Research Article

Comparative Genomics of Three-domain Multi-copper Oxidase Gene Family in Foxtail Millet (Setaria italica L.)  

Huilong Chen , Xiyin Wang , Weina Ge
College of Life Sciences, North China University of Science and Technology, Tangshan, 063000, China
Author    Correspondence author
Computational Molecular Biology, 2021, Vol. 11, No. 4   doi: 10.5376/cmb.2021.11.0004
Received: 10 Aug., 2020    Accepted: 11 Dec., 2020    Published: 21 Mar., 2021
© 2021 BioPublisher Publishing Platform
This article was first published in Molecular Plant Breeding in Chinese, and here was authorized to translate and publish the paper in English under the terms of Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Chen H.L., Wang X.Y., and Ge W.N., 2021, Comparative genomics of three-domain multi-copper oxidase gene family in foxtail millet (Setaria italica L.), Computational Molecular Biology, 11(4): 1-13 (doi: 10.5376/cmb.2021.11.0004)

Abstract

Three-domain multi-copper oxidase is the largest family in the supergene family of multi-copper oxidase and has multiple biological functions. In this paper, comparative genomics methods were used to analyze the whole genome three-domain multi-copper oxidase gene family in foxtail millet (Setaria italica). The results showed that the three-domain multi-copper oxidase gene family of foxtail millet had undergone a large-scale expansion, mainly caused by the tandem duplication which accured after the formation of foxtail millet species. The three-domain multi-copper oxidase genes of foxtail millet were divided into three groups: I, II, and III. Tandem repeat genes were mainly concentrated in Class I-7 subclass. Selection pressure analysis of this subclass revealed that 7 amino acid sites were positively selected. Foxtail millet three-domain multi-copper oxidase could respond to many abiotic stresses, the expression pattern showed up-regulated under ABA and PEG treatment; under heat stress, most of the gene were down-regulated, and the differentially expressed genes clusted some special subclass; under cold stress and salt stress, there were also many genes with significantly different expressions. This study revealed the evolutionary rules of the three-domain multi-copper oxidase gene family in foxtail millet and provides clues for further study of the gene's function.

Keywords
Foxtail millet; Three-domain multi-copper oxidase; Gene family; Comparative genomics; Abiotic stress

Multi-copper oxidase is a superfamily (McCaig et al., 2005), that contains a variety of enzymes, such as nitrite reductase, laccase, ascorbate oxidase, bilirubin oxidase, ferroxidase and ceruloplasmin, etc (Nakamura et al., 2003; Gorman et al., 2008; Ye et al., 2015). According to the different spectroscopic characteristics, there are three types of copper ions in the structure of multi-copper oxidase, named type1, type2 and type3, and the spectral values of the absorption peaks of the three copper ions are different (Nakamura et al., 2003). The mulit-copper oxidase contains 2, 3 or 6 repeated conserved domains, which are homologous with cupredoxin single domain, so the multi-copper oxidase is divided into two-domain multi-copper oxidase, three-domain multi-copper oxidase and six-domain multi-copper oxidase (Nakamura et al., 2003). In the above-mentioned conserved structure domain, there is a site that binds to type1 copper ion, called BCB domain (blue copper binding domain). In addition, there is an IDCB site (interdomain copper binding site) between different repeating units, which generally contains one type2 copper ion binding site and two type3 copper ion binding sites (Nakamura et al., 2003).

 

Three-domain multi-copper oxidase is the largest family in the multi-copper oxidase superfamily, including three conserved domains: Cu-oxidase (PF00394), Cu-oxidase_2 (PF07731) and Cu-oxidase_3 (PF07732), which are mainly composed of laccase (EC1.10.3.2) and Ascorbate oxidase (EC1.10.3.3) (Nakamura et al., 2003; Zhang et al., 2011). Laccase is closely related to the synthesis of lignin (Simões et al., 2020), and ascorbate oxidase plays an important role in plant growth and anti-aging processes (Pignocchi et al., 2003). At present, only the structural characteristics of three-domain multi-copper oxidase in model plant rice have been analyzed via bioinformatics (Wang et al., 2019), and the bioinformatics research on the three-domain multi-copper oxidase of other plants has not been reported.

