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Article

Gene Structural Specificity and Expression of MADS-Box Gene Family in Camellia chekiangoleosa

by
Pengyan Zhou
1,
Yanshu Qu
1,
Zhongwei Wang
2,
Bin Huang
3,
Qiang Wen
3,
Yue Xin
1,
Zhouxian Ni
1 and
Li’an Xu
1,*
1
Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics and Biotechnology Ministry of Education, Nanjing Forestry University, Nanjing 210037, China
2
Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China
3
Jiangxi Provincial Key Laboratory of Camellia Germplasm Conservation and Utilization, Jiangxi Academy of Forestry, Nanchang 330047, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(4), 3434; https://doi.org/10.3390/ijms24043434
Submission received: 9 December 2022 / Revised: 1 February 2023 / Accepted: 6 February 2023 / Published: 8 February 2023
(This article belongs to the Special Issue Advances in Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

:
MADS-box genes encode transcription factors that affect plant growth and development. Camellia chekiangoleosa is an oil tree species with ornamental value, but there have been few molecular biological studies on the developmental regulation of this species. To explore their possible role in C. chekiangoleosa and lay a foundation for subsequent research, 89 MADS-box genes were identified across the whole genome of C. chekiangoleosa for the first time. These genes were present on all the chromosomes and were found to have expanded by tandem duplication and fragment duplication. Based on the results of a phylogenetic analysis, the 89 MADS-box genes could be divided into either type I (38) or type II (51). Both the number and proportion of the type II genes were significantly greater than those of Camellia sinensis and Arabidopsis thaliana, indicating that C. chekiangoleosa type II genes experienced a higher duplication rate or a lower loss rate. The results of both a sequence alignment and a conserved motif analysis suggest that the type II genes are more conserved, meaning that they may have originated and differentiated earlier than the type I genes did. At the same time, the presence of extra-long amino acid sequences may be an important feature of C. chekiangoleosa. Gene structure analysis revealed the number of introns of MADS-box genes: twenty-one type I genes had no introns, and 13 type I genes contained only 1~2 introns. The type II genes have far more introns and longer introns than the type I genes do. Some MIKCC genes have super large introns (≥15 kb), which are rare in other species. The super large introns of these MIKCC genes may indicate richer gene expression. Moreover, the results of a qPCR expression analysis of the roots, flowers, leaves and seeds of C. chekiangoleosa showed that the MADS-box genes were expressed in all those tissues. Overall, compared with that of the type I genes, the expression of the type II genes was significantly higher. The CchMADS31 and CchMADS58 genes (type II) were highly expressed specifically in the flowers, which may in turn regulate the size of the flower meristem and petals. CchMADS55 was expressed specifically in the seeds, which might affect seed development. This study provides additional information for the functional characterization of the MADS-box gene family and lays an important foundation for in-depth study of related genes, such as those involved in the development of the reproductive organs of C. chekiangoleosa.

1. Introduction

Oil tea is one of the four major types of oil trees in the world and generally refers to any one of several Camellia plant species that are in the Theaceae family and have a high seed oil content. Oil tea species are the most important woody oil plant species in China. Camellia chekiangoleosa Hu, one of the main cultivars in China, has a short fruit ripening period, and its seeds have a high oil content. C. chekiangoleosa is rich in a variety of high-priced unsaturated fatty acids, which are collectively known as “oriental olive oil”. C. chekiangoleosa is also an industrial raw material for soaps, lubricants, and pharmaceuticals. In addition, it is a famous garden ornamental plant species because of its winter and spring flowering, long flowering period, and large and colorful flowers [1]. However, early research of C. chekiangoleosa mainly focused on germplasm resource conservation, genetic diversity analysis, molecular marker-assisted breeding, and other aspects [2,3,4]. There are few reports on the molecular biology of this species, such as that which drives developmental regulation.
Transcription factors (TFs) play a variety of roles throughout the life cycle of higher plants [5]. TFs bind to cis-acting regulatory sequences and are involved in plant growth, development, morphogenesis, and stress responses [6]. The MADS-box gene family is a large family whose members encode TFs, and four major MADS-box genes encoding TFs have been discovered: MCM1 of Saccharomyces cerevisiae [7], AG of Arabidopsis thaliana [8], DEF of Antirrhinum majus [9], and SRF of humans [7]. Based on their structure and evolutionary developmental state, MADS-box genes can be divided into two subtypes: SRF-like (type I) and MEF2-like (type II) [10]. The type I genes can be further divided into three subfamilies: Mα, Mβ, and Mγ. However, few type I genes have biological functions. Only a small number of type I genes, which affect the reproductive development of A. thaliana, have been reported. In contrast, the type II genes have been studied more in depth and extensively. Type II genes, which are also known as the MIKC type due to their common structure, can be further divided into two subtypes (MIKCC and MIKC*) based on different structural features [11]. The MIKCC subfamily can be divided into at least 14 different subclasses, such as STK, PI, AGL12, and SEP [12,13].
Previous studies have shown that MIKCC-type genes play an important role in plant floral organ development [14,15]. Molecular and genetic analysis subdivided these genes into five different classes (A, B, C, D, and E), to specify the identity of sepals (A), petals (A + B + E), stamens (B + C + E), carpels (C + E), and ovules (D) [12,15,16]. The genes belonging to the above five functional categories in A. thaliana include APETALA1 (AP1) and AP2 in class A, PISTILATA (PI) and APETALA3 (AP3) in class B, AGAMOUS (AG) in class C, SEEDSTICK/AGAMOUS-LIKE 1 (STK/AGL11) and SHATTERPROOF (SHP) in class D, and SEPALLATA (SEP1, SEP2, SEP3, SEP4) genes in class E [17,18,19,20,21].
The expression of type II genes in organs such as fruits and seeds is also common. Type II genes play an important role in seed development [22,23]. Type E (SEP) genes not only play a role in the development of flower organs and the maintenance of meristem characteristics in tomato (TM29) and strawberry (FaMADS9) but also regulate the development and maturation of their fruits [24,25]. The type D (AGL11/STK) gene STK promotes the association between seeds and fruits in A. thaliana [21]. After fruit ripening has occurred, the type A gene AP1 and the type D genes SHP1 and SHP2 together regulate valve separation of fruits, allowing seed dispersal [26].
Genome-wide identification analysis and functional characterization of the MADS-box gene family have been performed on many organisms [27,28]. However, there have been no reports of these on C. chekiangoleosa. In this study, we analyzed the whole-genome data of C. chekiangoleosa. With the help of a bioinformatics platform and real-time quantitative techniques, whole-genome identification and analysis of the MADS-box gene family of C. chekiangoleosa were carried out. Aiming to provide a basis for the reproductive organ development of C. chekiangoleosa at the genetic level, we also measured gene expression.

