate biosynthesis, and involves many genes transcription and regulation [22,23]. To the climacteric fruit like melon, the ripening process also accompanies with increasing in ethylene emission and burst of respiratory climacteric [24]. Recent research on tomato reveals that overexpression of SlbHLH22 resulting in early owering time, accelerates fruit ripening, accumulation of carotenoid by activation of the SlSFT or SlLFY genes, and exogenous ACC, IAA, ABA, and ethephon would upregulate the expression of SlbHLH22 [25].
Melon (Cucumis mleo L.) is an important horticultural crop worldwide, in 2017 whole world production of melon was more than 49 million tones, and China produced over one third of the melon (FAO). Melon is also an attractive model plant of the Cucurbitaceae family for studying the fruit development and ripening, especially for respiratory climacteric [26]. Although The bHLH genes have been suggested to being involved in a wide arrange of metabolic, physiological, and developmental processes in plant, very few studies efforts on bHLH genes in the regulation on fruit ripening. In this study, we identi ed the melon bHLH gene family, and using high-throughput sequencing data to research the gene expression of melon fruit growth, ripening, climacteric, and post-climacteric stage. Transgenic lines that overexpression of CmbHLH32 was generated to study the effect on fruit ripening, and yeast two hybrid was used to research the transcription activation activity of CmbHLH32. Our ndings shed light on the molecular properties and evolution patterns of the CmbHLH gene family and demonstrate biological function of CmbHLH32 gene in melon fruit growth and ripening.

Results
Identi cation of CmbHLH proteins and conserved domain alignment A total of 169 CmbHLH candidate protein sequences were obtained by HMM analysis, 213 protein sequences were found using blast method, then repetitive sequences were removed. The remaining sequences were searched against the CmbHLH proteins of the PlantTFDB database. After that 214 sequences were reserved and submitted to CDD domain search; then 159 sequences were found with a bHLH conserved domain above the minimum domain hit, and the redundant sequences of the 159 proteins were removed, nally, 118 sequences were left as the CmbHLHs gene models for the analysis and renamed based on their chromosome localization (Supplementary Table S1).
These putative CmbHLH lengths varied from 84 to 707 aa, the molecular weight ranges from 9.48 kDa to 75.97 kDa, and the theoretical isoelectric points (PI) range from 4.52 to 10.27 (Supplementary Table S1).
The CmbHLH proteins density (0.442) was lower than that in Cucumis sativus (0.527), which is also belongs to the Cucurbitaceae plant. The reason may be the smaller genome size of Cucumis sativus (203.0 Mb) compared to Cucumis melo (364.0 Mb) [27]. And the CmbHLH density was a little higher than that in Citrus sinensis (0.420) which has a similar genome size (Citrus sinensis, 367.0 Mb).
Multiple sequences alignment of the bHLH domain of CmbHLH proteins shows that 24 amino acid residues in their bHLH domains were conserved with more than 50% consensus ratio. The bHLH domain is highly conserved and comprises of two functionally distinct regions. The basic region of the bHLH domain determines the DNA-binding activity of target genes, gure 1 shows the bHLH domain logo of CmbHLH (Fig. 1), the basic region of CmbHLH proteins contains 5 conserved amino acids, and the HLH has 19 conserved amino acids. Previously described a classi cation of bHLH proteins that classi ed the bHLH proteins into four groups A, B, C and D. The classi cation was based on DNA-binding speci city as well as conservation of amino acids at certain positions [13]. According to the criterion, 8 CmbHLH classi ed into A group, 69 located in B group, 16 and 33 CmbHLH belonged to C and D group respectively.
Domain analysis of CmbHLH illustrated that there were two kinds of domains were found in CmbHLH except the bHLH domain (Fig. 2). One domain is bHLH_MYC_N, was found in 11 CmbHLH. All of the CmbHLHs bHLH_MYC_N domain located in the N-terminal of the proteins and bHLH domain in the Cterminal of proteins. Apart from the bHLH and bHLH_MYC_N domains, ACT (Aspartokinase, Chorismate, and TyrA) domain was also identi ed in eight CmbHLH (CmbHLH32, CmbHLH37, CmbHLH56, CmbHLH60, CmbHLH68, CmbHLH97, CmbHLH100, CmbHLH114), and all of the ACT domain located in the C terminal of the bHLH domain.