 

Foxtail millet (Setaria italica) is an important economic crop for both grain and grass, it is also a C4 model plant and a dry farming model plant in the grass family, and it has the characteristics of small genome and self-pollination (Sharma and Niranjan, 2018). With the completion of its genome sequencing in recent years, it has gradually become a new research hotspot (Bennetzen et al., 2012; Zhang et al., 2012). In order to understand the evolution and function of the three-domain multi-copper oxidase gene family in the foxtail millet genome, this study used the method of comparative genomics to identify and analyze the three-domain multi-copper oxidase of representative plants in plant evolution, in order to clarify evolutionary law of foxtail millet three-domain multi-copper oxidase, and transcriptome analysis of foxtail millet seedlings under various stresses, statistics of the expression of three-domain multi-copper oxidase genes in foxtail millet under different stresses, for in-depth study functions lay the foundation.

 

1 Results and Analysis

1.1 Identification of three-domain multi-copper oxidase gene family

Using the method of homology comparison, a total of 700 genes encoding three-domain multi-copper oxidase were identified in the selected 21 representative species (Figure 1). With the evolution of plants, the number of members of this gene family has gradually increased. For example, 1 and 3 three-domain multi-copper oxidase genes were identified in Coccomyxa subellipsoidea and Chlamydomonas reinhardtii, respectively. 12 were identified in Physcomitrella patens, 16 were identified in Selaginella moellendorfii, 22 were identified in Amborella trichopoda, and higher angiosperms contained more members of the family, suggest that the gene family is gradually expanding with the evolution of plants. It is speculated that the reason for the expansion may be related to plant’s WGD (whole genome duplication). However, in higher plants, the number of members of this gene family is quite different. For example, there are 60 in Brassica rapa, while there are only 38 in its related species, Brassica oleracea. It is speculated that a large amount of gene loss occurred in dicot plants after the WGD, and the loss situation in each species was different, resulting in large differences in the number of gene family members among different species. Among the six monocot plants studied, except for foxtail millet, the number of this gene family of other species is relatively close, all of which are more than 40. Although maize had a WGD event after speciation (Wang et al., 2015b), the number of members of the gene family did not change much. Foxtail millet did not have a WGD event after speciation, but the members of this gene family expanded significantly, which may be related to other gene duplication events such as segmental duplication or tandem duplication.

 

Figure 1 Quantitative distribution of three-domain multi-copper oxidase family genes in plants

 

1.2 Phylogeny and selection pressure analysis of three-domain multi-copper oxidase gene family

Based on the Neighbor-Joining method, phylogenetic trees of three-domain multi-copper oxidase gene family were constructed for six gramineae plants and foxtail millet single species, respectively. According to the topological structure of the phylogenetic trees, and referring to the classification criteria of the three-domain multi-copper oxidase gene family in rice by Wang et al. (2019), the multi-copper oxidase genes of both gramineae plants and foxtail millet are divided into I, II and III classes. Class I is divided into 7 subgroups, and Class II is divided into 2 subgroups. Some lone genes are topologically independent from the first and second classes, and they are classified as Class III (Figure 2; Figure 3A), only one gene Si5G086300.1 in foxtail millet belongs to this class. In the phylogenetic tree of the three-domain multi-copper oxidase gene family in Gramineae, the foxtail millet multi-copper oxidase genes are clustered in Class I-7, and in the phylogenetic tree of three-domain multi-copper oxidase gene family in foxtail millet, these genes are also clustered together, implying that the sequence similarity of these genes is high.