2. Results

2.1. Identification and Analysis of Physicochemical Properties of MADS-Box Proteins

According to the results of a BLAST alignment and the hidden Markov model (HMM), 111 MADS-box genes were preliminarily screened from the whole genome of C. chekiangoleosa. Based on the MADS-box model of A. thaliana, a total of 89 domain-intact MADS-box proteins were obtained. The protein sequence is shown in File S1 of the Supplementary Materials. They all had a conserved MADS domain at the N-terminus, and this domain consisted of approximately 59 amino acid sequences. Multiple sequence alignments of the MADS domains of the 89 proteins and sequence icons (Figure S1) revealed four highly conserved amino acids (aa) (aa 21, 25, 32, and 39). According to the alignment results (Figure S2), we found that the difference between type I and type II MADS-box domains mainly involved differences in N-terminal aa; moreover, the domain of type II MADS-box proteins was more conserved.
The physicochemical properties of the MADS-box proteins of C. chekiangoleosa showed that the aa of the 89 MADS-box proteins ranged from 102 to 1528 aa (Table 1). The sequence length of most CchMADS proteins (61.8%) was 200~300 aa. In total, 24.7% of the proteins were between 100 and 200 aa in length, and the rest were greater than 300 aa. CchMADS76 was the longest (1528 aa). The predicted molecular weight was from 11.521 to 176.716 kDa, and the predicted isoelectric point ranged from 4.92 to 10.48. Subcellular localization predicted that all the proteins were localized in the nucleus.

2.2. Phylogenetic Analysis of CchMADS Proteins

A phylogenetic tree was constructed based on the MADS-box genes of A. thaliana, Camellia sinensis, and C. chekiangoleosa (Figure 1), and the results showed that the CchMADS proteins could be divided into two categories: type I and type II. The type I proteins could be further divided into three subfamilies, namely, Mα (27), Mβ (2), and Mγ (9), and the type II proteins could be divided into two subfamilies, namely, MIKCC (45) and MIKC* (6). The type of MIKCC subfamily in CchMADS genes was shown in Table S1.
Although the total number of CchMADS genes in C. chekiangoleosa was similar to that in C. sinensis (83) (Table S2), C. sinensis had more type I genes, and there were fewer type II MIKCC genes than type II CchMADS genes. Moreover, there were significant differences in the number of genes in the Mβ and MIKCC subfamilies. In addition, although the total number of MADS-box genes (106) in A. thaliana was much higher than that in C. chekiangoleosa and the number of genes in each type I subfamily was higher, the number of type II MIKCC genes was lower in A. thaliana than in C. chekiangoleosa.

2.3. Gene Structure and Motif Analysis

Based on the results of our gene structure analysis (Figure 2c), it was found that among the 38 type I genes, 21 did not have introns. Four genes (CchMADS33, CchMADS39, CchMADS76, CchMADS87) contained 4-12 introns, and the remaining 13 genes contained 1~2 introns. Among the 51 type II genes, each contained at least one intron. Except for 8 genes (CchMADS11, CchMADS23, CchMADS35, CchMADS63, CchMADS70, CchMADS82, CchMADS83, CchMADS84) containing 1-4 introns, the number of introns of the remaining 43 genes were between 6 and 11. Overall, the average intron numbers of the type II genes (6.3) were much higher than those of the type I genes (1.6). In addition, we found that the length of introns for the different genes also varied greatly. The introns of CchMADS54, which were only 7 kb, were the longest among those of the type I genes. Among the type II genes, 39.2% of the introns were larger than 10 kb, and 11 genes, namely, CchMADS24, CchMADS31, CchMADS32, CchMADS36, CchMADS42, CchMADS44, CchMADS45, CchMADS55, CchMADS56, CchMADS72, and CchMADS80, had super large introns (≥15 kb). The length of these introns far exceeded that of the other genes.
In this study, a total of 10 conserved motifs of CchMADS proteins—motifs 1–10—were identified (Figure 2b). The results showed that motif 1, motif 2, and motif 4 were widely present in all CchMADS proteins. They were MADS domains, and motif 1 was the classic MADS domain. In addition, motif 3 only appeared in Mα and MIKCC. However, the type I proteins were quite different. Motif 6 was endemic to the Mγ subfamily, and motif 7, motif 9, and motif 10 were endemic to the Mα subfamily. The MIKCC proteins were more conserved. Motifs 5 and 8 were specific to MIKCC proteins. Motif 5 was a highly conserved K domain motif.