Phylogenetic, Motif Analysis and Gene Structure of CmbHLH
To evaluate the evolutionary relationships of the CmbHLH, we conducted a phylogenetic analysis based on full-length of protein sequences. Applying the ML method, we assigned the CmbHLH genes into 16 subfamilies and 4 orphan genes (Fig. 3). Subfamilies A, D and J were the largest groups, the smallest subfamily (L) had only 2 members. According to the phylogenetic tree, the CmbHLH binding activities was phylogenetically clustered which were consistent with the previously report. The evolutionary relationships of these CmbHLH proteins were also determined by conserved motifs. A total of 10 conserved motifs were characterized from CmbHLH proteins (Fig. 4A, B). Among these motifs, motif 1 and 2 were annotated to bHLH domain (IPR011598, IPR036638), motif 7 and 10 were annotated to transcription factor bHLH-MYC-N-terminal (IPR025610). The subfamily L contains the highest number of motifs (six motifs). CmbHLH14 and CmbHLH94 possess two and three motif-2 respectively. The motifs distribution and construction pattern exhibited similar model within subfamilies.
The exon-intron organizations of CmbHLH were examined to gain more insight into the evolution of the bHLH family in melon. The exon number of CmbHLH varied from 1 to 11 (Fig. 4C). Whereas, the exon-intron organizations were phylogenetically related. For example, The CmbHLH with one exon were clustered in two subfamilies (D and K), all the members of subfamily I have two exons. Intron distribution analysis of bHLH domain within all CmbHLH proteins exhibit 13 intron distribution patterns, and this pattern strongly related to the subfamilies of CmbHLH (Fig. 4D). As shown in gure 4, 85% of CmbHLH have intron insertion in their bHLH domain sequence region. Although the intron positions and lengths were varied, only ve intron insertion positions of CmbHLH were unconserved. Overall, the conserved motif arrangement and composition and the gene structure of CmbHLH genes, together with the phylogenetic analysis results, could strongly support the reliability of the classi cation.
Chromosomal distribution and collinearity analysis of CmbHLH CmbHLH genes were distributed unevenly among twelve chromosomes of melon (Fig. 5). However, the distribution of CmbHLH genes did not show either a chromosome length correlation or a phylogenetic correlation. Gene tandem duplication may involve in gene family enlargement and maintains of gene copy numbers. Thus, we analyzed the tandem duplication events of CmbHLH. Five genes were con rmed to be tandem duplicated genes. Three of them (CmbHLH81, CmbHLH82 and CmbHLH83) located on chromosome 8, and two of tandem duplicated genes (CmbHLH45 and CmbHLH44) located on chromosome 4.
To further infers the origin and phylogenetic relationships of bHLH genes, comparative collinearity analysis between Cucumis melo and other cucurbit species were conducted. Figure 6 displayed the collinearity relationship of CmbHLH genes with those in bottle gourd, cucumber, watermelon and Cucurbita maxima (Rimu) (Fig. 6). A total of 115 CmbHLH genes have orthologous in the four species, among them 95 CmbHLH genes were common in the four species. Interestingly, 43 CmbHLH genes have at least two orthologous in Rimu, and these genes spread out on the 12 chromosomes of melon. The reason maybe that Rimu genome underwent a whole-genome duplication (WGD) event, which was not observed in other four cucurbits (cucumber, melon, watermelon, and bitter gourd) [28]. Gene duplication events of CmbHLH in melon were also studied. Results show 38 CmbHLHs genes duplicated among CmbHLH genes (Supplementary Figure S1). Except chromosome 9, all the other chromosomes have duplication genes of CmbHLH. Most of the duplication genes were on chromosome 2, and 1 CmbHLH locates on chromosome 6, illustrating an uneven distribution of the duplication genes.

