Melon (Cucumismelon L.), as an important Cucurbitaceae crop, is widely distributed and has a long history of cultivation. It has high economic and nutritional value. China is a big melon producer, and its output has increased from 2×10⁶ tons in the 1950s and 1960s up to nearly 2×10⁷ tons, with an average annual growth rate of 3.68%, significantly higher than the world (2.75%). After the 21^st century, the status of China’s melon industry in the world has been continuously improved, and the total output gradually accounts for more than half of the world (Yang et al., 2019). Melon tastes sweet and juicy. It is rich in soluble sugar, amino acids, organic acids, vitamin C and other nutrients. With high nutritional quality, it can improve human immunity and promote human health (Xia et al., 2017). Biological and abiotic stresses are the main obstacles to melon production. Therefore, it is of practical significance for melon molecular breeding to study the response mechanism of melon to stress and to explore and utilize important gene resources.

As a second messenger, calcium (Ca²⁺) plays an important role in signal transduction of plant response to stress. It can regulate the activity of a variety of effector proteins and coordinate cell responses (Yang et al., 2019). There are three types of chiral calcium ion sensors in plants (Defalco et al., 2010): one is calmodulin, including calmodulin (CaM) and calmodulin-like (CML), the other is calcium dependent protein kinase (CDPK), and the third is calcineurin B-like protein (CBL).

Calmodulin-like (CML), as the most typical calcium binding protein, participates in a variety of physiological and biochemical processes in plants. It is a kind of protein that has been studied more at present. The similarity between CML and CaM amino acids is not less than 16% (Perochon et al., 2011). Each protein contains at least one conserved EF-hand chiral domain, and there are no other proven domains. This domain is a helix-ring-helix structure, and one Ca²⁺ can only bind one EF-hand. At the same time, there are differences in chiral molecular sequences among different calmodulin-like molecules. Mccormack et al. (2005) believed that this may reflect the small differences in the response of different family members to Ca²⁺ signals. CMLs are ubiquitous in plants. A variety of CML proteins have been identified in the genomes of Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum and Brassica pekinensis. They play an important role in growth, biological stress and abiotic stress (Cao et al., 2018). In Arabidopsis thaliana, Magnan et al. (2008) found that CML9 can enhance its tolerance to salt and drought stress and promote root growth. CML42 is also related to the formation of cell trichomes (Dobney et al., 2009), while CML24 is involved in encoding a potential Ca²⁺ sensor in response to ABA, sunshine and salt stress (Delk et al., 2005).

At present, there is no research on melon CML gene family. In this study, 60 CML family members were identified in the published melon genome by using bioinformatics methods. At the same time, the chromosomal location, gene structure, conserved protein domains, promoters and phylogenetic relationships of melon CML family members were analyzed, and the expression of some CML family genes induced by Fusarium oxysporum melon specialization was discussed. We hope that our study can provide reference and data basis for further research of melon CML family genes.

1 Results and Analysis

1.1 Analysis of basic characteristics of CML protein gene in melon

In this study, the proteins containing EF-hand but not other domains were selected as melon Calmodulins by bioinformatics. 60 melon calmodulin genes were screened and named CmCML1-60 according to their distribution and arrangement on chromosomes (Table 1). According to the analysis, the number of EF-hands of melon CML family members was 1~4, and the protein with 4 domains was the most, and most of them were EF-hand7. Except that the pI of CmCML1, 11, 16, 18 and 28 were greater than 7, the remaining 92% of the pI were between 3.99 (CmCML40)~6.57 (CmCML15), 96.7% of the amino acids were within 300, and the relative molecular weight was generally about 20 000 kD. It can be seen that the melon CML family proteins are mostly small acidic molecular proteins. The subcellular location of the 60 identified CmCML showed that CmCML were mainly distributed in cell membrane, vacuole, cytoplasm and nucleus. Among them, 53 genes were located on the cell membrane, accounting for 83.3%, followed by vacuole and cytoplasm. Only 4 CmCML genes were located in the nucleus, accounting for 6.7%.

1.2 chromosome location of CML gene in Melon

According to the location information of CmCML on the chromosome, MG2C online software was used to draw the physical map of melon CML chromosome and to locate the gene (Figure 1). 60 CmCML family members were distributed on 12 chromosomes, and the number of genes on each chromosome was mostly different.