 

Figure 2 Phylogenetic tree of three-domain multi-copper oxidase gene family in grass

 

Figure 3 Phylogenetic tree,protein domain, protein motif and gene structure of the three-domain multi-copper oxidase gene in foxtail millet

Note: A: Gene tree of foxtail millet three-domain multi-copper oxidase; B: Motif and domain of foxtail millet three-domain multi-copper oxidase; C: Gene structure of foxtail millet three-domain multi-copper oxidase

 

In view of the aggregation characteristics of foxtail millet multi-copper oxidase genes in Class I-7, selective pressure analysis was carried out for this subgroup. The NEB result of the site model showed that the 38, 40, 192, 357, 420, 458, 526 amino acids S, G, P, A, T, V, F of Class I-7 were positively selected, and the BEB result showed 357 amino acid A is subject to positive selection. Both NEB and BEB statistics showed that 357 position A is positively selected, and it is speculated that this position may be important for gene function.

 

1.3 Homology analysis of three-domain multi-copper oxidase gene between foxtail millet and rice

The genome of rice is the closest to gramineous ancestor species (Wang et al., 2015a), and rice is a model plant, and its gene function has been studied more. Therefore, we searched and analyzed the orthologous gene pairs of foxtail millet and rice in the genome homologous gene pairs of foxtail millet and rice, in order to have more enlightenment on the evolution and gene function of this gene family in foxtail millet. As a result, 73 pairs of orthologous genes were identified, 52 pairs of paralogous genes were identified in foxtail millet, and 24 pairs of paralogous genes were identified in rice (Figure 4). The 73 pairs of orthologous genes are all three-domain multi-copper oxidase, indicating that the orthologous genes of this gene family are relatively conserved in both species, and there are far more pairs of paralogous genes in foxtail millet than in rice. This indicates that gene duplication did occur in this gene family after the foxtail millet speciation. This is consistent with the results of the phylogenetic tree.

 

Figure 4 The homology of foxtail millet and rice three-domain multi-copper oxidase gene family

Note: The purple line indicates the orthologous gene pair between foxtail millet and rice; The green line indicates the paralogous gene pair in the rice; The yellow line indicates the paralogous gene pair in the foxtail millet

 

1.4 Analysis of gene duplication and loss

The expansion of gene families is always accompanied by gene duplication and loss (Song et al., 2018), so the Notung software was used to analyze the gene duplication and loss of this family in the evolution of grasses (Figure 5). The results showed that there was no loss of this family in the ancestral species of gramineae. However, after the differentiation of gramineae species, this gene family has undergone different degrees of duplication and loss in different species. Generally speaking, in gramineae, more genes of this family are lost than duplication. This result helps us better understand the expansion process of the gramineae family.

 

Figure 5 Duplication and loss of the three-domain multi-copper oxidase gene family in grass

 

There are many types of gene duplication, and these different types of duplication may lead to the expansion of gene families (Song et al., 2018). MCScanX software was used to analyze the five duplication origin types of members of the three-domain multi-copper oxidase gene family in gramineae (Table 1). The results showed that Dispersed contributed the most to the expansion of the family, implying that the gramineous three-domain multi-copper oxidase gene family mainly relied on Dispersed for duplication. In foxtail millet, except for Dispersed, Tandem's contribution is greater than that of other gramineous species. This may be the main reason why the number of foxtail millet family is larger than other gramineous species.

 

 

Table 1 Types of duplication of three-domain multi-copper oxidase family genes in grass

 