Expression pattern of CmbHLH in melon fruit development
Analysis of the expression data of PRJNA543288 exhibits 161 transcripts of 98 CmbHLH genes effectively expressed (expressed at least in two replicate libraries) in melon fruit Growth stage (G), Ripening stage (R), Climacteric stage (C), and Post-climacteric stage (P) samples. However, most of them were low expressed, 45 CmbHLH genes have an average expression higher than 10 FPKM, only 5 genes (CmbHLH23, CmbHLH32, CmbHLH41, CmbHLH67 and CmbHLH79) expression higher than 100 FPKM (Fig. 7A). Differential expression analysis shows 32 CmbHLH genes differentially expressed in G vs R, R vs C and C vs P stage samples. A total of 21 CmbHLH genes differentially expressed in G vs R samples (7 genes upregulated and 14 genes downregulated), CmbHLH32 was the highest expressed among these differential expression genes; 26 CmbHLH genes differentially expressed in R vs C samples, only 2 genes (CmbHLH9 and CmbHLH114) were up regulated, the others were down regulated. There were six CmbHLH genes differentially expressed in C vs P stage samples. Taken together, the expression of CmbHLH genes exhibits a down regulation trend from melon fruit G to P stage samples, suggesting most of the CmbHLH genes may function in early fruit developmental stage. Whereas, two genes (CmbHLH9 and CmbHLH114) up regulated in R vs C stage samples, indicating they may be involved in the regulation of fruit Climacteric.
Overexpression of CmbHLH32 leading to early ripen in melon fruit To further investigate the function of CmbHLH genes in fruit ripening, we generated the transgenic plant lines of overexpression CmbHLH32 (CmbHLH32-OE). The reasons for studying CmbHLH32 gene were: rst, CmbHLH32 was one of the highest expressed CmbHLH genes, second; CmbHLH32 was highest expressed among differential expression genes in G vs R stage samples; third, result in tissue expression analysis of CmbHLH32 illustrates CmbHLH32 was high expressed in female ower and early developmental stage of fruit (Fig. 7B); fourth, CmbHLH32 was identi ed homolog to AtbHLH93, which was proved to control Arabidopsis owering by repressing MAF5 [29]. However, blast analysis was failed to nd a homolog gene of MAF5 in melon, and CmbHLH32 also contains an ACT domain which is not nd in AtbHLH93, suggesting CmbHLH32 may have a different function in melon owering.
Transgenic T1 seeds that overexpression of CmbHLH32 was generated by the ovary injection method. Fruit ripening related phenotype observation of CmbHLH32-OE T1 plant that were PCR detection positive indicates that overexpression of CmbHLH32 results in early fruit ripening compared to the wild type (WT) melon fruit (Fig. 7C). The fruit ripening of CmbHLH32-OE line (about 38.7± 1.1 DAP) is in average 4 days earlier than that of WT melon fruit (about 42.6± 0.8 DAP). Quantitative RT-PCR analysis exhibits that the expression level of CmbHLH32 gene has increased an average about 5.5 times than WT fruit (Fig. 7D). Transcriptional activity of CmbHLH32 was also studied by yeast two hybrid, however, neither monomer nor homodimerizes of CmbHLH32 shows transcription activation activity in yeast (Fig. 8A).
Analysis of fruit weight, length, width and fruit soluble solids content shows no different between WT melon fruit and CmbHLH32-OE transgenic line fruits. However, CmbHLH32-OE transgenic plant fruits exhibit less rmness than WT melon fruits (Table 1). Expression correlation networks analysis using the transcriptome data suggesting 94 genes correlated with CmbHLH32 (Supplementary Table S2). Further Gene Ontology (GO) analysis of correlation genes revealed GO term plant-type cell wall biogenesis (GO:0009832) was enriched (Fig. 8B), suggesting CmbHLH32 function may affect fruit softening through plant cell wall synthesis. However, in melon CmbHLH proteins contained 24 conserved amino acid sites, the conservation pattern was similar to the bHLH proteins in tomato. They had more conserved amino acid in the second helix motif in the CmbHLH and SlbHLH proteins.
Proteins containing the HLH motif often form homo-or heterodimers with other bHLH proteins. Leu-23 and Leu-52 (Leu-60 in melon) residue of helix 1 and 2, respectively, are structurally necessary for dimer formation of plant bHLH and have been identi ed as the most highly conserved residues across plant bHLH [11,30]. Interestingly, in melon ve CmbHLH have mutant Leu-23 or Leu-60 or both. Leu-23 was mutated to Phe-23 and Ile-23 in CmbHLH31 and CmbHLH58 respectively. Leu-60 mutant to Met-60 in CmbHLH57 and CmbHLH91. In CmbHLH62, Leu-23 and Leu-60 was mutated to Val-23 and Glu-60 respectively. In Arabidopsis mutation on the two Leu site signi cantly affect the dimerization of the bHLH [31]. This result indicating a different functional mechanism may exist at least on these ve CmbHLH.