2.4. Chromosomal Localization and Duplication of CchMADS Genes

Chromosomal localization was based on data within gff3 annotation files. We found that the 86 CchMADS genes were unevenly distributed on 15 chromosomes (Figure 3). These genes were named CchMADS01 to CchMADS86 according to their chromosomal localization. Only three CchMADS genes (CchMADS87, CchMADS88, CchMADS89) could not be mapped to any chromosome. The results showed that the proportion of genes on the 15 chromosomes was between 2.25% and 14.61%. Chromosomes 3, 5, 13, and 15 had the fewest CchMADS genes (2), whereas chromosome 4 had the most genes (13).
There was one pair of tandemly duplicated genes (CchMADS41 and CchMADS42) (CchMADS52 and CchMADS53) (Table S3) on chromosomes 7 and 8, respectively. One pair (CchMADS88 and CchMADS89) of tandemly duplicated genes could not be mapped to any chromosome. In addition, 22.5% of segmentally duplicated genes were located on different chromosomes (Figure 4). Many duplicate sequences were detected on different chromosomes, which may be one of the driving forces of gene evolution. Their nonsynonymous (Ka) and synonymous (Ks) substitution rates were analyzed (Table S2), and it was found that all Ka/Ks values were less than 1, indicating that they evolved under purifying selection.

2.5. Cis-Acting Elements of MADS-Box Gene Family-Associated Promoters

To further study the regulatory mechanism of the MADS-box gene family in terms of the development of C. chekiangoleosa, cis-acting elements were analyzed (Figure S3). It was found that approximately 50 cis-acting elements could be effectively expressed, and the analysis revealed 21 elements with clear functions. Each gene had more than three light-responsive elements, which constituted the most abundant type (994), followed by hormone-responsive elements (509), including abscisic acid response elements, gibberellin response elements, methyl jasmonate (MeJA) response elements, etc. Most of the other response elements (303) were related to fruit and seed development, including circadian rhythm control, endosperm expression, and seed-specific regulation. This might mean that CchMADS genes play an important role in the reproductive growth of C. chekiangoleosa. The lowest number of cis-acting elements were involved in responses to abiotic stress (140), including drought stress, calli, etc.

2.6. Protein–Protein Interaction Network of CchMADSs

To elucidate the biological function and regulatory network of CchMADS proteins, a CchMADS protein–protein interaction network was predicted via the homologous MADS-box proteins of A. thaliana (Figure 5). Seventeen CchMADS proteins (CchMADS08, 31, 67, 40, 38, 82, 43, 60, 02, 47, 56, 70, 84, 58, 10, 35, 62, respectively) homologous to those in A. thaliana and 26 corresponding interacting functional genes (AP1, 3-Sep, AGL15, AGL24, AGL18, AGL65, AGL104, AGL12, PI, AGL2, AGL80, AGL6, AGL61, AGL38, AGL62, TT16, AP3, AGL42, AG, STK, SVP, AGL20, AGL8, AGL21, SHP2, AGL19, respectively) were identified. Among them, CchMADS40 and CchMADS67 were type I genes, and the rest belonged to the type II MIKCC subfamily. In addition, with the exception of CchMADS67, the remaining CchMADS proteins were found to interact with more than one protein, and seven homologous proteins (CchMADS62, CchMADS31, CchMADS82, CchMADS43, CchMADS47, CchMADS56, CchMADS84) could interact with more than 10 other MADS proteins. Most of these proteins, such as AGL6, SVP, and TT16, are mainly related to the development of flowers. These proteins (such as SHP2, STK, and AGL38) are not only related to flower development but also affect fruit development, maturity, and seed dispersal.

2.7. Expression of CchMADS Genes in Different Tissues

To further confirm the expression patterns of CchMADS genes in different organs and predict their potential role in plant growth and development, 18 genes were selected to explore their expression patterns in the roots, flowers, leaves, and seeds of C. chekiangoleosa. The information of primer sequence is shown in Table S4, the relative expression of CchMADS genes in four tissues is shown in Table S5. In Figure 6, the red block indicates high expression, and the blue indicates low expression. Overall, the expression of type I genes in all four tissues was lower than that of type II genes. Among the type I genes, the members of the Mβ and Mγ subfamilies (CchMADS13, CchMADS20, CchMADS40, CchMADS77) were expressed at very low levels in the four tissues. The genes of the Mα subfamily (CchMADS39, CchMADS7, CchMADS21) were slightly more highly expressed, especially CchMADS39 in the seeds, whose expression was relatively high. Among the type II genes, the expression of CchMADS12 in the leaves was relatively high, and CchMADS10 was highly expressed in the roots and leaves. Notably, CchMADS31 and CchMADS58 showed high expression specifically in the flowers, and similarly, CchMADS55 showed high expression specifically in the seeds, which might mean that these type II genes play important roles in the development of reproductive organs.