In order to understand the duplication history of the foxtail millet family in more detail, using MCScanX software to identify 7 pairs of segmental duplications and 14 pairs of tandem duplications in the foxtail millet (Figure 6), these 7 pairs of duplications are all located in the collinearity block inside the foxtail millet, and it was found that the tandem repetitive genes on chromosome 8 of foxtail millet were the most. This result once again proves that tandem duplications indeed have a great contribution to the evolution of the three-domain multi-copper oxidase gene family in foxtail millet. According to the evolution rate of plants, the divergence time of these repeated gene pairs was calculated (Table 2). The divergence time of the duplication gene pairs is about 80 million to 430 million years. Previous research results indicate that foxtail millet diverged from its ancestors about 510 million years ago (Wang et al., 2015b), which shows that the divergence of these gene pairs occurred after foxtail millet speciation. Eleven of the 14 tandem duplication gene pairs occurred after foxtail millet speciation. It is worth noting that there is a pair of tandem repeat genes Si8G211600.1-Si8G211700.1. Si8G211700.1 does not belong to the three-domain multi-copper oxidase gene family, indicating that duplication can indeed cause changes in gene structure. These results help to better understand the expansion and evolution of the three-domain multi-copper oxidase gene family.

 

Figure 6 The syntenic relationship and chromosomal location of the three domain multi-copper oxidase genes in foxtail millet

Note: The gray line represents the collinearity result inside the foxtail millet genome; The red line represents the three-domain multi-copper oxidase gene pair in the collinearity region inside the foxtail millet

 

 

Table 2 New ICT based fertility management model in private dairy farm India as well as abroad

 

1.5 Chromosome distribution, gene structure, motif and domain distribution characteristics of foxtail millet family genes

The chromosome distribution of the three-domain multi-copper oxidase genes in foxtail millet was drawn using circos software (Figure 6), and it was found that this family has a distribution on each chromosome and the number varies, of which chromosome 8 has the most distribution (26), accounting for 37.14% of the total. Chromosome 7 has the least distribution (2), accounting for 2.86% of the total. In addition, it was also found that the gene SiJ004900.1 exists on scaffold_11.

 

Through the analysis of the gene structure of the three-domain multi-copper oxidase of foxtail millet (Figure 3C), it is found that the gene length of this family is different, the average length is about 3 440 bp, the minimum length is 1 637 bp, and the maximum length is 9 331 bp. The number of cds is between 2-9, and most foxtail millet three-domain multi-copper oxidase genes have non-coding regions (UTR). The gene structure of Class I-7 is very similar, most of which have 6 cds and 5 introns. Class I-1 cds are the most (6~9). These results indicate that the three-domain multi-copper oxidase of foxtail millet has more intron insertions. It is worth noting that Si5G086300.1, the only gene belonging to Class III in foxtali millet, has only cds, which may be caused by the loss of some fragments during evolution.

 

Motif analysis showed that the motifs contained in most of the three-domain multi-copper oxidases of foxtail millet are more consistent (Figure 3B), but motifs are missing in each subgroup. For example, Si8G112300.1 in Class II-1 lacks motif 10. The absence of class III motifs is the most serious, with only 4 conservative motifs. In addition, the highly clustered Class I-7 genes all contain a high-confidence motif 8, suggesting that this subfamily may have unique biological functions. By comparing motif analysis and domain analysis, it is found that motif 10, motif 6, motif 1, and motif 7 are contained in the Cu-oxidase_3 domain, motif 9, motif 3 are contained in the Cu-oxidase domain, and motif 8, motif 4, Motif 2 and motif 5 are contained in the Cu-oxidase_2 domain. The above results are very helpful for understanding the structural characteristics of the three-domain multi-copper oxidase family genes of foxtail millet.

 