Figure 1  CmCML gene location on chromosome

CmCML1 and 2 were dispersed in the upper and lower arms of chromosome 1, CmCML3 was located in the upper arm of chromosome 2, CmCML4～6 were concentrated in the lower arm of chromosome 2, CmCML7～10 were evenly distributed on chromosome 3, and CmCML11～18 were clustered in the lower arm of chromosome 3. CmCML19, CmCML20～22, CmCML23～25, CmCML26 and 27 were distributed in the upper and lower arms of chromosomes 4 and 5 respectively, and CmCML28～32 was evenly distributed on chromosome 6. CmCML39 and 40, CmCML41 and 42, CmCML43 and 44, CmCML45 and 46, CmCML52 and 53, and CmCML54 were distributed in the upper and lower arms of chromosome 7, 8 and 10, respectively. There is only one chromosome of CmCML47 in the upper arm of chromosome 9, of which CmCML48～51 were distributed in the lower part. CmCML55 and 56, 57 and 58 were evenly distributed in the upper, middle and lower arms of chromosome 11, and CmCML59 and 60 were distributed in the middle and lower arms of chromosome 12. The unbalanced distribution of genes on 12 chromosomes indicated that the genetic variation of CmCMLs was in the process of evolution.

1.3 Analysis on structural characteristics of melon CML

1.3.1 Conservative motif analysis of melon CML family

The analysis of conserved domains plays an important role in determining gene functions. In this study, MEME, an online tool, was used to predict and analyze melon CML family proteins. At the same time, the maximum likelihood method was used to build a phylogenetic tree, and the results of conservative motif prediction were visualized with TBtools (Figure 2). The results showed that the melon CML family had 10 conserved motifs with a width between 15 and 50 amino acids (Table 2). Almost all CML proteins contain motif 1~5, which were EF-hand domains. Motif 6 and motif 8 mainly appeared in group I; Motif 10 mainly appears in group II. Members of phylogenetic tree with close relationship often contain the same motif, that is, CML proteins of the same group have certain similarity in function.

Figure 2 The prediction of conserved motifs in CmCML proteins

Table 2 Prediction of Conserved Motifs of CML Gene in Melon

1.3.2 Melon CML intron identification

Among the 60 identified CmCML, 19 family members contained introns, of which CmCML3, 46 and 55 contained only one intron, and CmCML57 contained the most introns, which was 9 (Figure 3). These introns may play an important role in regulating gene expression and promoting transcriptional expression. In this study, 68.3% of CmCML lack intron structure, which was also the main reason for the small length of melon CML family genes.

Figure 3 New ICT based fertility management model in private dairy farm India as well as abroad

1.4 Genetic evolution analysis of melon CML family

In order to reveal the homologous evolution relationship of CmCMLs, a phylogenetic tree was constructed with reference to Arabidopsis thaliana CML gene family. Phylogenetic analysis was carried out on 60 proteins of melon CML family and 50 proteins of Arabidopsis thaliana CML family (Figure 4). According to the genetic relationship and sequence similarity, the CML gene family was divided into 8 groups, I~VIII. Each group contained 4, 6, 14, 15, 6, 5, 6 and 4 members respectively. The genes of each group had high homology and close evolutionary relationship.

Figure 4 New ICT based fertility management model in private dairy farm India as well as abroad

1.5 Promoter analysis

In this study, the online software PlantCARE was used to analyze the sequence of 2000 bp upstream of the CmCML start codon, and the main promoters were displayed and plotted (Figure 5). A variety of cis-elements responding to environmental and hormonal signals were identified in the CmCML family. It can be seen that its expression regulation mechanism was complex. Melon CML family contained a variety of hormone response elements, including ABRES, TGA element, TCA element and ERE. TCA element was also involved in salicylic acid response. At the same time, it was found that CmCML could also respond to drought, light, temperature, hypoxia and salt stress.

Figure 5 New ICT based fertility management model in private dairy farm India as well as abroad

1.6 Analysis of gene expression characteristics of melon CML family induced by Fusarium oxysporum melon specialization

The expression characteristics of some genes of melon CML family induced by Fusarium oxysporum melon specialization were analyzed by fluorescence quantitative PCR. The expression levels of CmCML29, CmCML35 and CmCML45 were up-regulated under Fusarium oxysporum melon specialization, and the other genes were down-regulated. The expression levels of CmCML12, CmCML29 and CmCML35 genes in resistant and susceptible varieties were significantly different (Figure 6).