3. Discussion

3.1. Number and Characteristics of CchMADS Genes

After verification via the SMART program, 89 MADS-box gene family members (CchMADS1 to CchMADS89) with intact MADS domains were ultimately identified in C. chekiangoleosa. However, these genes were not evenly distributed on the chromosomes. Zhang [29] found a similar number of genes in C. sinensis (83), which was more than that in Salix suchowensis (60) [30] and Sesamum indicum (57) [31] but less than that in A. thaliana (106) [32] and poplar (105) [33]. The genome sizes of these species varied (C. sinensis, 3 Gb; C. chekiangoleosa, 2.73 Gb; S. suchowensis, 356 Mb; S. indicum, 337 Mb; A. thaliana, 207 Mb; poplar, 431 Mb). In general, the number of gene family members is related specifically to genome size. However, some species seem to be unrelated, which may be the result of complex historical events, such as genome duplication, but the specific reasons need to be further explored.
Our constructed phylogenetic tree that referred to the classification of A. thaliana [32] revealed that the proportion of type II genes in C. chekiangoleosa (57.3%) was significantly higher than that in C. sinensis (43.4%) and A. thaliana (47.4%). This meant that type II CchMADS genes experienced a higher duplication rate or a lower gene loss rate after genome duplication. Although the proportion of type II genes was quite different, the MIKCC subfamily (45 members) of C. chekiangoleosa had the most genes with homologs in C. sinensis and A. thaliana. This indicated that the genes of the MIKCC subfamily were conserved between different species. In addition, through the subfamily classification in the MIKCC group (Table S1), we found that the gene numbers of AGL17, SOC1, and SEP subfamilies in C. chekiangoleosa (8, 8, 5, respectively) was much higher than that of C. sinensis (3, 5, 2, respectively) [29]. There were no genes of the AGL6, AGL12, and Bsister subfamilies in C. sinensis. In previous research of A. thaliana, AGL17, SOC1, SEP, and AGL6 affect the flower organs [19,34,35], AGL12 affects root cell differentiation, and Bsister affects ovule and seed development [36,37]. These homologous genes in C. chekiangoleosa may also play a similar role, resulting in the characteristics of C. chekiangoleosa with large flowers and fruits.
Other subfamilies were more varied, and the most obvious was the Mβ subfamily. The Mβ gene family of C. chekiangoleosa was 1/6 that of C. sinensis and poplar and only 1/10 that of A. thaliana. Gene loss might have occurred during the evolutionary process because the Mβ genes in C. chekiangoleosa failed to play an important role. Similar results were found in sesame and soybean [31,38]. Therefore, compared with other CchMADS genes, the MIKCC genes may have undergone more duplication and differentiation in C. chekiangoleosa, while other CchMADS genes have been severely lost.
In addition, we found that the longest amino acid sequence of a MADS-box protein in C. chekiangoleosa was 1528 aa (CchMADS76), which was smaller than that in other species, such as Malus domestica (the longest being 593 aa) [22], Setaria italica L. (the longest being 477 aa) [28], and Solanum lycopersicum (the longest being 389 aa) [39]. In C. sinensis [29], a sequence of up to 2691 aa was found, which has not been reported in other species. Both genes belong to the Mα subfamily, which may be unique to the Camellia genus.

3.2. Expansion of the CchMADS Gene Family

Gene duplication was thought to be the product of errors in DNA replication and reconstruction. The copied genes may exert new functions and enhance the ability of plants to adapt to the environment [40,41]. In this study, both segmentally duplicated (18 pairs) and tandemly duplicated (3 pairs) gene pairs belonged to the Mα and MIKCC subfamilies. The proportion of Mα subfamilies (54%) was relatively high. This is similar to findings of a study of soybean, where tandem duplication and segment duplication of type I genes were found to have occurred more frequently than those of type II genes [38]. This may be because type I genes originated and differentiated later than did type II genes, and type II genes were more conserved. Ka and Ks values are considered important indicators for studying the selection pressure or strength of protein-coding genes [42]. By calculating the Ka and Ks values, we found that the CchMADS genes evolved under the pressure of purifying selection. Segmental duplication and tandem duplication may be driving forces of gene family expansion and play an important role in functional gene diversity [43].