Domain analysis of the CmbHLH illustrated eight proteins possessed an ACT domain. ACT domain is small regulatory domain involved in amino acid or purine metabolism and can bind to variety of ligands [32]. Mas-Droux et.al found a Lys and S-adenosylmethionine-sensitive Asp kinase isoform form a dimeric structure through ACT domain in Arabidopsis [33]. Kong et.al reveals that bHLH DNA-binding activity is suppressed if the C-terminal ACT domain is licensed to homodimerize, and this protein-protein interaction domain is important for the regulation of anthocyanin pigment biosynthesis in maize [34]. Eleven MYC type CmbHLH were found. In plant, MYC genes participate in growth and development, and are also key regulators of the jasmonic acid (JA) signaling pathway [35,36]. This result indicates CmbHLH may involve in a more complex regulation network.

Potential roles of CmbHLH genes in melon fruit development
In the past few decades, characterization and function of bHLH family on several species have been widely and extensively investigated. As one of the largest transcription factor family, bHLH functions involved in the regulation of plant growth and development, abiotic/biotic stress respond, hormone signaling, iron homeostasis, secondary metabolism. For example, in Arabidopsis, brassinosteroid (BR) and gibberellin can promote cell elongation by inhibiting an atypical bHLH transcription factor INCREASED LEAF INCLINATION1 BINDING bHLH1 (IBH1), and ectopic accumulation of IBH1 causes a dwarf phenotype in Arabidopsis [37]. In soybean, GmORG3-like gene enhances cadmium tolerance by increasing iron and reducing cadmium uptake and are transported from roots to shoots [38]. Whereas, studies on bHLH regulating fruit growth and development were less. Waseem et.al found that overexpression of SlbHLH22 leading to earlier fruit ripening, and produce more ethylene-producing phenotypes in tomato [25]. Zhao et.al used white-esh mutant strawberry identi ed seven FabHLH genes that are responsive to the fruit anthocyanin biosynthesis and hormone signaling transduction [39].
Using the transcriptome data, we studied the expression of CmbHLH in fruit different developmental stages. Three CmbHLH (CmbHLH14, CmbHLH32 and CmbHLH41) genes were high expressed (>100 FPKM) in G stage and down regulated in R stage. CmbHLH14 is 74% identity to UNE12 of Arabidopsis, which might be involved in the regulation of the speci c processes required for fertilization, and as a temperature-responsive SA immunity regulator [40,41]. CmbHLH23 was upregulated in R stage, and was homolog genes of AtbHLH68 were proposed to regulate lateral root elongation, and in the response to drought stress, likely through an ABA-dependent pathway in Arabidopsis [42]. CmbHLH59 showed signi cantly down regulation in R vs C stage samples. CmbHLH59 was an MYC type protein and differentially expressed from development to ripening stage in melon fruit. CmbHLH59 sequence is similar to ATbHLH13 in Arabidopsis, functions of ATbHLH13 are repressing Arabidopsis defense responses and regulating anthocyanin biosynthesis through JA signaling pathway [43,44]. CmbHLH114 was upregulated in R vs C stage samples, and CmbHLH114 is annotated to ICE1-like gene. in Arabidopsis, ICE1 is a multifunctional gene that responsive to cold stress, ABA signaling regulation, determination of seed dormancy and so on [45,46]. In banana, MaICE1 targeting to MaNAC1 during fruit cold storage and enhance fruit cold tolerance [47]. The expression of CmbHLH67 was down regulated in R vs C stage and upregulated in C vs P stage. CmbHLH67 is 59% identity to MYC2 of Arabidopsis, and response to a variety of JA-dependent functions including secondary metabolism, insect resistance and stress tolerance [48,49]. In apple fruit ripening, MdMYC2 is required for JA-induced ethylene biosynthesis, besides, MdMYC2 was also found interacting with MdERF2, which is a downstream molecular of ethylene signaling [50].