1.6 Expression analysis under different stress treatments

In order to better understand the function of the three-domain multi-copper oxidase gene in foxtail millet, especially under stress, the foxtail millet seedlings at the three-leaf and one-heart stage were treated with abscisic acid (ABA), PEG, high salt, heat and cold stress. A comparative transcriptome analysis was performed on the materials before and after the treatment, and the expression data of the three-domain multi-copper oxidase was extracted from the transcriptome data (Figure 7). The 26 genes have very high basic expression (FPKM value>3). The difference in FPKM value between the experimental group and the control group was 2 times or more as the standard of significant differential expression, and the differentially expressed genes under various treatments were statistically analyzed. Under ABA treatment, most of the three-domain multi-copper oxidase gene expression trends were up-regulated, of which 12 genes were significantly up-regulated, and 1 significantly down-regulated gene was found; under PEG treatment, most of the gene expression trends were also up-regulated, among which 11 genes were significantly up-regulated, and 2 genes were significantly down-regulated; under high salt stress, the three-domain multi-copper oxidase gene of foxtail millet did not show a clear trend of up-regulation or down-regulation, only 3 significantly up-regulated and 3 significantly down-regulated genes were identified. The three significantly up-regulated genes all gradually decreased with the extension of NaCl treatment time, and the three significantly down-regulated genes had the biggest difference when NaCl treatment for 1 h; Under heat shock treatment, the three-domain multi-copper oxidase gene of foxtail millet showed an overall downward-regulation trend. A total of 27 significantly differentially expressed genes were found, of which 22 were significantly down-regulated, 5 were significantly up-regulated, and the significant down-regulated genes were mainly concentrated in Class I-1 And Class I-6, implying that these two subfamilies may have similar functions. All genes that are significantly up-regulated after heat shock are increased with the extension of heat stress treatment time, and the maximum degree of upregulation is at 30 minutes of stress; Under cold stress, the three-domain multi-copper oxidase genes of foxtail millet did not show a clear trend of up-regulation or down-regulation. 5 significantly up-regulated genes and 10 down-regulated genes were found through difference comparison. Among the significantly up-regulated genes, except for Si4G261600, the differences increased with the extension of cold stress treatment time, the up-regulation degree was the largest at 24 h of cold stress treatment; among the 10 significantly down-regulated genes, except for Si8G212000 and Si8G213900, the other genes are all down-regulated with the extension of cold stress treatment time, and the most down-regulated level is at 24 h of cold stress. These results indicate that the three-domain multi-copper oxidase of foxtail millet can respond to a variety of adversity stresses, which is not only conducive to mining the potential stress resistance genes of foxtail millet, but also provides new clues for revealing the stress resistance mechanism of foxtail millet.

 

Figure 7 Gene expression of foxtail millet three domain multi-copper oxidase family genes under different stresses

Note: Orange represents the expression of family gene in the control group; Red represents the expression of family gene under ABA treatment; Green represents the expression of family gene under PEG treatment; Purple represents the expression of family gene under NaCl treatment; Blue represents the expression of family gene under heat treatment; Yellow represents the expression of family gene under cold treatment; The depth of the color corresponds to the level of the expression value (log2(FPKM+1)); The gray color block indicates that the expression was not detected

 

2 Discussion

With the evolution of plants from low to high, the three-domain multi-copper oxidase gene family of plants has a trend of continuous expansion. During the evolution of plants, there have been many whole genome duplication (WGD) events, The WGD event provides material for gene evolution (Wang et al., 2009), and it is speculated that the expansion of the three-domain multi-copper oxidase gene family in plants is also related to WGD. However, in higher plants, there is a large gap in the number of three-domain multi-copper oxidase gene family among closely related species, such as Brassica rapa and Brassica oleracea, foxtail millet and sorghum. It is speculated that all the genes produced by WGD cannot be preserved, there will be many losses, and the rate of gene loss in each species is different (Paterson et al., 2004), which led to the above results. The ancestral species of gramineae experienced a WGD approximately 700 million years ago (Wang et al., 2015b). After that, most of the gramineae species did not experience WGD again. Except for maize, a WGD occurred in maize after speciation (Wang et al., 2015b). After analysis, it is found that Dispresed is the main method of expansion of the three-domain multi-copper oxidase gene family in gramineae, but tandem repeat is also a main method of expansion in foxtail millet, this is the main reason why the number of three-domain multi-copper oxidase genes in foxtail millet is more than that of other grass species. The homology analysis of foxtail millet and rice also confirmed this point. For gramineae, rice largely retains the karyotype of the common ancestor of gramineae, and the genome structure has not undergone huge changes (Wang et al., 2015a), and compared with other grasses, the evolution of rice is slower, so rice is a good model for studying the evolution of grasses (Wang et al., 2015b). The homology analysis between foxtail millet and rice showed that after foxtail millet diverged with the rice species, the three-domain multi-copper oxidase gene family of foxtail millet had more duplications, mainly tandem duplication and most of the tandem duplication occurred after the species differentiation of foxtail millet. The tandemly repeated three-domain multi-copper oxidase genes in foxtail millet are concentrated on chromosome 8, most of which belong to the Class I-7 subclass. The structure of these genes is very conservative and has a unique motif 8. The selection pressure analysis of these genes found 7 amino acid sites subject to positive selection. The results suggest that these three-domain multi-copper oxidases newly produced by tandem repeats in foxtail millet have begun to gradually evolve towards new functions.