3.3. Intron Specificity of CchMADS Genes

The greater the number and length of introns, the more diverse the number of ways in which genes are spliced, thus affecting gene expression and protein activity [44,45,46]. The structure of type I genes of C. chekiangoleosa was simple, and most of them had no or only one intron, which is similar to the genes of A. thaliana. Compared with the type II genes of C. sinensis, the type I genes had more introns (average of 3.8). The reasons might be that this species has more type I genes and greater variation. The difference was that the number of introns (6.3) and the proportion of introns greater than 10 kb (39.2%) in type II CchMADS genes far exceeded those in C. sinensis (3.6 and 4.8%, respectively). A high proportion of type II genes was not found in other species, such as A. thaliana and S. suchowensis. Therefore, these type II genes may play important role in C. chekiangoleosa.
It has been suggested that genes with super large introns and transposons are more highly expressed [47,48]. Compared to only 3 MIKCC genes in C. sinensis, more than 10 MIKCC genes with super large introns (≥15 kb) in C. chekiangoleosa were found, but this was not the case in other species. MIKCC genes play an important role in plant flower organ development, flowering time duration, and sex determination of male and female flowers [49]. The presence of super large introns in Camellia plant species, especially in C. chekiangoleosa, may have contributed to the specificity of Camellia plants. The morphology and size of the flowers and fruits of C. chekiangoleosa are quite different from those of other species, and the number of introns and longer length of its type II genes mean that the expression of genes is relatively diverse. These characteristics may affect the expression of MIKCC genes in the reproductive organs of C. chekiangoleosa and play an important role in the formation of flower morphology and size.

3.4. Gene Expression and Potential Function

According to the results of qPCR, the AGL6-like gene CchMADS31 and PI-like gene CchMADS58 were highly expressed, specifically in flowers. The AGL104-like gene CchMADS55 was expressed specifically in the seeds, and all of these genes were type II. It is believed that AGL6 is involved in the gene regulation of floral meristems. PI affects the development of petals and stamens and is responsible for regulating the expression of genes associated with flower development [50,51]. AGL104 double mutants have defects in pollen viability and pollen tube growth, resulting in delayed germination and reduced fertility [52]. Therefore, CchMADS31 and CchMADS58 may regulate the flower meristem of C. chekiangoleosa and affect the size of the petals, and CchMADS55 may affect seed development. In addition, the type II genes CchMADS10 and CchMADS12 were also highly expressed in the roots and leaves, which meant that some type II genes not only play important roles in the reproductive organs of C. chekiangoleosa but also participate in the development of roots, leaves, and other tissues.

4. Materials and Methods

4.1. Identification of C. chekiangoleosa MADS-Box Genes

The C. chekiangoleosa genomic data were published by our team in 2022 and are publicly accessible at https://ngdc.cncb.ac.cn/gwh (accessed on 17 January 2022). The A. thaliana MADS-box protein sequences were obtained from The Arabidopsis Information Resource (TAIR) database (http://www.arabidopsis.org/, accessed on 10 January 2022) and utilized for BLASTP (E value = 1 × 10−20) searches against the sequences of proteins of C. chekiangoleosa. Moreover, the HMM profile of the MADS-box domain (Pfam accession PF00319) was downloaded from the Pfam database (http://pfam.xfam.org/, accessed on 11 January 2022) and then used to retrieve the MADS-box protein sequences from all the annotated genes of the C. chekiangoleosa genome via the HMMER program version 3.0 [53].
All the candidate protein sequences were assessed based on the presence of the conserved domain via the SMART program (http://smart.embl-heidelberg.de/, accessed on 20 January 2022) [54], Conserved Domain Database (CDD) search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 21 January 2022), and PlantTFDB (http://planttfdb.gao-lab.org/, accessed on 22 January 2022) [55]. Sequences that incorrectly occupied or did not carry an entire domain were removed from the MADS-box genes. In addition, ExPASy (https://web.expasy.org/protparam/, accessed on 10 February 2022) [56] was used to analyze the physical and chemical properties of the MADS-box proteins, and Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 12 February 2022) [57] was used for subcellular localization predictions.

4.2. Multiple Alignment and Phylogenetic Analysis

A sequence logo of the identified C. chekiangoleosa MADS-box genes was generated using WebLogo3 (http://weblogo.threeplusone.com, accessed on 16 February 2022) with the default parameters [58]. We subsequently used ClustalX version 2.1 to perform a multisequence alignment of the MADS-box domains, and ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi, accessed on 18 February 2022) was then used to visualize the resulting alignment.
A phylogenetic tree was constructed by the maximum likelihood (ML) method in MEGA version 11.0 [59]. The MADS-box protein sequences of C. sinensis were acquired from the article of Zhang [29]. Finally, the network profile of the phylogenetic tree was visualized by iTOLs version 6 (https://itol.embl.de/, accessed on 12 March 2022) [60].

4.3. Chromosomal Localization and Gene Structure Analysis

The distribution of the 89 MADS-box genes and gene density were visualized using TBtools version 1.098745 [61]. The conserved motifs were analyzed using MEME (http://meme-suite.org/, accessed on 20 March 2022) [62]. The parameters were set to a repeat motif site of any number, a maximum number of motifs of 10, and a width of each motif ranging from 6 to 60 residues. An exon/intron map was constructed in the Gene Structure Display Server program (http://gsds.cbi.pku.edu.cn/, accessed on 21 March 2022) [63].

4.4. Gene Duplication and Promoter Cis-Acting Regulatory Element Analysis

MCScanX [64] was used to analyze gene duplication. Multi-sequence and BLASTp alignments (E-value = 1 × 10−20) were performed to obtain the similarities between these CchMADS genes. The major criteria used for analyzing potential gene duplications included the following: (a) the length of sequence that can be aligned covers 75% of the longer gene, and (b) the similarity of aligned regions covers 75% [65]. When a duplicated gene pair constituted two consecutive genes on the same chromosome, it was considered a tandemly duplicated gene pair. The Ka and Ks values were determined via KaKs_Calculator [42].
The upstream regions (2000 bp) of the start codon (ATG) of the MADS-box genes were used as the gene promoter sequence and retrieved from the C. chekiangoleosa genome, and its cis-acting elements were analyzed using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 2 April 2022) online software. The results were visualized by TBtools.

