Abstract
To reveal the molecular function of elongation family of very long chain fatty acids(ELO) protein in Cyrtotrachelus buqueti, we have identified 15 ELO proteins from C.buqueti genome. 15 CbuELO proteins were located on four chromosomes. Their isoelectric points ranged from 9.22 to 9.68, and they were alkaline. These CbuELO proteins were stable and hydrophobic. CbuELO proteins had transmembrane movement, and had multiple phosphorylation sites. The secondary structure of CbuELO proteins was mainly α-helix. A total of 10 conserved motifs were identified in CbuELO protein family. Phylogenetic analysis showed that molecular evolutionary relationships of ELO protein family between C. buqueti and Tribolium castaneum was the closest. Developmental transcriptome analysis indicated that CbuELO10, CbuELO13 and CbuELO02 genes were key enzyme genes that determine the synthesis of very long chain fatty acids in pupae and eggs, CbuELO6 and CbuELO7 were that in the male, and CbuELO8 and CbuELO11 were that in the larva. Transcriptome analysis under different temperature conditions indicated that CbuELO1, CbuELO5, CbuELO12 and CbuELO14 participated in regulating temperature stress responses. Transcriptome analysis at different feeding times showed CbuELO12 gene expression level in all feeding time periods was significant downregulation. The qRT-PCR experiment verified expression level changes of CbuELO gene family under different temperature and feeding time conditions. Protein-protein interaction analysis showed that 9 CbuELO proteins were related to each other, CbuELO1, CbuELO4 and CbuELO12 had more than one interaction relationship. These results lay a theoretical foundation for further studying its molecular function during growth and development of C. buqueti.
Similar content being viewed by others
Introduction
Cyrtotrachelus buqueti belongs to Coleoptera. This insect is mainly distributed in the southwest of China, coastal areas of Guangdong and Shanghai, and other countries in South east Asia, such as Vietnam and Thailand [1,2,3]. The insect is completely metamorphosed and needs to go through four stages: adult, egg, larva and pupa [4]. Adults supplement nutrition by feeding on bamboo shoots. The eggs of the insect are laid under the shells of the bamboo shoots. After hatching, the larvae feed on the bamboo shoots and bite through the bamboo shoots. They pupate in the soil and overwinter as adults. It only takes 15 days, and C. buqueti is an oligophagous insect, mainly feeding on the tips of young bamboo shoots that grow in clusters [5,6,7,8]. It is one of the main pests of bamboo forests, and it was listed as one of the main pests in my country’s forestry by the State Forestry Administration in 2003 [9]. At present, the research on C. buqueti focuses on the digestive system structure studied by Luo et al [10], the respiratory system structure studied by Deng et al [11], the reproductive system structure studied by Li et al [12], the habits studied by Díaz-García et al [13], the morphological structure studied by Li et al [14], and the control methods studied by Yang et al [15], but there is a lack of genome-wide identification of elongation family of very long chain fatty acids protein of C. buqueti.
Elongation family of very long chain fatty acids protein is called FAE in plants [16], in insects and fungal yeast, called ELO [17], and in fish and mammals, named ELOVL [18], it is the rate-limiting condensing enzyme in the first step of the fatty acid extension reaction [17]. The condensation and extension process of the carbon chain during the synthesis of very long-chain fatty acids (VLCFA) is a cyclic process similar to the β-oxidative reverse reaction. The four-step cycle of condensation, hydrogenation, dehydration, and rehydrogenation completes the extension of carbon chain, among which the extension of fatty acid carbon chain mainly occurs in mitochondria, endoplasmic reticulum, and peroxisome [19]. One of the condensation reactions is catalyzed by ELO, where malonic acid monoacyl CoA provides two carbon atoms to extend the fatty acid carbon chain, and the distance between the active site and the lysine residue determines the length of the ELO fatty acid [20]. This shows that ELO plays a crucial role in the regulation of fatty acid metabolism and the biosynthesis of lipids. Among insect field studies, Chertemps et al. reported the ELO gene of insects for the first time and named it elo68α and an ELO specifically expressed in female Drosophila, named eloF [16,17,18,19,20,21]. Studies on ELO have focused on Drosophila melanogaster, which mainly include effects in ELO on fertility during the formation of fertilized eggs [22, 23], on epidermal functions [24], on locomotor functions, reducing their viability [25], on oenocyte [26], and on the synthesis of pheromones such as esters, hydrocarbons and alcohols in pheromone synthesis. It was found that ELO also had an effect on epidermal transcriptional function in Apis mellifera [27]. Juárez studied the function of ELO in the epidermis of Blatella germanica [28]. It has also been slightly reported in Tenebrio molitor, among which ELO can interfere with the genes of Tenebrio larvae, affecting the growth of T. molitor and resulting in an increase in T. molitor mortality [29].
At present, the research on C. buqueti mainly focuses on the morphological structure, living habits, and control methods. However, there are relatively few studies on the genetics of C. buqueti, mainly the karyotype analysis of Wang et al [30]. So far, apart from our research team completing the whole genome sequencing of C. buqueti and publishing its genome sequence, there is no research report on elongation family of very long chain fatty acids protein(CbuELO) in C. buqueti genome. This study aims to systematically identify and investigate the molecular mechanisms of CbuELO gene family in its fatty acid synthesis process and its involvement in temperature stress response and energy conversion processes. In this study, we have three objectives. Firstly, we have completed the identification of ELO gene family in the entire genome of C. buqueti. Secondly, we systematically analyze the genetic characteristics, protein characteristics, and molecular evolutionary relationships of CbuELO gene family. Thirdly, transcriptome sequencing was performed under different temperature and feeding time treatments to reveal the molecular mechanisms by which the CbuELO gene family regulates its temperature response and energy conversion. In this study, the physicochemical properties, subcellular localization, transmembrane domain structure, protein hydrophilicity and hydrophobicity, phosphorylation site situation, protein secondary structure, protein tertiary structure, conserved structure, chromosomal location and molecular relationship of CbuELO proteins were systematically analyzed by using bioinformatics software and websites. These research results will lay a certain theoretical foundation for further studying the biological functions of C. buqueti.
Materials and methods
Data acquisition and experimental design
All the whole genome sequences, protein sequences, gene annotation files, transcriptome sequencing data of adults and larvae of C. buqueti under different temperature conditions, and transcriptome sequencing and proteome sequencing data of C. buqueti at different feeding times were downloaded from these data (NCBI genome and transcriptome accession number: PRJNA675312, PRJNA719467 and PRJNA718062) submitted to NCBI database by Chun Fu et al [31]. The adults and larval samples of C. buqueti used in this study were collected in Danan Town, Muchuan County, Leshan City, Sichuan Province, and the adults and larvae of C. buqueti were used for genome sequencing, transcriptome sequencing and proteome sequencing, respectively. Adults (female and male) and larvae of C. buqueti were treated at different temperature (25 °C in the control group, 4 °C, 42 °C and 50 °C in the treatment group) and different feeding times (no feeding in the control group (0 h), 0.5 h after feeding fresh bamboo shoots, 1 h after feeding, and 2 h after feeding in the treatment group) were all carried out in the Molecular Biology Laboratory of Key Laboratory of Sichuan Province for Bamboo Pests Control and Resource Development of Leshan Normal University from July 15 to August 15, 2020. The ELO gene sequences and protein sequences of all insect species used in this study were downloaded from the InsectBase2.0 database (http://v2.insect-genome.com/) by searching for them and used for bioinformatics analysis [32] (Supplementary Table S1). The expression data of ELO gene family in C. buqueti genome comes from the expression data in the developmental transcriptome of C. buqueti studied by Yang et.al [33]. The total RNA was extracted from muscle tissue samples of C. buqueti under different temperature treatments and intestinal contents samples of C. buqueti under different time conditions after feeding. Reverse transcription of purified RNA into cDNA using a reverse transcription kit, and reverse transcribed cDNA was used for qRT-PCR to verify the expression of CbuP450 genes in C. buqueti under different temperature treatments and different time conditions after feeding. The qRT-PCR experiments in this study were all completed on fluorescence quantitative PCR instrument (qToWer3 G) of the Analytick Jena AG. The number of replicates of biological samples in each treatment group and control group is 3, and the number of machine replicates on fluorescence quantitative PCR is 3.