 

Expression analysis under adversity stress showed that the three-domain multi-copper oxidase family genes of foxtail millet can respond to a variety of different adversity stresses. The expression patterns of the family genes under ABA and PEG treatments are relatively similar, and the overall expression pattern is up-regulated. Under heat stress, the gene expression of this family showed an overall down-regulation pattern, and among the 22 genes that significantly down-regulated genes, there was an obvious clustering phenomenon, mainly clustering in Class I-1 and Class I-6. This indicates that the genes clustered together in the phylogenetic tree may have similar biological functions. The gene expression of this family under cold stress and salt stress did not show obvious up-regulation or down-regulation as a whole, but several significantly differentially expressed genes were also found. Under the five treatments, there were 27 significantly differentially expressed genes in response to heat shock treatment, and only 6 significantly differentially expressed genes in response to high salt stress, indicating that this family of genes had a strong response to heat stress and a weak response to salt stress. Different genes may show different expression patterns under different stresses, indicating that members of this family in foxtail millet may respond to different stress treatments through different mechanisms of action.

 

The results of this study enable people to better understand the origin, evolution and expansion of the three-domain multi-copper oxidase gene in foxtail millet. At the same time, combined with the expression analysis under adversity stress can provide a theoretical basis for in-depth study of the function of the three-domain multi-copper oxidase gene in foxtail millet, and provide certain clues to improve the stress resistance of foxtail millet.

 

3 Materials and Methods

3.1 Data collection

We downloaded from the JGI database (https://genome.jgi.doe.gov/portal/) 21 representative species in the evolution of plants (5 green algae, 1 Bryophyte, 1 Pteridophyta, 1 basal Angiosperm, 6 monocots and 7 dicots) genome data. And we downloaded the hidden Markov models of Cu-oxidase (PF00394), Cu-oxidase_2 (PF07731) and Cu-oxidase_3 (PF07732) domains from the Pfam database (http://pfam.xfam.org/) (Finn et al., 2013), Using HMMER 3.2.1 software (Mistry et al., 2013), based on the default parameters to search 21 species of homologous proteins containing these three domains, extract the intersection of the three identification results, and search the Pfam and SMART databases (http://smart.embl-heidelberg.de/), only proteins containing these three domains at the same time are identified as members of the three-domain multi-copper oxidase gene family.

 

3.2 Phylogenetic tree construction

Using MAFFT 7.037b (Katoh and Standley, 2013) to compare the amino acid sequence of the three-domain copper oxidase gene family in gramineous plants (maize, foxtail millet, rice, sorghum, barley, and Brachypodium). BMGE software (Criscuolo and Gribaldo, 2010) selected the sequence alignment region suitable for constructing phylogenetic tree inference, and finally we used the NJ method in MEGA7 software (Kumar et al., 2016) to construct the phylogenetic tree of the three-domain multi-copper oxidase gene family of gramineae species, the parameters are set as follows: possion model, Pairwise deletion and 1000 bootstrap duplication. The same method was used to construct the phylogenetic tree of the three-domain multi-copper oxidase gene family of foxtail millet single species.

