Molecular Cloning and Expression Profiling of Minus-C Odorant Binding Proteins from Apocheima cinerarius (Lepidoptera: Geometridae)1
Based on transcriptome data of adults and pupae, 3 Minus-C OBPs (odorant binding proteins) genes were cloned and analyzed in various tissues (including head, thorax, abdomen, legs of female and male adults, and wings of males) of Apocheima cinerarius Erschoff (Lepidoptera: Geometridae). Expression patterns were examined across the developmental stages of egg, larva (1st–5th instars), pupa (non-diapause and diapause), and adults. The qRT-PCR results revealed a significant up-regulation of OBP8 in 3rd and 4th larval instars, with the highest expression level observed in 4th-instar larvae. OBP14 exhibited higher expression levels in diapausing pupal and adult stages, with highest levels in the female adult. Conversely, OBP18 displayed highest expression in the non-diapausing pupae, while exhibiting extremely low expression levels among other stages. The tissue expression profile revealed a gradual increase in the expression of OBP14, with highest levels in the male legs, whereas the expression pattern of OBP18 exhibited an inverse trend with maximum expression observed in the male head (including antennae). OBP8 exhibited high expression levels in the abdomen of the female adult, while OBP14 showed high expression in the legs of the male adult. Additionally, OBP18 displayed high expression in the female head. These findings suggest the crucial role played by OBP genes in both olfactory and non-olfactory tissues of A. cinerarius. The results provide a fundamental basis for further investigation into the recognition of odorant binding in A. cinerarius.Abstract
Apocheima cinerarius Erschoff (Lepidoptera: Geometridae) is primarily distributed in Northern China, Southeast Russia, and Central Asia (Hu et al. 2001, Liu et al. 2014) and is a pest of poplar (Populus bonatii Levl), willow (Salix matsudana Koidz), locust (Sophora japonica L.), mulberry (Morus alba L.), and other tree species, as well as Korshinsk pea shrub (Caragana korshinskii Kom) and Russian olive (Elaeagnus angustifolia L.). Larvae are foliage feeders with continuous feeding leading to host plant death and eventual destruction of the forest landscape (Furniss and Carolin 2015, Millar et al. 1990).
Of special concern is its impact on C. korshinskii which is highly suitable for mitigating wind erosion in the central and western regions of Inner Mongolia. These areas suffer from severe soil desertification, resulting in low productivity and a fragile ecological environment. In recent years, infestations of A. cinerarius have significantly impacted the livelihoods of farmers and herdsmen, exacerbating destruction of an already vulnerable grassland ecosystem. Control of the pest in such areas is not cost effective and presents additional environmental problems. Ecologically friendly and cost-effective methods for the prevention and control of A. cinerarius must be immediately addressed.
The pest impact of A. cinerarius is closely associated with specific physiological behaviors, including aggregation, migration, feeding, robust reproductive capacity, and evasion of natural enemies. These behaviors are intricately linked to its highly evolved olfactory sensory system.
Olfaction is essential for the survival and reproduction of insects. It is involved in insect feeding, mate finding, oviposition site selection, and the completion of life activities such as individual and group communication (Su et al. 2009). It includes odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs), ionotropic receptors (IRs), sensory neuron membrane proteins (SNMPs), and odorant degradation enzymes (ODEs). These proteins are highly coordinated to complete the process of recognition and assimilation of environmental information (Yang et al. 2019).
The process of recognition of the external environment initiates with interactions of OBPs and chemical substances present in the surrounding environment (Feng and Prestwich 1997, Liu et al. 2015; Swarup et al. 2011,). Based on conserved cysteine residues in their amino acid sequences, OBPs can be categorized into Classical OBP, Min, and OBP (Du Yali et al. 2020). At present, more than 150 OBPs have been identified from 35 species of 13 lepidopteran families (Zhou et al. 2010). Studies with many insects, such as Anoplophora glabripennis Motschulsky (Hu et al. 2016; Wang et al. 2017, 2019), Ectropis obliqua Warren (Ma 2016, Yan et al. 2021), and Cydia pomonella (L.) (Tian 2016), have been conducted on the types, functions, and olfactory mechanisms of olfactory-related proteins. Many have shown that antennae are an important part of OBP expression, but OBPs are also expressed in other tissues such as the head, thorax, and abdomen (Zhang et al. 2017). Expression of OBPs related to gender and developmental activities also vary.
Relevant studies have indicated that OBPs play roles in recognition, but they may also serve non-olfactory functions. For example, approximately one-half of the OBP transcripts identified in Sesamia inferens Walker are also weakly expressed in non-chemoreception organization (thorax and abdomen), suggesting that these OBPs may possess other functions (Zhang et al. 2013). Among the 38 SlitOBP transcripts of Spodoptera litura F., 17 exhibit unique or predominant expression in male and female antennae and, thus, potential involvement in chemoreception (Gu et al. 2015). The expression of litOBP11 in larval antennae at higher levels than that in adult antennae suggests potential involvement in the olfactory process of larvae (Luo et al. 2021). The antennae of Eogystia hippophaecolus Hua, Chou, Fang & Chen exhibit the highest expression levels of most OBPs, with OBP6 showing a male reproductive bias expression, identification, and combination of pheromones for mating. In addition, OBP1 has the highest expression level in the tarsus, which may have the function of identifying host plant volatiles (Hu et al. 2016). The OBPs genes of Pieris rapae L. showed gender-biased expression. PrapOBP1, 2, 4 and 13 were expressed in the male antennae, while PrapOBP7 and PrapOBP10 were expressed in female antennae (Li et al. 2020).