Identification, chromosomal localization and gene structure of ELO gene family in C. buqueti
The Pfam model of ELO gene family was used to retrieve all the protein sequences of ELO gene family in C. buqueti genome, and then combined with SMART tool to retrieve the conserved structural domains of all the protein member sequences identified by Pfam searching, and the sequences without ELO conserved structural domains were removed to be the ELO gene family. Only by searching for Pfam model of ELO gene and searching for its conserved domain, can the gene sequence of both be preliminarily identified as a member of the ELO gene family. At the same time, the preliminarily identified ELO gene sequence needs to be verified in conjunction with ELO gene members in the genome of the model insect Drosophila melanogaster, and the qualified sequence is the true member of CbuELO gene family. The chromosomes and gene start positions of ELO genes were obtained from the gff information of C. buqueti, and then the chromosomal localization analysis of CbuELO gene family was performed using MapChart software, and the maps were saved in PDF format. Gene Structure Display Server 2.0 (http://gsds.gao-lab.org), an online website, was used to analyze the exons and introns in the gene structure.
Analysis of physicochemical properties and subcellular localization of CbuELO proteins
The EXPASY-ProtParam tool (https://web.expasy.org/protparam/) was used to predict physicochemical properties of CbuELO proteins. The molecular weight size, number of amino acids, instability index, aliphatic amino acid index, isoelectric point, total average hydrophilicity, and total number of positively and negatively charged residues of the protein can be analyzed. Subcellular localization was performed by using CELLO: Subcellular (http://cello.life.nctu.edu.tw/).
Transmembrane domain, hydrophilic analysis and phosphorylation site prediction of CbuELO proteins
Transmembrane domain analysis of CbuELO proteins was performed by using TMHMM-2.0-Services (https://services.healthtech.dtu.dk/service.), and EXPASY-Proscale (https://web.expasy.org/protscale/) was used for hydrophilic analysis of CbuELO proteins [34]. Phosphorylation sites were predicted using NetPhos-3.1-Services (https://services.healthtech.dtu.dk/service.) with input in protein sequence fasta format, default parameters, and output saved in PDF format [35].
Analysis of secondary structure, tertiary structure, and conserved structure of CbuELO proteins
The secondary structure of CbuELO proteins was predicted by using SOPMA oneline tool (https://npsa-prabi.ibcp.fr/), the tertiary structure of CbuELO proteins was predicted by using SWISS-MODEL (https://swissmodel.expasy.org/interactive) and MEME-tool (https://meme-suite.org/) was used to analyze the conserved structure of CbuELO proteins, with the initial number of motifs set to 10 and the input protein sequence in fasta format, and finally saved in PDF format [36, 37].
Phylogenetic tree construction of CbuELO proteins
In order to understand evolutionary relationship between CbuELO genes and those of other species, the protein sequences of ELO genes in the same target species were downloaded from the online website InsectBase, and the evolutionary tree was constructed by MEGA11 software, whose protein sequences were in fasta format. The phylogenetic tree of ELO protein sequences of multiple species was constructed using the maximum likelihood method (Maximum Likelihood, ML) [38,39,40].
SSR and codon bias analysis of CbuELO genes
SSR sites of CbuELO genes were identified by using MISA (https://webblast.ipk-gatersleben.de/misa/) and default parameters (SSR modes: 1–10, 2–6, 3–5, 4–5, 5–5, and 6 − 5; The maximum sequence length that can be registered as a composite SSR between two SSRs is 100 bp). CodonW software was used to analyze codon preference parameters of CbuELO genes, including the GC ratio of codon, the proportion of ATGC (A3s, T3s, G3s, T3s) on the third position of the codon, the GC (GC3S) ratio of the 3rd position of the synonymous codon, the codon bias index (CBI), the codon adaption index (CAI), The results of frequency of optimal codon (FOP), effective number of codon (ENC, NC) and relative synonymous codon usage (RSCU), which were analyzed by Origin and ggplot2 softwave.
Analysis of ELO gene expression in C.buqueti
The first experimental design was: a series of temperatures (25 °C, 4 °C, 42 °C, 50 °C) were set to treat the larvae, females and males of C.buqueti for 1 h, and set up 3 biological replicates respectively. The second experimental design was: both adults and larvae of C.buqueti were fed fresh bamboo shoots, while the control group was not fed (0 h), and the treatment group was fed at 0.5 h, 1 h, and 2 h after feeding, with three biological replicates set up. All samples from the treatment and control groups of two experiments were used for transcriptome sequencing and qRT-PCR analysis. Transcriptome sequencing data and gene expression data validated by qRT-PCR experiment of all muscle tissue and intestinal contents samples of C.buqueti were plotted by OmicShare Tools (https://www.omicshare.com/tools/Home/Task). All CbuELO gene primers designed by TBtools Batch qRT-PCR primer design tool used in qRT-PCR validation experiment in this study are shown in Supplementary Table S2. The qRT-PCR primers used in this study were synthesized by Sangon Biotech (Shanghai) Co., Ltd on commission. The relative gene expression levels of CbuELO gene family obtained through qRT-PCR under different temperatures and feeding conditions were plotted in Excel and presented in a bar chart to visualize the relative gene expression levels.
Protein-protein interaction analysis of CbuELO proteins
The transcription and translation of genes into proteins to exercise biological functions requires further research on whether the proteins encoded within individual gene family members will regulate or interact with each other. Moreover, studying protein-protein interactions within individual gene family members can help understand how protein members directly or indirectly interact with each other and what role they play in regulating gene expression. The ELO protein sequences of C.buqueti were imported into the string website (https://cn.string-db.org/), and the protein interaction diagram was obtained by selecting the reference species of Tribolium castaneum with the default parameters.
Results
Identification and chromosomal localization of ELO genes in C. buqueti
Through the identification of ELO genes in C. buqueti, it was known that there were 15 ELO genes in C. buqueti, which were named as CbuELO01-CbuELO15 according to the gene order (Supplementary Table S3). Chromosomal localization analysis of CbuELO gene family by using MapChart software showed that CbuELO genes were located on chr0, chr5, chr8 and chr9, among which chr0 accounted for the most, with 11 CbuELO genes, whose genes were CbuELO01-CbuELO11, respectively. CbuELO12 was located on chr5, CbuELO13 and CbuELO14 were located on chr8, and CbuELO15 was located on Chr9, respectively (Fig. 1) [41].
Chromosome mapping of ELO gene family in C. buqueti
The analysis rusults of CbuELO gene structure showed that the sequence length and coding length of each CbuELO gene were not identical to each other, with the sequence length of 15 CbuELO genes ranging from 1958 to 31756 bp, and the coding length ranging from 863 to 29913 bp. From Fig. 2, it can be seen that all CbuELO genes contained exons (CDS); all CbuELO genes had introns except CbuELO12 which had no introns; all CbuELO genes had non-coding regions except CbuELO04 which had no non-coding region (UTR). Each CbuELO gene contained 1 to 10 exons, 0 to 9 introns and 0 to 2 non-coding regions. Among them, CbuELO14 contained the largest number of exons and introns, 10 and 9, respectively (Fig. 2).
Gene structure of ELO gene family in C. buqueti
Physicochemical properties analysis of CbuELO proteins
Based on the amino acid sequence of CbuELO, the physicochemical properties (molecular weight, isoelectric point, instability, etc.) of CbuELO proteins were analyzed by using EXPASY-ProtParam tool. The results showed that the number of amino acids in CbuELO proteins ranged from 219 to 368 aa, CbuELO protein containing the highest number of amino acids was CbuELO03 with 368 aa. CbuELO protein containing the lowest number of amino acids was CbuELO02 with 219 aa, while the average amino acid content of CbuELO protein family was 285.4 aa. the molecular weights of CbuELO protein family ranged from 25.99 to 43.33 kD. The largest molecular weight was CbuELO03 with 43.33 kD. the smallest molecular weight is CbuELO with 25.99 kD. while the average molecular weight of the whole CbuELO protein family was 33.67 kD. the theoretical isoelectric point of CbuELO protein family was between 9.22 and 9.68. It can be concluded that the whole CbuELO protein family was basic, and this result was consistent with ELO gene family of T.olitor [29]. Based on the theoretical instability index greater than 40 were unstable proteins and less than 40 are stable proteins. From this, six of the unstable proteins were CbuELO03, CbuELO04, CbuELO06, CbuELO07, CbuELO10 and CbuELO12. The rest were all stable proteins, and overall the number of stable proteins was greater than that of unstable proteins. The overall content of aliphatic amino acid index ranged from 72.28 to 120.22, indicating that the thermal stability of CbuELO protein family varied widely among them. Analyzed in terms of the total average hydrophilicity, negative values represent hydrophilicity, positive values represent hydrophobicity, and absolute values represent their hydrophilic size [42]. Except for CbuELO03 protein, which was hydrophilic, all other CbuELO proteins were hydrophobic, and the overall average coefficient of hydrop hilicity of CbuELO protein family was 0.433, indicating that it was hydrophobic [43]. From the analysis of the number of positively and negatively charged residues, the total number of positively charged residues (Arg + Lys) of the overall CbuELO protein family was much larger than the total number of negatively charged residues (Asp + Glu), indicating that the overall CbuELO protein family was positively charged. The subcellular localization analysis results showed that CbuELO protein family was localized on plasma membrane, indicating that CbuELO protein family played a biological role on the cell membrane(Table 1).