In this study, we observed that overexpression of CmbHLH32 leading to early fruit ripening. This result suggesting CmbHLH32 may participate in growth and ripening of melon fruit, however, detailed study is still needed. In apple leaf senescence and the expression of senescence related genes is promoted by MdbHLH93 (homolog to AtbHLH93), and leaf senescence was delayed when an ABA-responsive protein, MdBT2, interacted directly with MdbHLH93 [51]. In Arabidopsis, double mutants of bHLH93 failed to ower under short day (SD) condition and bHLH93 plays a major role in regulation Arabidopsis SD owering [29]. Tissue expression analysis illustrates high expression level of CmbHLH32 in female ower, suggesting CmbHLH32 may play a regulation role in owering. However, melon is day-neutral plant, suggesting CmbHLH32 may have different function of bHLH93 in Arabidopsis. Yeast two hybrid analysis indicates transcriptional activation activity of CmbHLH32 may depend on other proteins. Melon CmbHLH32 contains an ACT domain, ACT domain was rst identi ed as a ligand-binding domain, and was found to suppress bHLH DNA-binding activity [34,52]. This could explain homodimerizes of CmbHLH32 did not show transcriptional activation activity in vitro.

Conclusions
This study was focused on the identi cation of melon bHLH gene family, and function of bHLH genes in fruit growth and ripening. We identi ed the CmbHLH gene family in melon. We demonstrated the CmbHLH classi cation, the bHLH domain characters and intron patterns, and phylogenetic relations of CmbHLH and other four cucurbit species. Expression characters of CmbHLH genes using the transcriptome data suggesting most of these CmbHLH genes were low/ no expressed in fruit development. And CmbHLH genes tend to expressed in early stage of fruit development, CmbHLH32 was the most prominent among them. Expression of CmbHLH32 was high in female owers and early fruit growth stage. Transgenic plant lines of overexpression of CmbHLH32 result in early fruit ripening compared to the wild type fruits. These ndings clarify the members and characters of CmbHLH gene family, and provide new insights into the role of melon bHLH genes in regulating fruit development.

Sequence retrieval and identi cation of bHLH proteins in melon
The Hidden Markov Model method and blast method were used to identify the melon bHLH protein sequences. Melon protein sequences (CM3.5.1_protein) were downloaded from the Cucurbit Genomics Database (CuGenDB, http://cucurbitgenomics.org/) [53]. The protein sequences of A. thaliana bHLH (AtbHLH, Araport11_genes.201606.pep.fasta) were retrieved from The Arabidopsis Information Resource (TAIR), (https://www.arabidopsis.org/download/index-auto.jsp?dir=/download_ les/Proteins), on www.arabidopsis.org. The Hidden Markov Model (HMM) le of the HLH domain (PF00010) was downloaded from the Pfam database (version 32.0; http://pfam.xfam.org/) [54]. The HMM software (version3.2.1; http://www.hmmer.org/) was used to search against the melon protein sequence data using default parameters [55]. The Arabidopsis bHLH protein sequences were used to blast against the melon protein sequences using Blast software by default parameters [56]. Then sequences were compared to the CmbHLH proteins downloaded from PlantTFDB (http://planttfdb_v4.cbi.pku.edu.cn/), and repeated sequences were removed. After that these protein sequences were submitted to the online Batch CD-search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) to verify the existence and integrity of the conserved bHLH domain [57,58]. The sequences above the minimum threshold bit score were kept, and the redundant sequences were removed. These representative sequences of putative CmbHLHs were named based on their chromosome location. Transcriptome data using previously published data from our laboratory (PRJNA543288).

Identi cation of conserved motifs and tandem repeat genes
The molecular weight (Mw) and theoretical isoelectric point (pI)-values for these CmbHLH protein sequences were determined by the Compute pI/Mw tool on the ExPASy server (http://web.expasy.org/compute_pi/) [59]. Conserved motifs of CmbHLHs were identi ed by MEME (http://meme-suite.org/) server with maximum number of motifs set at 10 and optimum width of motifs Plant material and growth condition Melon (Cucumis melo cv. Hetao) plants were grown in green house at Dengkou (N40°19′46.07″, E107°0′11.46″), Inner Mongolia Autonomous Region, China. Hetao melon is a conventional melon variety planted in the western part of Inner Mongolia. It has been cultivated for more than 70 years in the local area. The fruit of Hetao melon is respiratory climacteric fruit, small fruit and medium early maturing variety, which can be cultivated in eld or in greenhouse. The melon plants that we used to construct transgenic plant were using the above-mentioned cultivars, after our laboratory 11 generations of selfpollination breeding selection and obtained typical and stable inbred lines of Hetao melon. The owers were self-pollinated, the pollination time was recorded, and the date of pollination was recorded as 0 days after pollination (DAP). Only one fruit was kept for each plant.