4.5. Protein–Protein Interaction Network Analysis

The MADS-box protein interaction network of C. chekiangoleosa was analyzed using the online website String (https://string-db.org/, accessed on 12 April 2022). The protein interaction network was visualized using Cytoscape version 3.9.1 software [66].

4.6. Plant Materials and Expression Analysis in Different Tissues

The experimental qPCR materials, which included 5-year-old live seedlings, were derived from the germplasm resource garden of the Zhongshan Botanical Garden of Jiangsu Province (118°69′ N, 32°51′ E). Seeds were collected in August 2021, and roots, flowers, and leaves were collected in March 2022. Total RNA was isolated using an RNA kit (RNAprep Pure Plant Kit, Tiangen, Beijing, China), the extraction procedures could be found in the manufacturer’s instructions. The quality and concentration of RNA samples were determined by the NanoDrop 2000 c spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and 1% agarose gel electrophoresis. cDNA was synthesized from 1000 ng of total RNA in a 20 μL reaction volume using PrimeScriptTM RT Master Mix (TaKaRa, Dalian, China). We took 3 different biological replicates per tissue and froze them in liquid nitrogen. Then, we stored them in a −80 °C freezer. The 2−ΔΔCT method was used to calculate the relative expression in the various tissues. The expression levels were log10 standardized, and Heml version 1.0 software (http://hemi.biocuckoo.org/down.php, accessed on 20 May 2022) was used to construct an expression profile heatmap of the CchMADS genes.

5. Conclusions

In this study, a total of 89 MADS-box genes were identified in C. chekiangoleosa. Fragment duplication and tandem duplication were the driving forces for the expansion of MADS-box family members. These genes could be divided into type I and type II, of which there were 38 and 51 genes, respectively. The proportion of type II genes in C. chekiangoleosa was higher than that in the other species analyzed. Through phylogenetic, conserved motif, and other analyses, we found that the structure of the type II genes is more conserved than that of the type I genes. The presence of superlong amino acid sequences may be an important feature of the Camellia genus. Compared with type I genes, type II genes are present in a higher proportion, have a higher number of introns, and have longer introns in the genome of C. chekiangoleosa. The superlong introns of many genes of the MIKCC subfamily may indicate increased gene expression. Further qPCR analysis found that the overall expression level of the type II genes was significantly higher than that of the type I genes. Some type II genes were highly expressed specifically in the reproductive organs, indicating that these genes may be involved in regulating their developmental process. In addition, we found evidence of the expression of the MADS-box genes in the roots and leaves. This study provides additional information for the functional characterization of the MADS-box gene family. At the same time, it establishes an important foundation for the in-depth study of reproductive organ development and other related genes of C. chekiangoleosa.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24043434/s1.