Transmembrane domain, hydrophobicity, and phosphorylation site prediction of CbuELO proteins
The transmembrane domain analysis of CbuELO protein family showed that each member of CbuELO protein family had some number of transmembrane structures, with proteins CbuELO01, CbuELO003, CbuELO05 ~ CbuELO08, and CbuELO15 contained the largest number of transmembrane structures. Therefore, it can be inferred that CbuELO protein family was transmembrane proteins(Table 2; Fig. 3).
The transmembrane domain analysis of CbuELO proteins from C. buqueti
The hydrophilicity and hydrophobicity analysis results of CbuELO protein family showed that the maximum hydrophobicity of this protein family ranged from 2.311 to 3.378, and CbuELO05 had the maximum value of 3.378. While the maximum hydrophilic range was between − 3.344 and − 2.067, CbuELO01 had the smallest value of -3.344 (Supplementary Table 4). From the absolute value of the score representing the hydrophilicity and hydrophobicity, it can be seen that the number of hydrophobic amino acid residues was much larger than that of hydrophilic amino acid residues (Supplementary Fig. S1). It can be seen that CbuELO proteins were hydrophobic proteins, so it can be inferred as an insoluble protein.
The prediction of phosphorylation sites showed that CbuELO protein family had several different phosphorylation sites, and overall the protein family had 247 serine phosphorylation sites, 125 threonine phosphorylation sites, and 102 tyrosine phosphorylation sites. CbuELO03 protein contained the most phosphorylation sites, 51, while CbuELO10 contained the least phosphorylation sites with 15. The most serine phosphorylation site was CbuELO12 with 29, while the least number was member CbuELO13 with 9. The most likely phosphorylation site was located on 27 and 276 with a value of 0.996 (much larger than the threshold value of 0.500); the most threonine phosphorylation sites was CbuELO13 and CbuELO14 with 13, and the least abundant was CbuELO10 with only 1, of which the most likely phosphorylation site was located on 85 with a value of 0.964; the most abundant tyrosine phosphorylation site was CbuELO03 with 16, while the least abundant was CbuELO10 with only 1, of which the most likely phosphorylation site was located on 147 with a value of 0.973 (Table 3).
Secondary structure, tertiary structure and conserved structure of CbuELO proteins
The secondary structure analysis results of CbuELO proteins showed that overall secondary structure of CbuELO protein family was dominated by alpha helix, consisting of 2064 amino acids, accounting for 48.51% of the whole; the extended strand consists of 802 amino acid residues, accounting for 18.72% of the whole; beta turn consists of 126 amino acid residues, accounting for 2.98% of the whole; and the random coil consists of 1289 amino acid residues, accounting for 29.79% of the whole. Among α-helix, CbuELO10 protein accounted for the highest proportion, with 55.84% being α-helix, composed of 129 amino acid residues, while the least was CbuELO14 protein, 42.39% was α-helix, composed of 156 amino acid residues; In the extended strand structure, CbuELO04 protein accounted for the highest proportion, with 24.64% of the extended strand structure composed of 65 amino acid residues, while CbuELO13 with the smallest proportion was only 15.13% of extended strand structure, composed of 46 amino acid residues Among β-turns, CbuELO12 had the highest proportion, with 4.53% being β-turn, consisting of 13 amino acid residues; while the least proportion was CbuELO01, with only 1.67% being β-turn, consisting of 5 amino acids residue composition; in random coils, CbuELO05 had the highest proportion, with a content of 35.92%, consisting of 111 amino acid residues, while the smallest proportion was CbuELO10, with a content of 22.08%, consisting of 51 amino acid residues composition (Supplementary Table S5, Supplementary Fig. S2).
The tertiary structures analysis results showed that the tertiary structures of CbuELO proteins were divided into four categories according to the degree of structural similarity, and each graph was represented by A, B, C, D, E, F, G, H, I, G, K, L, M, N, and O in order for easy observation. Firstly, the first category was I, J, K, L, M, N, O, with the largest number of seven, corresponding to proteins CbuELO04, CbuELO06-CbuELO08, CbuELO10, CbuELO13, CbuELO15; the second category was E, F, G, H, corresponding to proteins CbuELO09, CbuELO11, CbuELO12, CbuELO14; the third category was B, C and D, corresponding to proteins CbuELO01, CbuELO03 and CbuELO05, respectively; the fourth category A has only one corresponding to protein CbuELO02. From the tertiary structure diagram, it can be seen that the α-helix and random coil were the main structural elements in CbuELO protein family, while β-turn was scattered sporadically, which point of structural prediction was consistent with the secondary structure analysis [44] (Supplementary Fig. S3).
The conserved structures analysis result of CbuELO proteins showed that 10 conserved motifs were identified in CbuELO protein family, with motif lengths ranging from 10 to 50 aa. The four most important conserved motifs in CbuELO protein family were motif1, motif2, motif3 and motif4, among which motif4 was present in each CbuELO protein. CbuELO06-CbuELO09 contain the most conserved motifs, all with nine, while the least conserved motif is protein CbuELO12 with only one conserved motif [45]. This suggests that CbuELO12, a member of CbuELO gene family, may be a pseudogene as it has lost an important conserved motif module. The molecular function of CbuELO12 gene may have lost its structural gene function during its evolution, and may play a regulatory role in the gene. In the previous literature, histidine-box HXXHH was reported as important motif, the study found that only CbuELO4 and CbuELO12 do not contain histidine-box HXXHH.(Supplementary Table S6, Supplementary Fig. S4).
Phylogenetic evolution and expression analysis of CbuELO genes
The CbuELO protein family evolutionary tree was constructed by MEGA11 software with the calibration parameter Bootstrap repeated 1000 times. The results showed that the 15 CbuELO proteins were divided into 3 subfamilies (labeled as Group1, Group2 and Group3 accordingly) according to their aggregation degree in the evolutionary tree. Among them, Group1 had the most family members with six CbuELO proteins, accounting for 40% of the whole; while Group2 had the least family members with four CbuELO proteins, accounting for 26% of the whole. The homology of the genes can be obtained according to the bootstrap values of the evolutionary tree, and it can be seen that CbuELO02 and CbuELO10, CbuELO06 and CbuELO07, CbuELO11 and CbuELO15 had 100% bootstrap values. In the whole family Group2 was the most primitive, while Group3 was the fastest evolving. In CbuELO gene family of C. buqueti genome, CbuELO06 and CbuELO07 had the fastest evolutionary rate and were the least conserved. CbuELO02 and CbuELO10 had the slowest evolutionary rate and were the most conserved(Fig. 4).
Phylogenetic tree of ELO genes in C. buqueti
Phylogenetic trees were constructed from ELO protein sequences of C. buqueti and Dendroctonus ponderosae. The evolutionary relationships between the two were divided into four groups (labeled Group1, Group2, Group3, and Group4). There were four CbuELO members in Group 1 as CbuELO01, CbuELO08, CbuELO09 and CbuELO14; four CbuELO members in Group 2 as well as CbuELO03, CbuELO06, CbuELO07 and CbuELO10; and three in Group 3 as CbuELO05, CbuELO09 and CbuELO14. There were 4 CbuELO proteins in Group 4, namely CbuELO02, CbuELO04, CbuELO11 and CbuELO15. while the number of ELO proteins in Dendroctonus ponderosae was 3, 1, 4, and 1 in Groups 1 to 4, respectively. Homologous gene pairs, such as CbuELO08 with DpoELO07, CbuELO05 with DpoELO05 and DpoELO06, and CbuELO12 with DpoELO04, were found based on evolutionary tree bootstrap values, and the bootstrap values of these gene pairs were greater than 95%. Moreover, CbuELO08 and DpoELO07 had the fastest evolutionary rate, CbuELO04 and DpoELO08 had the slowest evolutionary rate and were the most conserved(Fig. 5).
Phylogenetic tree of ELO gene family in C. buqueti and Dendroctonus ponderosae
Phylogenetic trees were constructed from the ELO protein sequences of C. buqueti and Rhynchophorus ferrugineus. Based on the evolutionary analysis, the evolutionary relationships between the two were divided into four groups (labeled Group1, Group2, Group3, Group4). There were five CbuELO members in Group 1 as CbuELO01, CbuELO08, CbuELO09, CbuELO12, CbuELO13; two CbuELO members in Group 2 also as CbuELO02, CbuELO05; two CbuELO members in Group 3 as CbuELO03, CbuELO10, and the number of ELO proteins in R. ferrugineus was 5, 3, 1 and 4 in Groups 1 to 4, respectively. CbuELO12 and RfeELO03, CbuELO13 and RfeELO04, CbuELO09 and RfeELO10, RfeELO12, CbuELO05 and RfeELO01, RfeELO07, RfeELO08 had bootstrap values greater than 99%, Moreover, CbuELO12 and RfeELO03 had the fastest evolutionary rate, CbuELO06 and RfeELO11 had the slowest evolutionary rate and were the most conserved (Fig. 6).