The wild-type melon seeds, which were from the above mentioned stable inbred lines of Hetao melon, sterilized by mercuric chloride were planted in 1/2MS medium at 25 ℃, light (1000lx) for 16 hours, dark treatment for 8 hours, and cultured for 30 days to obtain the root, stem and leaf tissues of melon. The male and female owers of wild type melon without pollination which were planted in greenhouse were collected and preserved immediately in liquid nitrogen.

Gene cloning and transgenic plant generation
An open reading frame of 1023bp of CmbHLH32 (MELO3C011110) gene was ampli ed by RT-PCR from melon total RNA using upstream primer: 5'-atggagagctcacgaacgtctt-3' with a Kpn restriction site and downstream primer: 5'-ctacagcagcttcctcatac-3' with a Xba restriction site. The PCR products were cloned into the vector of pMD-19T (Takara Bio, Shiga, Japan) by TA cloning. Subsequently, the fragment was inserted into the overexpression vector pPZP221 applying Kpn and Bam HI sites. The plasmid pPZP221-CmbHLH32 was diluted with 2×SSC (pH 7.0) solution to 100ng/μl. After 7 hours of arti cial pollination, 100ng/μl of plasmid solution was injected into the pistil ovary by ovary injection method to obtain transgenic plant. T1 transgenic plants were identi ed by PCR test using two primer sets, one is CaMV35S upstream primer: 5'-CAGAAAGAATGCTAACCC-3' and downstream primer: 5'-TTCTTCTTGTCATTGAGTCGTA-3'; another is upstream primer: 5'-TTTCGGTCGTGAGTTCGGAG-3' and downstream primer: 5'-CACTTCTTCCCGTATGCCCA-3'. Only if the plant that was detected by both of these primers will be preserved for later observations. Quantitative real-time PCR analysis A quantitative real-time PCR (qRT-PCR) assay was performed to validate the expression of CmbHLHs. Total mRNAs were reverse transcribed using PrimeScript™ RT reagent Kit with gDNA Eraser kit (Takara Bio, Shiga, Japan) following the manufacturer. The qRT-PCRs were performed using SYBR® Premix Ex Taq™ II (Takara Bio, Shiga, Japan) by a 96-well Chromo4 Real-Time PCR system. The qRT-PCR conditions were as follows: a predenaturation of 30 s at 95°C, followed by 35 cycles of 5 sec at 95°C and 30 sec at 60°C. The 2 -ΔΔCT method was used to analyze the relative mRNA expression level.
Transcriptional activation activity analysis in yeast Full length of CmbHLH32 was cloned and inserted into pGBKT7 and pGADT7 vectors (TaKaRa) to obtain pGBKT7-CmbHLH32 and pGADT7-CmbHLH32 vectors. The pGBKT7-CmbHLH32 was transformed into the yeast strain AH109 (TaKaRa) with pGADT7-T vector for transcriptional activation activity test. Homodimerizes transcriptional activation activity of CmbHLH32 used cotransformation of pGBKT7-CmbHLH32 and pGADT7-CmbHLH32 vectors. Cotransformation of pGBKT7-53 + pGADT7-T and pGBKT7-Lam + pGADT7-T were used as positive control and negative control respectively. The transformed yeast cells were streaked on SD/-Trp/-His/Ade/3-AT solid medium in different dilutions (10 -1 , 10 -2 and 10 -3 ). After 3-day incubation at 30 ℃, the transcriptional activation activity of CmbHLH32 were evaluated according to growth status of transformed yeast cells.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The datasets analyzed during the current study are available in the SRA (https://www.ncbi.nlm.nih.gov/sra), SRA accession: PRJNA543288.

Competing interests
The research was conducted in the absence of any potential con ict of interest.  The domain distribution and types of CmbHLH.