Author Contributions

Conceptualization, L.X. and P.Z.; methodology, Y.Q. and P.Z.; formal analysis, P.Z.; investigation, Q.W. and B.H.; resources, Z.W.; data curation, Z.N. and Y.X.; writing—original draft preparation, P.Z.; writing—review and editing, P.Z.; visualization, P.Z.; supervision, L.X.; project administration, L.X.; funding acquisition, Q.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX22_1112), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the National Natural Science Foundation of China (32201592), the Key Research and Development Program of Jiangxi Province, China (20201BBF61003), the Science and Technology Innovation Bases Program of Jiangxi Province, China (20212BCD46002), the Basic Research and Talent Development Project of Jiangxi Academy of Forestry (2022511001), the Doctor Initial Project of JiangXi Academic of Forestry (2021521001), and Oil-tea special research project of Jiangxi Provincial Department of Forestry (YCYJZX20220103).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Ijms 24 03434 g001 550
Figure 1. Phylogenetic tree comprising MADS-box proteins of C. chekiangoleosa, C. sinensis, and A. thaliana. The red circles represent CchMADS proteins. The light pink color indicates the Mβ subfamily, the light orange color indicates the Mγ subfamily, the light grey color indicates the MIKC* subfamily, the pink color indicates the Mα subfamily, and the light green color indicates the MIKCC subfamily.
Figure 1. Phylogenetic tree comprising MADS-box proteins of C. chekiangoleosa, C. sinensis, and A. thaliana. The red circles represent CchMADS proteins. The light pink color indicates the Mβ subfamily, the light orange color indicates the Mγ subfamily, the light grey color indicates the MIKC* subfamily, the pink color indicates the Mα subfamily, and the light green color indicates the MIKCC subfamily.
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Figure 2. (a) The phylogenetic classification of the CchMADS proteins. (b) Motif analysis. The different colors of boxes represent different motif numbers. The length of a box indicates the motif length. (c) Structure of CchMADS genes.
Figure 2. (a) The phylogenetic classification of the CchMADS proteins. (b) Motif analysis. The different colors of boxes represent different motif numbers. The length of a box indicates the motif length. (c) Structure of CchMADS genes.
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Figure 3. Chromosomal locations of CchMADS genes. The number of each chromosome is given above the lines.
Figure 3. Chromosomal locations of CchMADS genes. The number of each chromosome is given above the lines.
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Figure 4. Fragment duplication of CchMADS genes, with the red lines connecting fragmentally duplicated gene pairs.
Figure 4. Fragment duplication of CchMADS genes, with the red lines connecting fragmentally duplicated gene pairs.
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Figure 5. Protein–protein interactions between CchMADSs. (a) Blue color indicates A. thaliana genes homologous to the CchMADS genes. (b) Red color represents CchMADSs.
Figure 5. Protein–protein interactions between CchMADSs. (a) Blue color indicates A. thaliana genes homologous to the CchMADS genes. (b) Red color represents CchMADSs.
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Figure 6. Relative expression of CchMADS genes in different tissues. The color scale represents relative expression levels from high (orange color) to low (blue color).
Figure 6. Relative expression of CchMADS genes in different tissues. The color scale represents relative expression levels from high (orange color) to low (blue color).
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Table
Table 1. Amino acid composition and physiochemical properties of CchMADS proteins.
Table 1. Amino acid composition and physiochemical properties of CchMADS proteins.
Gene NameSequence IDLocationLength (aa)MW (kDa)pISubcellular LocalizationTypeIntron
CchMADS01Cole01G001968.1Chr122225.8236.47NucleusMIKCC6
CchMADS02Cole01G003080.1Chr122925.9975.75NucleusMIKCC7
CchMADS03Cole01G003276.1Chr152258.7235.46NucleusMIKC*11
CchMADS04Cole01G003279.1Chr134939.5034.96NucleusMIKC*10
CchMADS05Cole01G003924.1Chr120824.2669.48NucleusMIKCC6
CchMADS06Cole01G004072.1Chr125229.4368.76NucleusMIKCC6
CchMADS07Cole01G004142.1Chr119321.3339.37NucleusMα0
CchMADS08Cole02G001598.1Chr226329.7949.01NucleusMIKCC5
CchMADS09Cole02G002131.1Chr230734.6145.54NucleusMIKC*8
CchMADS10Cole02G002392.1Chr224628.2338.93NucleusMIKCC7
CchMADS11Cole02G002395.1Chr222325.6379NucleusMIKCC4
CchMADS12Cole02G002503.1Chr232937.9346.64NucleusMIKC*8
CchMADS13Cole02G003948.1Chr252459.9646.19NucleusMβ1
CchMADS14Cole02G004095.1Chr224228.2038.44NucleusMIKCC6
CchMADS15Cole02G004285.1Chr224928.6529.02NucleusMIKCC7
CchMADS16Cole02G004286.1Chr224829.0037.08NucleusMIKCC7