Phylogenetic tree of ELO gene family in C. buqueti and Rhynchophorus ferrugineus
Phylogenetic tree was constructed from ELO protein sequences of C. buqueti and Tribolium castaneum, and their evolutionary relationships were studied. According to the evolutionary analysis, the evolutionary relationship of ELO protein sequences of C. buqueti and T.castaneum was divided into 5 groups (marked as Group1, Group2, Group3, Group4, Group5). In Group 1, there was 1 CbuELO member, CbuELO05; in Group 2, there were also 3 CbuELO members, namely CbuELO08, CbuELO12, and CbuELO13; in Group 3, there were 5 CbuELO members, namely CbuELO04, CbuELO06, CbuELO07, CbuELO011, and CbuELO15; in Group 4, there was 1 with CbuELO02; in Group 5, there were also 5 CbuELO members, namely CbuELO01, CbuELO03, CbuELO09, CbuELO10 and CbuELO12. The number of ELO proteins of T. castaneum in Groups 1 to 5 were 8, 1, 4, 1, and 4, respectively. Among them, Group2 was the most primitive, and Group1 evolves the fastest. According to the bootstrap value of the phylogenetic tree, the orthologous gene pairs of C. buqueti and Tribolium castaneum, such as CbuELO05 and TcaELO14, CbuELO12 and TcaELO02, and CbuELO09 and TcaELO01. The bootstrap values of these gene pairs were all 100%, Moreover, CbuELO05 and TcaELO14 had the fastest evolutionary rate, CbuELO09 and TcaELO01 had the slowest evolutionary rate and were the most conserved (Fig. 7).
Phylogenetic tree of ELO gene family in C. buqueti and Tribolium castaneum
Construction of phylogenetic tree between ELO family proteins of C. buqueti and that of D. ponderosae, R. ferrugineus, Hypothenemus hampei, Elaeidobius kamerunicus, Ips nitidus, Ips typographus, and Sitophilus oryzae. According to the evolutionary analysis, the evolutionary relationship between them was divided into 5 groups marked as (Group1, Group2, Group3, Group4, Group5). In Group 1, there were 2 CbuELO members, CbuELO05 and CbuELO12; in Group 2, there were also 5 CbuELO members, namely CbuELO06, CbuELO07, CbuELO11, CbuELO13, and CbuELO15; in Group 4, there were 5 CbuELO members, CbuELO01CbuELO4, CbuELO09; in Group 5, there are 3 CbuELO proteins namely CbuELO10, CbuELO08, and CbuELO14. From the whole phylogenetic tree, the ELO family proteins of C. buqueti had the closest relation ship with T. castaneum, in addition to D. ponderosae and S. oryzae [46] (Fig. 8).
Phylogenetic tree of ELO gene family in C. buqueti and all species of Curculionidea
The phylogenetic tree was constructed from ELO family proteins of C. buqueti and all the ELO sequences of Coleoptera, and a total of eight subfamilies (tagged with Group1, Group2, Group3, Group4, Group5, Group6, Group7 and Group8, respectively) were classified according to the evolutionary relationships between CbuELO and all the ELO family proteins of Coleoptera. Among them, CbuELO family proteins had 3 members in Group1; 1 member in Group3; 2 members in Group5, 3 members in Group7; Group8 contained the most members with 6. As can be seen from Supplementary Fig. S5, Group1 evolved the fastest, while Group8 was the most primitive. Moreover, these results showed that CbuELO gene family was closely related to ELO gene family of the red palm weevil.
SSR analysis of CbuELO gene family
A total of 40 SSRs were found in 15 unigenes, distributed in 11 unigenes, and the total length of the sequences was less than 174906 bp, including 10 sequences containing more than 1 SSR. There were only three types of transcriptome SSRs, and the frequency of each type was also different, with the largest number of single nucleotide repeats, with a total of 34, all of which were A/T (85.00%), followed by dinucleotides and trinucleotides, with 3 respectively, 2 (5.00%) AT/AT, 1 (2.50%) AG/CT, and 3 (7.50%) AAT/ATT. In addition, it was found that there was also a certain variation in the number of repeats of SSRs, with the highest frequency of 10 repetitions (27), 7 repetitions of 11 repetitions, followed by 5 repetitions and 6 repetitions, both of which were 3, and the frequency of repetitions of 7, 8, and 9 was the lowest (Supplementary Table S7,Supplementary Table S8).
Codon bias analysis of CbuELO gene family
CodonW software was used to analyze the codon bias of ELO genes in C. buqueti, and the content of codon at the third position of different genes was different, the total GC ratio was 31.20%~37.50%, the mean value was 34.75%, the proportion of GC3s was 28.00%~36.40%, the mean value was 32.33%, and the proportions of T3s, C3s, A3s and G3s were 36.67%~46.14%, 18.65%~24.99% and 36.84%~48.48%, respectively, 18.42%~ 26.10%, the mean values were 42.50%, 21.55%, 43.43% and 22.27%, respectively, A3s > T3s > G3s > C3s, indicating that the content of the 3rd base AT of the synonymous codon in the genome of C. buqueti was high. The CAI value was 0.152 ~ 0.185, the mean value was 0.169, the CBI was − 0.153~-0.088, the mean value was − 0.111, the Fop value was 0.315 ~ 0.370, the mean value was 0.347, the Nc value was 49.47 ~ 56.57, and the mean value was 53.87, all of which were greater than 35, indicating that the codon preference in the genome of C. buqueti was weak (Fig. 9).
There were 33 codons with RSCU values ≥ 1.00, which were UUU, UUA, UUG, CUU, AUU, AUA, AUG, GUU, GUA, UCU, UCA, CCU, CCA, ACU, ACA, GCU, GCA, UAU, UAA, CAU, CAA, AAU, AAA, GAU, GAA, UGU, UGG, CGU, CGU, AGU, AGA, GGU and GGA, 14 of which ended in A, 12 ended in U, and only 3 ended in G, UUA had the highest RSCU value, was 1.92, these codons were the preferred codons in C. buqueti genome, indicating that the high-frequency codons in C. buqueti genome prefer to end in U or A. At the same time, the RSCU values of 31 codons < 1, and most of the low-frequency codons end in C (16) or G (13) (Fig. 10).
Codon bias-related parameters of CbuELO genes
RSCU values of CbuELO genes
Gene expression analysis of CbuELO genes under developmental transcriptome, different temperature and feeding time conditions
The developmental transcriptome analysis results of tissues and organs in different developmental stages of C. buqueti, including male, female, larva, pupae and egg, showed that among 15 ELO genes of C. buqueti, the gene expression contents of CbuELO10, CbuELO13 and CbuELO02 genes in pupa and egg were high, that of CbuELO6 and CbuELO7 in male were high, The expression contents of CbuELO01, CbuELO03, CbuELO04, CbuELO05, CbuELO09, CbuELO12, CbuELO14 and CbuELO15 in females were high, and the expression contents of CbuELO8 and CbuELO11 in larvae were high. It can be seen that CbuELO10, CbuELO13 and CbuELO02 genes were the key enzyme genes that determine the synthesis of very long chain fatty acids in pupae and eggs, CbuELO6 and CbuELO7 were the key enzyme genes that determine the synthesis of very long chain fatty acids in the male of C. buqueti, and CbuELO8 and CbuELO11 were the key enzyme genes that determine the synthesis of very long chain fatty acids in the larva of C. buqueti, The remaining eight ELO genes were the key enzyme genes that determine the synthesis of very long chain fatty acids in the female of C. buqueti(Supplementary Fig. S6).
The larvae, females and males of C. buqueti were treated at different temperatures (4 °C, 25 °C, 42 °C, 50 °C), and their muscle tissues were taken for transcriptome sequencing after 1 h, and the obtained data were used as heat maps. The results showed that the average value of larvae was 15.11 at 25 °C, and changed to 14.49 at 4 °C, and the expression of larvae for temperature reduction was down-regulated, with CbuELO12 being the most significant down-regulation, from 86.61 at 25 °C to 81.47 at 4 °C, followed by CbuELO14, from 30.60 at 25 °C to 25.59 at 4 °C, and CbuELO5 and CbuELO1, which were significantly down-regulated, with 3.46 and 3.21 respectively.