Figure 6 New ICT based fertility management model in private dairy farm India as well as abroad

2 Discussion

A total of 60 melon CML family proteins were identified in this study, which were consistent with the studies on 50 Arabidopsis thaliana CML (Delk et al., 2005), 52 Lycopersicon esculentum CML (Munir et al., 2016), 68 Glycine max CML (Chen et al., 2015) and 60 Gossypium CML (He 2015, Huazhong Agricultural University, pp.3-7). Melon CML proteins were mostly acidic small molecular proteins (Table 1), with large differences in the number of exons between genes (Figure 3), and relatively complex gene structures. Therefore, CmCML was easy to be cut during replication, resulting in instability of gene structure.

Only one EF-hand domain can be predicted in CMLs, indicating the importance and necessity of this domain. EF-hand plays an important role in Ca^2＋ binding and transport. Through comparison, it was found that CmCML contained 1~4 EF-hand domains, which were similar to the characteristics of Arabidopsis thaliana. Therefore, the whole CML gene families of melon and Arabidopsis thaliana were analyzed by phylogenetic tree and divided into 8 subfamilies (Figure 4), indicating that melon CML family had many protein members, different origins and diverse functions. Through analysis, it was found that CMLⅠ, Ⅳ, Ⅴ, Ⅵ and Ⅷ contain roughly the same amount of CML in the two species, but only in the three groups of CMLⅡ, CMLⅢ and CMLⅦ had great differences, indicating that CML had a relatively close evolutionary relationship among plant genes. In the same subfamily or smaller branch structure, different genes basically played similar functions (Wang et al., 2018). In promoter analysis (Figure 5), about 97% of melon CMLs contain MYC, so it was predicted that CML family proteins had strong drought resistance. In addition, cis-elements and a large number of hormone response elements under hypoxia, low temperature and salt stress had been identified. It can be seen that hormones play a very important role in plant stress resistance. Based on the fluorescence quantitative PCR technology, it was found that the expression of some melon CML family genes changed significantly under the induction of Fusarium oxysporum melon specialization (Figure 6). Therefore, it can be inferred that CmCMLs are conducive to inhibiting the occurrence of Melon Fusarium Wilt and alleviating continuous cropping obstacles, but the specific mechanism and mode of action need to be further studied.

CMLs were the most conserved calcium sensing proteins in all eukaryotes (Yang et al., 2019). They play a key role in the cellular signal network by regulating various targets, thus responding to plant stress response and phylogeny (Perochon et al., 2011). This study preliminarily clarified the basic bioinformatics characteristics of melon CML gene, verified that CML gene was involved in the formation of Melon Fusarium Wilt resistance, and provided data reference and theoretical basis for further study on the biological function of the family members.

3 Materials and Methods

3.1 Identification and physicochemical properties analysis of melon CML protein gene

In order to screen and obtain CML family genes in melon, the TAIR genome database (https://www.arabidopsis.org/) was used to download 50 Arabidopsis thaliana CML genes, and the Cucurbitaceae database (http://cucurbitgenomics.org/) was used to obtain the candidate protein coding sequences of melon CML family genes by blast search and comparison (E-value was e^-5, Identity>60%). Then the online database Pfam and SMART were used to analyze and identify the above candidate proteins (E-value<1.0), and the genes without domains were eliminated. Finally, the candidate proteins were manually screened and identified by Markov model. A series of bioinformatics analyses were carried out on all the identified members of the melon calmodulin family, and ExPASy (http://web.expasy.org/protparam/) was used to analyze the biochemical properties such as isoelectric point, amino acid number and relative molecular weight.

3.2 Prediction of gene structure of melon CML family members

MG2C online software (http://mg2c.iask.in/mg2c_v2.0/) was used to draw the physical map of chromosome and locate the genes. Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) online software was used to predict subcellular localization. Finally, TBtools local tools were used to analyze and map the gene structure.

3.3 Conservative motif analysis of melon CML family

MEME online tool was used to predict and analyze the conserved motifs of melon CML family genes (Bailey et al., 2009). The parameters were set as follows: the minimum width of conserved motifs was set to 6, the maximum was set to 50, the number of conserved motifs was set to 10, and other parameters were set by default. Download the result file mast.xml. The phylogenetic tree of melon CML family genes was constructed by using the maximum likelihood method (ML). The amino acid replacement model was LG+G, and the phylogenetic analysis was carried out. The Bootstrap method was used to repeat 1000 times to evaluate the support rate of each node, and the Partial deletion value was set to 80%. The results of prediction and conservative motifs were visualized by TBtools (https://www.biorxiv.org/content/10.1101/289660v2.abstract).