 

3.3 Identification of homologous gene pairs

OrthoMCL 2.0.9 software (Li et al., 2003) was used to identify the homologous gene pairs of foxtail millet and rice genomes, and the all-against-all BLASTP algorithm was used to calculate the similarity (Blast+2.10.0) (Camacho et al.) al., 2009), parameter settings: e value is set to 1e-10, percent Match Cutoff is set to 75%, and sequences less than 50 amino acids are filtered out. And writing a python script to extract the homologous gene pairs of the three-domain multi-copper oxidase gene between foxtail millet and rice, and finally writing a python program to visualize the results.

 

3.4 Duplication and loss of family genes

MCScanX software (Wang et al., 2012) was used to analyze the duplication patterns of three-domain multi-copper oxidase genes in gramineous plants. And writing a python program to extract the WGD/segmental and tandem gene pairs in foxtail millet, and using the circos 0.96-6 software (Krzywinski et al., 2009) to visualize the results. We used MAFFT 7.037b software to align the amino acid sequences of repeated gene pairs, and import the amino acid sequence alignment file and the corresponding CDS sequence into the PAL2NAL website (http://www.bork.embl.de/pal2nal/) (Suyama et al., 2006), using amino acid sequence alignment to guide the corresponding coding sequence alignment, and finally we used KaKs_calculator 2.0 software (Wang et al., 2010) to calculate the replicated gene pair's non-synonymous substitution rate (Ka) and synonymous substitution rate (Ks) based on the NG method (Nei and Gojobori, 1986) and the coding sequence alignment file. Then we calculated the divergence time of replicated gene pairs according to the formula T=Ks/2r, where r represents the evolution rate of plants, and the annual synonymous replacement of gramineous plants is 6.5ⅹ10-9 (Gaut et al., 1996; Wang et al., 2015b; Yadav et al., 2015; Chai et al., 2018;). Finally, the software Notung 2.9.1.3 (Chen et al., 2000) was used to analyze the duplication and loss of the gene family of grasses.

 

3.5 Selection pressure

Using the Codeml tool in PAML 4.9i software (Yang, 1997) to analyze the selection pressure of Group I-7 family genes based on the site model. M1, M2 and M7, M8 models are used for testing.

 

3.6 Gene structure, motif and domains of the three-domain multi-copper oxidase gene family in foxtail millet

The MEME website (http://meme-suite.org/) (Bailey et al., 2009) was used to analyze the motif of the three-domain multi-copper oxidase gene family in foxtail millet. Parameter setting: Any Number of Repetitions (anr), the number of motif search is set to 10 (Wang et al., 2019). According to the foxtail millet genome physical location file (GFF3), Pfam domain prediction results and MEME prediction results, the self-made CFVisual software was used to graph the results.

 

3.7 The expression of three-domain multi-copper oxidase gene in foxtail millet under different treatments

Five treatments were performed on foxtail millet seedlings at the three-leaf and one-heart stage, namely 100 mmol/L ABA treatment, and the materials were taken at 30 min, 1 h, and 3 h; 20% PEG treatment, and the materials were taken at 30 min, 1 h and 3 h, 1 h; 100 mmol/L NaCl treatment, and the materials were taken at 30 min, 1 h, and 3 h; heat shock treatment at 42°C, and the materials were taken at 5 min and 30 min; cold stress treatment at 4°C, and the materials were taken at 3 h and 24 h. All the materials were whole seedlings, and the experiment biology was repeated 3 times.

 

Authors’ contributions

Wang Xiyin and Chen Huilong are the experimental designers and executives of the experimental research; Chen Huilong and Ge Weina completed the data analysis and the writing of the first draft of the paper; Ge Weina participated in the experimental design and analysis of the experimental results; Wang Xiyin was the person in charge of the project, guiding the experimental design and data Analysis and essay writing and revision. All authors read and approved the final manuscript.

 

Acknowledgments

This research was funded by the Natural Science Foundation of Hebei Province (C2016209097).

 

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