For adults, the temperature increased to 42 °C, the temperature continued to increase, and the temperature decreased to 4 °C. For females, the average value at 25 °C was 10.51 and 42 °C was 14.72, with the most significant upward adjustment being CbuELO14 (25.99) and CbuELO12 (32.58). The gene expression of males changed more significantly with temperature, with an average value of 0.27 at 4 °C, and the most significant down-regulation was CbuELO12 (65.02), followed by CbuELO14 (14.71). The average value at 42 °C was 32.58, with the most significant upward revision being CbuELO13 with an upward revision of 165.03, followed by CbuELO11 with an upward revision of 125.29. The average value at 50 °C was 5.75, with the most significant downward revision being CbuELO12 with a downward revision of 54.22, followed by CbuELO14 with a downward revision of 16.03.
In terms of individual genes, the expression of CbuELO12 was the highest at different temperatures compared to other genes, and although there was a large change in temperature increase, it was still greater than that of other genes. The female and male worms were 15.11 at 25 °C, and the female and male worms at 42 °C were 14.72 and 32.58, respectively, compared with 25 °C, the low temperature had little effect on the overall gene expression of larvae, and the male worms were more tolerant to high temperature than females, and the gene expression of males showed an upward trend with the increase of temperature, and the gene expression of males had a higher expression for the increase of temperature (Fig. 11).
Heat map of gene expression analysis of CbuELO genes under different temperature
Taking the time interval (0 h, 0.5 h, 1 h, 2 h) after feeding (0 h, 0.5 h, 1 h, 2 h) of female and male elephants as variables, three parallel experiments were performed in each group, and the intestinal contents were taken for transcriptome sequencing to obtain their transcriptional expression, and the data were used as heat maps.
In males, the gene with high expression was CbuELO14, with a maximum value of 9118.33 in 2 h after eating, and the gene was up-regulated with the increase of feeding time interval, followed by CbuELO12, with a maximum value of 8491.67 after 2 h of feeding. Compared with other genes, the gene expression of CbuELO7 gene was the weakest, indicating that it may have little effect on the degradation of bamboo fiber. Compared with the genes that had just eaten for 0 h, the down-regulated genes accounted for the majority, and there were three genes that had the largest transcriptome gene expression at 0.5 h after eating, including CbuELO1, CbuELO6 and CbuELO7, among which the gene CbuELO1 was the most significantly up-regulated. It was up-regulated by 319.00, CbuELO6 by 93.00 and CbuELO7 by 5.33, and 12 genes were down-regulated, including CbuELO13 by 1899.00, CbuELO14 by 1515.00 and CbuELO11 by 1311.67. There were 7 genes with the largest transcriptome gene expression 1 h after eating, including CbuELO1, CbuELO3, CbuELO6, CbuELO7, CbuELO8, CbuELO12, and CbuELO14, among which CbuELO12 was the most significantly up-regulated. It was revised up by 834.33, followed by CbuELO1 with 440.33, and CbuELO13 by 681.67 and CbuELO11 by 714.00. There were 11 genes that were up-regulated 2 h after eating, including CbuELO1, CbuELO2, CbuELO3, CbuELO4, CbuELO5, CbuELO6, CbuELO7, CbuELO8, CbuELO12, and CbuELO14, and CbuELO12 was significantly up-regulated. It was raised by 4809.33, CbuELO14 by 4399.00, CbuELO3 by 747.33, CbuELO12 by 529.00, and CbuELO11 by 675.00.
In females, the genes with high expression were CbuELO12 and CbuELO14, the maximum value of CbuELO12 was 12963.33 after 0 h of feeding, the maximum value of CbuELO14 was 5975.00 after 0.5 h of feeding, and the gene with low expression was CbuELO7. Compared with the 0 h after eating, there were 11 genes with the largest transcriptome gene expression at 0.5 h after eating, including CbuELO3, CbuELO4, CbuELO5, CbuELO6, CbuELO7, CbuELO8, CbuELO9, CbuELO10, CbuELO11, CbuELO14 and CbuELO15, among which CbuELO14 was the most significant up-regulated. It was up-regulated by 934.00, followed by CbuELO9 with 186.33, and the other genes were slightly up-regulated, with CbuELO12 down-regulated by 3156.67 and CbuELO13 down-regulated by 1139.33. There were 10 genes with the largest transcriptome gene expression 1 h after eating, including CbuELO1, CbuELO2, CbuELO6, CbuELO7, CbuELO8, CbuELO9, CbuELO10, CbuELO11, CbuELO14, and CbuELO15, among which CbuELO was the most significantly up-regulated 14, 843.67 was up-regulated, followed by CbuELO9, 324.33, and the other genes were very small, and CbuELO12 was down-regulated by 5849.67. There were 12 genes up-regulated 2 h after eating, including CbuELO1, CbuELO2, CbuELO3, CbuELO4, CbuELO6, CbuELO7, CbuELO8, CbuELO9, CbuELO10, CbuELO11, CbuELO14, and CbuELO15, among which the most significant up-regulation was CbuELO9, which was 363.00, followed by CbuELO2, It was raised by 284.00, CbuELO6 by 261.33 and CbuELO 14 by 204.67, while CbuELO12 was lowered by 4823.33 and CbuELO13 by 315.00.
In summary, the CbuELO gene was the most up-regulated in females and males at 2 h after feeding, and CbuELO12, CbuELO14 and CbuELO13 were highly expressed in females, CbuELO14 was up-regulated, CbuELO12 and CbuELO13 were down-regulated, CbuELO12 was the highest in males, CbuELO12, CbuELO14 and CbuELO13 were high-expression genes, and CbuELO12, The expression of CbuELO14 was up-regulated, and CbuELO13 was down-regulated. CbuELO14 was up-regulated in both females and males, suggesting that this gene may be involved in the degradation of bamboo fibers (Fig. 12).
Gene expression of CbuELO genes under different feeding time conditions
Taking the time interval (0 h, 0.5 h, 1 h, 2 h) after feeding (0 h, 0.5 h, 1 h, 2 h) of female and male adult as variables, three parallel experiments were performed in each group, and the intestinal contents of the insects were taken for protein sequencing, and the obtained data were used as heat maps. The results showed that most of the results were 0, only CbuELO14 protein had results, and the protein expression of males was slightly higher than that of females, in males, the protein expression was the highest at 2 h after eating, with an average value of 1.788, followed by 1 h after eating, with a mean value of 1.629, and in females, the protein expression was the highest at 2 h after eating, with a mean value of 0.809, followed by 0.5 h after eating, with a mean value of 0.461 (Supplementary Fig. S7).
qRT-PCR validation analysis of CbuELO gene expression under different temperature and feeding time
The larvae, females and males of C. buqueti were treated at different temperatures (4 °C, 25 °C, 42 °C, 50 °C), and their muscle tissues were extracted from RNA after 1 h, reverse transcribed into cDNA, and cDNA was used as a template for qRT-PCR to detect the expression level of each gene. The results showed that with the increase of temperature, the general trend was as follows: 4 °C ~ 25 °C increased first, 25 °C ~ 50 °C increased and then decreased, the maximum was at 42 °C, compared with the control group (25 °C), the largest change was in the male of CbuELO13 at 42 °C, the smallest change was in the male of CbuELO1 at 50 °C, the larvae of CbuELO7, CbuELO8 and CbuELO9 at 4 °C, and the male of CbuELO7 at 42 °C. Compared with the control group (25 °C), the largest up-regulated genes were males with CbuELO13 at 42 °C, the smallest up-regulated genes were males with CbuELO10 at 50 °C, the most down-regulated genes were males with CbuELO12 at 4 °C, and the smallest down-regulated genes were CbuELO4 larvae at 4 °C. In summary, the validation results are basically consistent with the heat map results, CbuELO12 was the highest expression at different temperatures compared with other genes, although there is a large change in temperature increase, compared with 25 °C, low temperature has little effect on the overall expression of larvae genes, males are more tolerant to high temperature than females, and the gene expression of males increases with the increase of temperature, in summary, the gene has higher expression for temperature increase(Fig. 13).
The time interval (0 h, 0.5 h, 1 h, 2 h) after feeding of female and male adult was taken as variables, and 0 h was used as the control group, and three parallel experiments were performed in each group, and the intestinal contents were extracted from RNA, reverse transcribed into cDNA, and cDNA was used as a template for qRT-PCR to detect the expression level of each gene. The results showed that although the expression of CbuELO7 increased with the interval between meals, its expression level was lower than that of other genes, and the highest expression genes were CbuELO12, CbuELO13 and CbuELO14. For females, 0 h ~ 2 h decreases first and then increases, and reaches the maximum value in 2 h, but the increased value is always lower than the expression level of the control group (0 h). The expression of males decreased first at 0 h ~ 0.5 h, increased at 0.5 h ~ 2 h, and reached the maximum value at 2 h, which was higher than that of the control group. Among the genes up-regulated in females, CbuELO14 was the largest, CbuELO7 was the smallest, CbuELO12 was the most down-regulated, and CbuELO4 was the smallest. Among the genes upregulated in males, CbuELO12 was the largest, CbuELO7 was the smallest, CbuELO13 was the most down-regulated, and CbuELO15 was the smallest. In summary, the validation results were basically consistent with the heat map results, and the CbuELO14 was the most up-regulated gene at 2 h after feeding in females and males, CbuELO12, CbuELO14 and CbuELO13 were highly expressed in both females and males, and CbuELO14 was up-regulated in both females and males, indicating that this gene may be involved in the degradation of bamboo fiber. For males, with the increase of feeding intervals, the gene expression level of males decreased first and then increased, reaching the maximum value at 2 h after feeding, while the female increased first and then decreased, and then increased, reaching the maximum value at 2 h after feeding. The overall trend of CbuELO14 was not large, and the expression level of CbuELO14 was also small(Fig. 14).