3.4 Phylogenetic analysis of melon CML

the MUSCLE program in MEGAX was used to perform multiple sequence alignment for the identified melon CML family genes (60) and Arabidopsis thaliana CML family genes (50), where the Max Iterations parameter was set to 100, and other parameters were set by default. The Neighbor-Joining (NJ) method was adopted. The Bootstrap value was set to 1000, and the Partial deletion value was set to 80%. Other parameters were set by default.

3.5 Prediction of melon CML cis-elements

2 000 bp upstream DNA sequences of 60 CML family members were extracted from melon genome database as promoter sequences and submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The possible cis-acting elements in the promoter sequence were analyzed.

3.6 Analysis of gene expression characteristics of melon CML family induced by Fusarium oxysporum melon specialization

3.6.1 Activation of Fusarium oxysporum melon specialization

The activated pathogens were cultured in PDA medium and Armstrong medium. The specific method was as follows: the mycelium was inoculated on PDA (200 g potato, 20 g glucose, 20 g agar, 1 l distilled water) potato culture medium, dark cultured at 25 ℃ for 7 days, cut into 0.5 cm² cubes after the culture, inoculated on Armstrong (1.1 g KH₂PO₄, 1.6 g KCl, 5.9 g Ca(NO₃)₂, 0.4 g MgSO₄·7H₂O, 0.2 mg ZnSO₄, 0.2 mg MnSO₄, 0.2 mg FeCl₃, 20 g glucose, Inoculate in Armstrong (1.1 g KH₂PO₄, 1.6 g KCl, 5.9 g Ca(NO₃)₂, 0.4 g MgSO₄·7H₂O, 0.2 mg ZnSO₄, 0.2 mg MnSO₄, 0.2 mg FeCl₃, 20 g glucose, add distilled water to 1 L) liquid medium, and culture at 27 ℃ and 120 rpm for 3 days.

3.6.2 Inoculation of melon seedlings

The tested melon varieties were M4-45 and MR-1. Before sowing, the soil and seeds were disinfected. When the tested seedlings grew to the third leaf stage, the root soaking inoculation method was adopted, and the inoculation concentration was 1×10⁶/mL spore suspension. Seedlings with the same growth were selected to cut the roots and soak the roots in the bacterial solution for 1 quarter of an hour, and shake them for 2~3 times. After root soaking, it was planted in a nutrient bowl, and the one inoculated with clean water was used as the control. Buckle the shed to keep warm and moist. Root tissues were taken at 0 h (control), 12 h and 72 h after inoculation. The samples were named M4-45CK, M4-45-12h, M4-45-72h, MR-1CK, MR-1-12h and MR-1-72h. Each treatment was repeated for 3 times, with 10 strains for each repetition, a total of 18 samples. Clean it repeatedly with distilled water, dry the residual distilled water on the surface with filter paper, and then freeze it with liquid nitrogen at -80 ℃.

3.6.3 Analysis on gene expression characteristics of melon CML family

Total RNA was extracted with Trizol reagent of Invitrogen company, and cDNA synthesis was performed with the reverse transcription kit TRUEscript 1st Stand cDNA Synthesis Kit of Aidlab company. Melon actin gene was used as the internal reference gene (Sense:5'-TCTATTCCAGCCATCTCTC-3', Antisense:5'-GACCCTCCAATCCAAAC-3''), and candidate gene specific primers were designed with Primer Premier v6.0 software (Table 3), QRT PCR experiments were conducted on iQ5Gradient Real Time PCR system (Bio-Rad, CA, USA). The reaction conditions were as follows: 95 ℃ for 30 s, 95 ℃ for 15 s, 60 ℃ for 30 s, 72 ℃ for 30 s, and for 40 cycles, 55 ℃~95 ℃, +0.5 ℃/cycle, 5 s, and stored at 4 ℃. The reaction system was carried out according to the instructions of SYBR Green Realtime PCR Master mix (TOYOBO, OSAKA, JAPAN), and the results were determined by relative quantity 2^-ΔΔCT method, and the variance analysis was performed on SAS local software at significant level α=0.05.