CchMADS17Cole02G004715.1Chr224027.7377.75NucleusMIKCC6
CchMADS18Cole03G000406.1Chr328933.3888.89NucleusMIKCC6
CchMADS19Cole03G001914.1Chr321625.3796.77NucleusMIKCC6
CchMADS20Cole04G000929.1Chr412113.5919.83NucleusMα0
CchMADS21Cole04G000931.1Chr421924.9059.49NucleusMα0
CchMADS22Cole04G000947.1Chr421924.2479.08NucleusMα0
CchMADS23Cole04G001061.1Chr411412.8339.61NucleusMIKCC2
CchMADS24Cole04G001062.1Chr423927.4756.41NucleusMIKCC7
CchMADS25Cole04G001125.1Chr421825.0048.61NucleusMIKCC7
CchMADS26Cole04G001870.1Chr420624.2179.2NucleusMγ1
CchMADS27Cole04G001878.1Chr423026.6469.55NucleusMγ1
CchMADS28Cole04G002096.1Chr419722.1977.69NucleusMα0
CchMADS29Cole04G003100.1Chr426230.0476.13NucleusMIKCC7
CchMADS30Cole04G003823.1Chr425228.9189.77NucleusMγ0
CchMADS31Cole04G004469.1Chr424828.1818.83NucleusMIKCC7
CchMADS32Cole04G004470.1Chr421724.8246.47NucleusMIKCC6
CchMADS33Cole05G000003.1Chr533238.97310.14NucleusMγ4
CchMADS34Cole05G000013.1Chr510612.35910.12NucleusMγ0
CchMADS35Cole06G000488.1Chr613114.6249.16NucleusMIKCC2
CchMADS36Cole06G000572.1Chr621324.6288.8NucleusMIKCC6
CchMADS37Cole06G000576.1Chr621724.7957.05NucleusMIKCC7
CchMADS38Cole06G002198.1Chr625929.6669.2NucleusMIKCC8
CchMADS39Cole06G002899.1Chr628932.7058.65NucleusMβ10
CchMADS40Cole07G000640.1Chr732637.2739.82NucleusMγ2
CchMADS41Cole07G001913.1Chr721725.0536.39NucleusMIKCC6
CchMADS42Cole07G001920.1Chr721725.0426.18NucleusMIKCC6
CchMADS43Cole07G004098.1Chr724428.5478.44NucleusMIKCC7
CchMADS44Cole07G004743.1Chr720823.7099.49NucleusMIKCC6
CchMADS45Cole07G004746.1Chr725328.6158.74NucleusMIKCC7
CchMADS46Cole08G000724.1Chr826128.4326.76NucleusMα0
CchMADS47Cole08G000781.1Chr823326.7119.78NucleusMIKCC8
CchMADS48Cole08G002218.1Chr820222.7498.45NucleusMα0
CchMADS49Cole08G002233.1Chr810311.5219.56NucleusMα0
CchMADS50Cole08G002238.1Chr820523.5516.72NucleusMα0
CchMADS51Cole08G002611.1Chr810711.8539.96NucleusMα1
CchMADS52Cole08G002612.1Chr821924.0539.32NucleusMα0
CchMADS53Cole08G002613.1Chr821123.1329.12NucleusMα0
CchMADS54Cole08G002614.1Chr814115.7259.94NucleusMα1
CchMADS55Cole09G000962.1Chr934238.9055.65NucleusMIKC*10
CchMADS56Cole09G001241.1Chr923727.3049.38NucleusMIKCC6
CchMADS57Cole09G001278.1Chr932335.8386.85NucleusMIKCC9
CchMADS58Cole09G001941.1Chr920824.3729.11NucleusMIKCC6
CchMADS59Cole09G001965.1Chr930635.9939.35NucleusMIKCC9
CchMADS60Cole09G002445.1Chr924528.2759.22NucleusMIKCC9
CchMADS61Cole09G004317.1Chr925028.5924.92NucleusMγ0
CchMADS62Cole10G000146.1Chr1024227.6838.96NucleusMIKCC7
CchMADS63Cole10G001610.1Chr1012814.4449.9NucleusMIKCC1
CchMADS64Cole10G003622.1Chr1024427.6148.24NucleusMIKCC7
CchMADS65Cole11G000155.1Chr1120823.1415.22NucleusMα0
CchMADS66Cole11G000294.1Chr1126330.2979.35NucleusMα2
CchMADS67Cole11G000296.1Chr1111412.38110.48NucleusMα1
CchMADS68Cole11G000299.1Chr1113615.1997.92NucleusMα1
CchMADS69Cole11G000477.1Chr1121324.6867.66NucleusMIKCC6
CchMADS70Cole11G003071.1Chr1115217.3259.32NucleusMIKCC2
CchMADS71Cole12G000125.1Chr1219922.8387.02NucleusMIKCC6
CchMADS72Cole12G000456.1Chr1227931.4389NucleusMIKCC8
CchMADS73Cole12G000684.1Chr1228232.2918.63NucleusMIKCC7
CchMADS74Cole12G000760.2Chr1224626.9726.54NucleusMα0
CchMADS75Cole12G001299.1Chr1221924.1019.25NucleusMα0
CchMADS76Cole12G001525.1Chr121528176.7169.12NucleusMα12
CchMADS77Cole12G002412.1Chr1225729.3445.7NucleusMγ0
CchMADS78Cole12G002414.1Chr1221324.1745.3NucleusMγ1
CchMADS79Cole12G003350.1Chr1218420.5166.45NucleusMα1
CchMADS80Cole13G001797.1Chr1322625.8526.09NucleusMIKCC6
CchMADS81Cole13G003210.1Chr1314917.17110.44NucleusMα1
CchMADS82Cole14G000617.1Chr1412414.3149.67NucleusMIKCC3
CchMADS83Cole14G000851.1Chr1410212.0189.78NucleusMIKC*1
CchMADS84Cole14G001528.1Chr1411313.2349.84NucleusMIKCC2
CchMADS85Cole15G000774.1Chr1521924.6829.72NucleusMα0
CchMADS86Cole15G000995.1Chr1514115.6486.32NucleusMα0
CchMADS87Cole00G064455.1ChrN43948.725.9NucleusMα7
CchMADS88Cole00G064458.1ChrN18420.5055.75NucleusMα1
CchMADS89Cole00G064466.1ChrN14115.5366.32NucleusMα0
MW: molecular weight (kDa), PI: isoelectric point.
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MDPI and ACS Style

Zhou, P.; Qu, Y.; Wang, Z.; Huang, B.; Wen, Q.; Xin, Y.; Ni, Z.; Xu, L. Gene Structural Specificity and Expression of MADS-Box Gene Family in Camellia chekiangoleosa. Int. J. Mol. Sci. 2023, 24, 3434. https://doi.org/10.3390/ijms24043434

AMA Style

Zhou P, Qu Y, Wang Z, Huang B, Wen Q, Xin Y, Ni Z, Xu L. Gene Structural Specificity and Expression of MADS-Box Gene Family in Camellia chekiangoleosa. International Journal of Molecular Sciences. 2023; 24(4):3434. https://doi.org/10.3390/ijms24043434

Chicago/Turabian Style

Zhou, Pengyan, Yanshu Qu, Zhongwei Wang, Bin Huang, Qiang Wen, Yue Xin, Zhouxian Ni, and Li’an Xu. 2023. "Gene Structural Specificity and Expression of MADS-Box Gene Family in Camellia chekiangoleosa" International Journal of Molecular Sciences 24, no. 4: 3434. https://doi.org/10.3390/ijms24043434

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