Protein interaction analysis of CbuELO proteins
By constructing the interaction diagram of 15 CbuELO proteins in C.buqueti, the results showed that 9 of 15 CbuELO proteins were related to each other, and most of the members had more than one interaction relationship, such as CbuELO01, CbuELO04 and CbuELO12, indicating that there was a direct or indirect regulatory effect between them, and one protein was independent, which was CbuELO05. The results showed that CbuELO06, CbuELO07, CbuELO08, CbuELO09 and CbuELO10 had no interaction with other proteins of CbuELO protein family. According to the following types of proteins involved in the interaction: CbuELO01 and CbuELO12 were involved in the biosynthesis of long-chain fatty acids. CbuELO01, CbuELO02, CbuELO03, CbuELO11, CbuELO12, CbuELO13 and CbuELO15 were involved in the elongation of fatty acids. CbuELO1 and CbuELO04 were involved in pheromone metabolism. CbuELO05 was involved in the metabolism of organic matter (Fig. 15).
Gene expression level of CbuELO gene verified by qRT-PCR at different temperatures
Gene expression level of CbuELO gene verified by qRT-PCR at different feeding intervals
Protein interaction analysis of CbuELO proteins
Discussion
Bioinformatics is a discipline that intersects with medicine, statistics, mathematics, physics, and computer science based on the development of both information science and molecular biology [47, 48]. Compared with traditional studies, bioinformatics methods are cost-effective and efficient, and the methods have been widely used to predict and analyze the structure, function, and other biological properties of proteins [49]. C. buqueti occurs once a year in Sichuan and overwinters as an adult in a pupal chamber in the soil [50]. Adults insect are less active in the early morning but become more active and more capable of flight as the temperature rises [51]. Both larvae and adults feed on bamboo shoots, resulting in a large number of severed and deformed bamboos, and the adults have a pseudomortality and generally live from 50 to 70 d. The adults become extinct in bamboo forests in early October [52, 53]. C. buqueti is a major forestry pest, with damage rates of up to 50-80% and up to 100% in severe cases [54]. It mainly affects the bamboo shoots of Bambusa textilis, Neosino Calamusaffinis and other clumped bamboos [55].
Elongation family of very long chain fatty acids protein(ELO) are involved in the production of long-chain fatty acids (C16, C18 and C20) [56], which are formed in the presence of ELO and this reaction requires energy from NADPH [57].ELO is widely found in plants, insects and animals. Only in recent years has the specific role of ELO in fatty acids gained widespread attention. In mammalian studies, ELOVL family proteins not only control the length of fatty acid carbon chains, but also ELOVL2 has an effect on sperm maturation in male rats [58,59,60]. In addition to this study, ELOVL family proteins were found to be associated with diseases such as diabetes [61], obesity [62], and cancer [63]. In Saccharomyces cerevisiae, three genes encoding fatty acid elongases were identified and their functions were investigated and they were found to catalyze C14 into long-chain fatty acids of different lengths [64, 65]. In insects, ELOs are involved in life activity processes such as mating, reproduction, pheromone biosynthesis, and epidermal formation [66]. Different ELOs have different substrate preferences and tissue-specific expression in the same insect. Today, studies on ELO in insects have focused on Drosophilid, where 20 ELO family proteins have been identified and ELO is a key gene in Drosophilid development [67, 68]. Studies on Aedes albopictus ELO revealed that the ELO family proteins are important for egg retention and resistance to desiccation [59]. In Nilaparvata lugens, 20 ELO family proteins were identified, with different express ion sites and expression profiles, thus performing different biological functions [62]. Although ELO genes have been intensively studied in some insects, there is still a gap in the study of ELO genes in C. buqueti [69].
In this study, comprehensive analysis of CbuELO gene family in C. buqueti was conducted using bioinformatics. The identification of its gene family revealed that there were 15 CbuELO proteins, and 15 CbuELO proteins were located on four chromosomes. Gene structure analysis results revealed that all CbuELO proteins contained exons (CDS); all family members had introns except for CbuELO12, which had no introns; all family members had non-coding regions (UTR) except for CbuELO04. The physical and chemical properties of CbuELO protein family were predicted by EXPASY-ProtParam, and the theoretical isoelectric points of CbuELO protein family were found to be between 9.22 and 9.68. The overall number of stable proteins was greater than that of unstable proteins, and the lipid coefficients were between 72.28 and 120.22. The average coefficient of hydrophilicity of CbuELO protein family was 0.433, indicating that they are hydrophobic proteins, which is consistent with the prediction of Chertemps et al [70]. The total number of Arg + Lys of overall CbuELO protein family was much larger than that of Asp + Glu, indicating that the overall CbuELO protein family is positively charged. Subcellular localization analysis showed that CbuELO protein family was fully localized to plasma membrane. The transmembrane domain analysis of CbuELO protein family showed that each member of the protein family had a transmembrane structure, so it can be inferred that the CbuELO protein family is a transmembrane protein, which is consistent with the prediction of Uttaro et al. study [71]. The prediction of phosphorylation sites using the online software NetPhos revealed that the protein family has multiple phosphorylation sites. The tertiary structure of the proteins was predicted using SWISS-MODEL and found that the α-helix and random coil was the main structural element in CbuELO protein family. The conserved structures of the proteins were analyzed using MEME, and the results showed that 10 conserved motifs were identified in the CbuELO protein family, among which motif4 was present in each member, which was consistent with the prediction of Zheng et al. study [72]. The phylogenetic tree of CbuELO protein family was constructed by MEGA11 software, and the homology of ELO family proteins of C. buqueti. to several gene sequences of T. castaneum reached 100%. In addition, it is closest to the D. ponderosae and Elaeidobius kamerunicus. The expression analysis of ELO gene family in C. buqueti genome in different developmental organs showed that different ELO genes determined the synthesis of very long chain fatty acids in different tissues and organs of C. buqueti.
To sum up, this study carried out a systematic bioinformatics study on the elongation family of very long chain fatty acids proteins in C. buqueti. The results laid a foundation for revealing the molecular mechanism of C. buqueti, and also provided a new target for the green control of pests [73].
Conclusion
In this study, we have identified 15 ELO proteins in C.buqueti genome. 15 CbuELO proteins were located on four chromosomes. Phylogenetic analysis showed that molecular evolutionary relationships of ELO protein family between C. buqueti and Tribolium castaneum was the closest. Developmental transcriptome analysis indicated that CbuELO10, CbuELO13 and CbuELO02 genes were the key enzyme genes that determine the synthesis of very long chain fatty acids in pupae and eggs, CbuELO6 and CbuELO7 were that in the male, and CbuELO8 and CbuELO11 were that in the larva. Transcriptome analysis under different temperature conditions indicated that CbuELO1, CbuELO5, CbuELO12, and CbuELO14 participated in regulating temperature stress responses. Transcriptome analysis at different feeding times showed CbuELO12 gene expression level in all feeding time periods was significant downregulation. The qRT-PCR experiment verified the expression level changes of CbuELO gene family under different temperature and feeding time conditions. Protein-protein interaction analysis showed that 9 CbuELO proteins were related to each other, and CbuELO1, CbuELO4 and CbuELO12 had more than one interaction relationship. These results lay a theoretical foundation for further studying its molecular function during growth and development of C. buqueti.
Data availability
The genome assembly data and RNA-seq data used in this study were retrieved from the NCBI Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra) under the accession code PRJNA675312, PRJNA719467 and PRJNA718062.
References
Yang H, Liu YL, Tao YY, et al. Bioinformatic and biochemical analysis of the key binding sites of the pheromone binding protein of C. Buqueti Guerin-Meneville (Coleoptera: Curculionidea). PeerJ. 2019;7:e7818.
Baca SM, Gustafson GT, Toledo M, Miller KB. Revision of the neotropical burrowing water beetle genus Liocanthydrus Guignot (Coleoptera: Noteridae: Noterinae: Noterini) with the description of two new species. Zootaxa. 2014;3793:231–46.