Authors’ Contributions

LL and WXZ were the executors and designers of this experimental study; LL, SXY and SL completed writing the first draft of the manuscript; GP and LY directed the writing of the manuscript; WXZ, the conceiver and principal of this study, directed the data analysis and revision of the manuscript. All authors read and approved the final manuscript.

Acknowledgments

This study was jointly funded by National Natural Science Foundation of China (31772333) and Post Expert of National Melon Industrial Technology System of China (CARS-026-02).

References

Bailey T.L., Boden M., Buske F.A., Frith M., Grant C.E., Clementi L., Ren J., Li W.W., and Noble W.S., 2009, MEME SUITE: tools for motif discovery and searching, Nucleic Acids Research, 37(S2): W202-W208.

https://doi.org/10.1093/nar/gkp335

Cao S.Y., Wang Y.F., Su W.Y., Zhang L., Zhang Y.H., and Xu J.Q., 2018, Research progress on functions of calmodulin-like proteins in processes of plant growth and developments and stresses, Zhiwu Shengli Xuebao (Plant Physiology Journal), 54 (10): 1517-1526.

Chen C., Duanmu H.Z., Zhu D., Liu A.L., Xiao J.L., and Zhu Y.M., 2015, Bioinformatics analysis of GmCML genes in Soybean Genome, Dadou Kexue (Soybean Science), 34(6): 957-963.

Defalco T.A., Bender K.W., and Snedden W.A., 2010, Breaking the code: Ca²⁺ sensors inplant signalling, Biochemical Journal, 425(1): 27-40.

https://doi.org/10.1042/BJ20091147

Delk N.A., Johnson K.A., and Chowdhury N.I.J., 2005, CML24, regulated in expression by diverse stimuli, encodes a potential Ca^{2 +} sensor that functions in responses to abscisic acid, daylength,and ion stress, Plant Physiology, 139(1): 240-253.

https://doi.org/10.1104/pp.105.062612

Dobney S., Chiasson D., Lam P., Smith S.P., and Snedden W.A.,2009, The calmodulin-related calcium sensor CML42 plays a role in trichome branching, Journal of Biological Chemistry, 284(46): 31647-31657.

https://doi.org/10.1074/jbc.M109.056770

Magnan F., Ranty B., Charpenteau M., Sotta B., Galaud J.P., and Aldon D., 2008, Mutations in AtCML9, a calmodulin-like protein from Arabidopsis thaliana, alter plant responses to abiotic stress and abscisic acid,The Plant Journal, 56(4): 575-589.

https://doi.org/10.1111/j.1365-313X.2008.03622.x

Mccormack E., Tsai Y., and Braam J., 2005, Handling Calcium signaling: arabidopsisCaMs and CMLs, Trends in Plant Science, 10(8): 383-389.

https://doi.org/10.1016/j.tplants.2005.07.001

Munir S., Khan M.R.G., Song J., Munir S., Zhang Y., Yea Z., and Wang T., 2016, Genome-wide identification, characterization and expression analysis of calmodulin-like (CML) proteins in tomato (Solanum lycopersicum), Plant Physiologyand Biochemistry, 102: 167-179.

https://doi.org/10.1016/j.plaphy.2016.02.020

Perochon A., Aldon D., Galaud J.P., and Ranty B., 2011, Calmodulin and calmodulin-like proteins in plant calcium signaling, Biochimie, 93(12): 2048-2053.

https://doi.org/10.1016/j.biochi.2011.07.012

Wang C.N., Zhu Q.L., Cui H.N., Cui B.M., Wang X., and Luan F.S., 2018, Identifcation and characteristic analysis of CDPK gene family, Beifang Yuanyi (Northern Horticulture), (17): 1-6.

Xia M.L., Lu X.M., and Qian C.T., 2017, Research progress on cultivation techniques of improving the main nutritional quality of melon, Zhongguo Guacai (China Cucurbits and Vegetables), 30(6): 1-5.

Yang N., Wang W.Y., Cao C.Y., and Wu J.X., 2019, Analysis on the Development Status and Trend of Melon Industry in China, Zhongguo Guacai (China Cucurbits and Vegetables), 32(8): 50-54.

Yang X., Xu Y.C., Yang F.F., Cai X. Y., Hou Y.Q., Wang Y.H., Wang X.X., Wang K.B., Liu F., and Zhou Z.L., 2019, Mianhua Xuebao (Cotton Science), 31(4):307-318.