Li L, Sun W, Guo C, Guo H, Lili L, Yu P. Mathematical model and nanoindentation properties of the claws of Cyrtotrachelus Buqueti Guer (Coleoptera: Curculionidae). IET Nanobiotechnol. 2022;16(6):211–24.
Li X, Guo C. Wing-kinematics measurement and flight modelling of the bamboo weevil C. Buqueti. IET Nanobiotechnol. 2020;14(1):53–8.
Liu L, Wang F, Yang W, Yang H, Huang Q, Yang C, Hui W. Molecular and functional characterization of pheromone binding protein 2 from Cyrtotrachelus buqueti (Coleoptera: Curculionidae). Int J Mol Sci. 2023;24(23):16925.
Fu C, Yang D, Long WC, Xiao X, Wang H, Jiang N, Yang Y. Genome-wide identification, molecular evolution and gene expression of P450 gene family in Cyrtotrachelus buqueti. BMC Genomics. 2024;25(1):453.
Li X, Guo C. Microstructure and material properties of Hind wings of a bamboo weevil Cyrtotrachelus Buqueti (Coleoptera: Curculionidae). Microsc Res Tech. 2019;82(7):1102–13.
Luo C, Li Y, Liao H, et al. De novo transcriptome assembly of the bamboo snout beetle C. Buqueti reveals ability to degrade lignocellulose of bamboo feedstock. Biotechnol Biofuels. 2018;11(1):1–20.
Li X, Guo C, Li L. Functional morphology and structural characteristics of the Hind wings of the bamboo weevil Cyrtotrachelus Buqueti (Coleoptera, Curculionidae). Anim Cells Syst (Seoul). 2019;23(2):143–53.
Luo C, Li Y, Chen Y, et al. Bamboo lignocellulose degradation by gut symbiotic microbiota of the bamboo snout beetle C. Buqueti. Biotechnol Biofuels. 2019;12(1):1–16.
Díaz-García L, Huang S, Spröer C, et al. Dilution-to-stimulation/extinction method: a combination enrichment strategy to develop a minimal and versatile lignocellulolytic bacterial consortium. Appl Environ Microbiol. 2021;87(2):e02427–20.
Li X, Guo C. Structural characteristics analysis of the Hind wings in a bamboo weevil (Cyrtotrachelus Buqueti). IET Nanobiotechnol. 2019;13(8):850–6.
Wang J, Wang F, Wang R, et al. Modeling the effects of bioclimatic characteristics and distribution on the occurrence of C. Buqueti in the Sichuan Basin. Global Ecol Conserv. 2019;17:e00540.
Li X, Guo C. Structural characteristics analysis of the Hind wings in a bamboo weevil (C. Buqueti). IET Nanobiotechnol. 2019;13(8):850–6.
Yang WJ, Yang DX, Xu KK, Cao Y, Meng YL, Wu Y, Li GY, Zhang GZ, Wang YW, Li C. Complete mitochondrial genome of the bamboo snout beetle, Cyrotrachelus buqueti (Coleoptera: Curculionidae). Mitochondrial DNA B Resour. 2018;3(1):88–89.
Haslam TM, Kunst L. Extending the story of very-long-chain fatty acid elongation. Plant Sci. 2013;210:93–107.
Xiang M, Zhang HZ, Jing XY et al. Sequencing, expression, and functional analyses of four genes related to fatty acid biosynthesis during the diapause process in the female Ladybird, Coccinella septempunctata L. Front Physiol, 2021: 1249.
Jakobsson A, Westerberg R, Jacobsson A. Fatty acid elongases in mammals: Teir regulation and roles in metabolism. Prog Lipid Res. 2006;45:237–49.
Nugteren DH. The enzymic chain elongation of fatty acids by rat-liver microsomes. Biochim Biophys Acta. 1965;106:280–90.
Sassa T, Kihara A. Metabolism of very long-chain fatty acids: genes and pathophysiology. Biomol Ter. 2014;22:83–92.
Chertemps T, Duportets L, Labeur C, et al. A new elongase selectively expressed in Drosophila male reproductive system. Biochem Biophys Res Commun. 2005;333(4):1066–72.
Pei XJ, Chen N, Bai Y, et al. BgFas1: a fatty acid synthase gene required for both hydrocarbon and cuticular fatty acid biosynthesis in the German cockroach, Blattella germanica (L). Insect Biochem Mol Biol. 2019;112:103203.
Szafer-Glusman E, Giansanti MG, Nishihamar et al. A role for very-long-chain fatty, acids in furrow ingression during cytokinesis in Drosophila spermatocytes. Curr Biology 2008,18 (18):1426–31.
FalcónT, Ferreira-Caliman JF, Franco Nunes FM, et al. Exoskeleton formation in Apis mell ifera: cuticular hydrocarbons profiles and expression of desaturase and elongase genes during pupal and adult development. Insect Biochem Mol Biol. 2014;50(11):68–81.
Kniazeva M, Sieber M, McCauley S, et al. Suppression of the ELO-2 FA elongation activity results in alterations of the fatty acid composition and multiple physiological defects, including abnormal ultradian rhythms, in Caenorhabditis elegans. Genetics. 2003;163(1):159–69.
Parvy J-P, Napal L, Rubin T, et al. Drosophila melanogaster acetyl-CoA-Carboxy lase sustains a fatty acid-dependent remote signal to waterproof the respiratory system. PLoS Genet. 2012;8(8):e1002925.
Zadravec D, Tvrdik P, Guillou H, et al. ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis, a prerequisite for male fertility and sperm maturation in mice. J Lipid Res. 2011;52:245–55.
Juárez M. Fatty acyl-CoA elongation in Blatella Germanica integumental microsomes. Arch Insect Biochem Physiol. 2004;56(4):170–8.
Chung H, Loehlin DW, Dufour HD, et al. A single gene affects both ecological divergence and mate choice in Drosophila. Science. 2014;343:1148–51.
Yang H, Liu L, Wang F, Yang W, Huang Q, Wang N, Hu H. The Molecular and functional characterization of sensory Neuron membrane protein 1b (SNMP1b) from Cyrtotrachelus buqueti (Coleoptera: Curculionidae). Insects. 2024;15(2):111.
Fu C, Long W, Luo C, Nong X, Xiao X, Liao H, Li Y, Chen Y, Yu J, Cheng S, Baloch S, Yang Y. Chromosome-Level Genome Assembly of C. Buqueti and Mining of its specific genes. Front Ecol Evol. 2021;9:729100.
Yang Mei D, Jing S, Tang X, Chen H, Chen H, Duanmu Y, Cong M, Chen X, Ye. Hang Zhou, Kang He, Fei Li*. InsectBase 2.0: a comprehensive gene resource for insects. Nucleic Acids Res. 2021, gkab1090.
Yang H, Su T, Yang W, Yang C, Lu L, Chen Z. The developmental transcriptome of the bamboo snout beetle C. Buqueti and insights into candidate pheromone-binding proteins. PLoS ONE. 2017;12(6):e0179807.
Wang H, Zou Z, Gong M, Gene, Cloning. Bioinformatic Analysis and preliminary functional characterization of Gene Encoding Galactinol Synthase 3 in Jatropha curcas. Biotechnol Bull. 2015;31(7):91.
Xiang Y, Liu X, Sun Q, Liao K, Liu X, Zhao Z, Feng L, Liu Y, Wang B. The development of cancers research based on mitochondrial heat shock protein 90. Front Oncol. 2023;13:1296456.
Shabbirahmed AM, Haldar D, Dey P, Patel AK, Singhania RR, Dong CD, Purkait MK. Sugarcane bagasse into value-added products: a review. Environ Sci Pollut Res Int. 2022;29(42):62785–806.
Saral Sariyer A. Three new inhibitors of class A β-lactamases evaluated by molecular docking and dynamics simulations methods: relebactam, enmetazobactam, and QPX7728. J Mol Model. 2022;28(4):1–13.
Xiaoli L, Hagey JV, Park DJ, Gulvik CA, Young EL, Alikhan NF, Lawsin A, Hassell N, Knipe K, Oakeson KF, Retchless AC, Shakya M, Lo CC, Chain P, Page AJ, Metcalf BJ, Su M, Rowell J, Vidyaprakash E, Paden CR, Huang AD, Roellig D, Patel K, Winglee K, Weigand MR, Katz LS. Benchmark datasets for SARS-CoV-2 surveillance bioinformatics. PeerJ. 2022;10:e13821.
Shanmugasundaram K, Singha H, Saini S, et al. 16S rDNA and ITS sequence diversity of Burkholderia mallei isolated from glanders-affected horses and mules in India (2013–2019). Curr Microbiol. 2022;79(1):1–13.
Wang J, Zhu Q, Cao D, Peng Q, Zhang X, Li C, Zhang C, Zhou BO, Yue R. Bone marrow-derived IGF-1 orchestrates maintenance and regeneration of the adult skeleton. Proc Natl Acad Sci U S A. 2023;120(1):e2203779120.
Zuo W, Li C, Luan Y, et al. Genome-wide identification and analysis of elongase of very long chain fatty acid genes in the silkworm, Bombyx mori. Genome. 2018;61(3):167–76.
Johnson GP, English AM, Cronin S, Hoey DA, Meade KG, Fair S. Genomic identification, expression profiling, and functional characterization of CatSper channels in the bovine. Biol Reprod. 2017;97(2):302–12.
Oeo-Santos C, López-Rodríguez JC, García-Mouton C, et al. Biophysical and biological impact on the structure and IgE-binding of the interaction of the olive pollen allergen ole e 7 with lipids. Volume 1862. Biochimica et Biophysica Acta (BBA)-Biomembranes; 2020. p. 183258. 6.
Wei W, Wang L, Zhou P, et al. Bioinformatic prediction and identification of immunogenic epitopes of the antigenic 14-3-3 protein of Echinococcus Multilocularis. Acta Trop. 2021;220:105955.
Finck J, Berdan EL, Mayer F, et al. Divergence of cuticular hydrocarbons in two sympatric grasshopper species and the evolution of fatty acid synthases and elongases across insects. Sci Rep. 2016;6(1):1–13.
Zhou L, Xie Z, Zhang Y. The complete mitochondrial genome of the Siganus canaliculatus (Perciformes: Siganidae). Mitochondrial DNA DNA Mapp Seq Anal. 2016;27(2):1111–2.
Gauthier J, Vincent AT, Charette SJ, et al. A brief history of bioinformatics. Brief Bioinform. 2019;20(6):1981–96.
Li H, Tian S, Li Y, et al. Modern deep learning in bioinformatics. J Mol Cell Biol. 2020;12(11):823–7.
CamejoA GDA, Lu D, et al. Identification of three novel Toxoplasam gonii rhoptry proteins[J]. Int J Parasitol. 2014;44(2):147–60.
Luo C, Liu A, Long W, et al. Transcriptome analysis of C. Buqueti in two cities in China. Gene. 2018;647:1–12.
Li Y, Lei L, Luo R, et al. Morphological structures of bamboo (Bambusa emeiensis) shoot shells and trichomes and functions in response to herbivory. J Plant Growth Regul. 2021;40(4):1400–8.
Tumlinson JH, Lait CG. Biosynthesis of fatty acid amide elicitors of plant volatiles by insect herbivores. Arch Insect Biochem Physiol. 2005;58(2):54–68.
Dey M, Brown E, Charlu S, Keene A, Dahanukar A. Evolution of fatty acid taste in drosophilids. Cell Rep. 2023;42(10):113297.
Yang H, Su T, Yang W, et al. The developmental transcriptome of the bamboo snout beetle C. Buqueti and insights into candidate pheromone-binding proteins. PLoS ONE. 2017;12(6):e0179807.
Lei G, Zhou H, Chen Y, Vasseur L, Gurr GM, You M, You S. A very long-chain fatty acid enzyme gene, PxHacd2 affects the temperature adaptability of a cosmopolitan insect by altering epidermal permeability. Sci Total Environ. 2023;891:164372.
Guillou H, Zadravec D, Martin PG, et al. The key roles of elongases and desaturases in mammalian fatty acid metabolism: insights from transgenic mice. Prog Lipid Res. 2010;49:186–99.
Zhu G, Shi X, Cai X. The reductase domain in a type I fatty acid synthase from the apicomplexan Cryptosporidium parvum: restricted substrate preference towards very long chain fatty acyl thioesters. BMC Biochem. 2010;11(1):1–8.
Ohno Y, Suto S, Yamanaka M, et al. ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis. Proc Natl Acad Sci USA. 2010;107:18439–44.
Sassa T, Ohno Y, Suzuki S, et al. Impaired epidermal permeability barrier in mice lacking elovl1, the gene responsible for very-long-chain fatty acid production. Mol Cell Biology. 2013;33:2787–96.
Lee SH, Stephens JL, Englund PT. A fatty-acid synthesis mechanism specialized for parasitism. Nat Rev Microbiol. 2007;5(4):287–97.
Liu Y, Wang F, Yu XL, et al. Genetic analysis of the ELOVL6 gene polymorphism associated with type 2 diabetes mellitus. Brazilian J Med Biol Res. 2013;46:623–8.
Zadravec D, Brolinson A, Fisher RM, et al. Ablation of the very-long-chain fatty acid elongase ELOVL3 in mice leads to constrained lipid storage and resistance to diet-induced obesity. Faseb J. 2010;24:4366–77.
Naganuma T, Sato Y, Sassa T, et al. Biochemical characterization of the very long-chain fatty acid elongase ELOVL7. FEBS Lett. 2011;585:3337–41.
Oh CS, Toke DA, Mandala S, et al. ELO2 and ELO3, Homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation. J Biol Chem. 1997;272(28):17376–84.
Toke DA, Martin CE. Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae. J Biol Chem. 1996;271(31):18413–22.
Kniazeva M, Crawford QT, Seiber M, et al. Monomethyl branched-chain fatty acids play an essential role in Caenorhabditis elegans development. PLoS Biol. 2004;2(9):e257.
Holze H, Schrader L, Buellesbach J. Advances in deciphering the genetic basis of insect cuticular hydrocarbon biosynthesis and variation. Heredity. 2021;126(2):219–34.
Jung A, Hollman M, Schafer MA. The fatty acid elongate NOA is necessary for viability and has a somatic role in Drosophila sperm development. J Cell Sci. 2007;120(16):2924–34.
Li DT, Chen X, Wang XQ, et al. The fatty acid elongase gene family in the brown planthopper, Nilaparvata lugens[J]. Insect Biochem Mol Biol. 2019;108:32–43.
Chertemps T, Duportets L, Labeur C et al. A female-biased expressed elongase involved in long-chain hydrocarbon biosynthesis and courtship behavior in Drosophila melanogaster. Proceedings of the National Academy of Sciences, 2007, 104(11): 4273–4278.
Uttaro AD. Acquisition and biosynthesis of saturated and unsaturated fatty acids by trypanosomatids. Mol Biochem Parasitol. 2014;196(1):61–70.
Zheng T, Li H, Han N, et al. Functional characterization of two elongases of very long-chain fatty acid from Tenebrio molitor L.(Coleoptera: Tenebrionidae). Sci Rep. 2017;7(1):1–10.
Zhao X, Yang Y, Niu N, et al. The fatty acid elongase gene LmElo7 is required for hydrocarbon biosynthesis and cuticle permeability in the migratory Locust, Locusta Migratoria. J Insect Physiol. 2020;123:104052.
Acknowledgements
We would like to thank thank BIOMARKER TECHNOLOGIES Co.,Ltd.for providing the BMKCloud Service Platform(https://www.biocloud.net/) to analyze transcriptome data of C. buqueti.
Funding
This research was funded by National Key Research and Development Program of China (2022YFD1901405), Science and Technology Program of Leshan Normal University (2022SSDJ005, KYPY2023-0006, XJR17005, LZD010). Opening Foundation of Key Laboratory of Sichuan Province for Bamboo Pests Control and Resource Development(ZLKF202202).
Author information
Authors and Affiliations
Contributions
C.F. participated in the formal data analysis, data interpretation and scientifc paper writing. C.F., T.Y. participated in formal data analysis, data paper writing, omics data management and scientifc paper writing. H.L., W.C.L. and H.Y.W. participated in the conceptualization and investigation methodology. Y.L.H. and N.J. participated in the conceptualization of the project. C.F. and Y.J.Y. participated in the conceptualization, supervision, investigation, data analysis and interpretation, funding acquisition, project administration and scientifc paper writing. All authors read and approved the fnal manuscript.
Corresponding authors
Ethics declarations
Ethics approval and consent to participate
The adults and larval samples of C. buqueti used in this study were collected in Danan Town, Muchuan County, Leshan City, Sichuan Province. All experiments were conducted within ethical limits and were conducted safely in the Molecular Biology Laboratory of Key Laboratory of Sichuan Province for Bamboo Pests Control and Resource Development of Leshan Normal University from July 15 to August 15, 2020. All experiments do not involve humans.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it.The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Fu, C., Yang, T., Liao, H. et al. Genome-wide identification and molecular evolution of elongation family of very long chain fatty acids proteins in Cyrtotrachelus buqueti. BMC Genomics 25, 758 (2024). https://doi.org/10.1186/s12864-024-10658-8
Received:
Accepted:
Published:
DOI: https://doi.org/10.1186/s12864-024-10658-8