The objectives of this study were to (1) clone 3 Minus-C OBPs through RT-PCR, utilizing transcriptome data from both adults and pupae of A. cinerarius, (2) identify their sequence signature, physicochemical properties, and bioinformatics as well as the distribution patterns of AcinOBP8, AcinOBP14, and AcinOBP18 across different developmental stages and tissues of A. cinerarius using qRT-PCR, and (3) identify efficient and environmentally friendly target sites for management of this pest.
Materials and Methods
Experimental design
In the winter of 2021, A. cinerarius overwintering pupae were collected and cultured under controlled laboratory conditions (22 ± 1°C, 18:6 h L:D photo regime, 55–59% relative humidity). Upon emergence, male and female adults were paired and reared on C. korshinskii. After oviposition, they were collected and divided into 2 groups. One group was quick-frozen in liquid nitrogen for future use, while the other group underwent further rearing. Larvae were continuously reared using C. korshinskii until reaching the pupal stage. The experimental treatments were: (1) developmental stages of eggs, larvae (1–5 instars) (2 days after molting), pupae (diapause and non-diapause), and adults; and (2) tissues from head (including antennae), thorax, abdomen, legs of male and female adults, and wings of male adults.
The experiment included 3 biological replicates, with each replicate containing 30 eggs, 30 first-instar larvae, 20 second-instar larvae, 3rd–5th instar larva, pupae, and male and female adults (n = 3), 30 heads, 5 thoraxes and 5 abdomens. Additionally, 50 leg samples were taken for each type of the leg (e.g., foreleg, midleg, and hindleg), and 20 male wing samples. The samples were quick-frozen in liquid nitrogen and stored at a temperature of −80°C.
RNA extraction
The RNA extraction kit, reverse transcription kit for cDNA synthesis of strand 1, PCR reaction MIX, and pMD19-T connection vector were procured from Dalian Bao Biological Technology Company (Dalian, Liaoning, China). The 5α competent cells were obtained from Beijing Tiangen Biochemical Technology Company (Changping District, Beijing, China). The GoTaq® qPCR Master Mix (2×) was purchased from Promega Corp (Madison, WI).
Total RNA extraction and synthesis of the first strand cDNA were accomplished by placing insects in a pre-cooled mortar that had been sterilized at high temperature and cooled with liquid nitrogen. The RNA extraction procedure followed the instructions of the TaKaRa RNA extraction kit. The quality and concentration of RNA extraction were detected using 1.0% agarose gel electrophoresis and NanoPhotometer-N50 ultra microspectrophotometer. Subsequently, the first strand of cDNA was synthesized through reverse transcription following successful evaluation.
Cloning
Based on the OBP genes identified from the transcriptome data of adults and pupae of A. cinerarius in our laboratory, 3 Minus-C OBPs were identified for experiments, and primers were designed using Primer Premier 5.0 software (Table 1). The protein coding region of the OBP genes was amplified using the synthesized cDNA as the amplification template. The PCR reaction system (25 μL) consisted of 1 μL cDNA amplification template, 1 μL forward and 1 μL reverse primers, 12.5 μL PCR Mix, and RNase-free water to fill the remaining volume. The PCR reaction procedure included pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 62°C for OBP8, 66°C for OBP14, 62°C for OBP18, extension at 72°C for 1 min, repeated for a total of 30 cycles, filled and extended at 72°C for 10 min, and finally stored at 4°C. The size of the destination strip was adjusted to match the desired specifications, was connected to the pMD19-T vector, and transformed into DH5α E. coli using 1% agarose gel electrophoresis detection. Subsequently, the white-blue plaque selection was conducted. After culturing at 37°C, 3 randomly selected positive colonies of the expected size were sent to Biotechnology Limited Company of Shanghai for sequencing.
Bioinformatics analysis
The ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder) of the NCBI online website was used to predict the coding protein region of the OBP gene of A. cinerarius. The DNAMAN 6.0 (Lynnon Biosoft, Quebec, Canada) software was used to analyze the sequence identity of the OBP gene between the selected other insects and the A. cinerarius. SignalIP5 (http://www.cbs.dtu.dk/services/SignalP/ online prediction tool) was used to predict the N-terminal signal peptide of odorant binding protein of A. cinerarius. ExPASy-ProtParam tool (http://web.expasy.org/protparam/ software) was used to predict the physicochemical properties of the OBP gene of A. cinerarius. The phylogenetic tree was constructed by MEGA6.0 software, and the neighbor-joining (NJ) and p-distance were used to repeat the process 1,000 times.
Expression profile
Based on the cloned OBPs gene sequence, primers were designed using the Primer3 online website (Table 1). Amplification length was between 80–250 bp, and the optimal dilution of cDNA template was selected, with Actin as the reference gene. qRT-PCR was performed to amplify the 3 OBP target genes and reference genes. The reaction system consisted of 20 μL: template cDNA (2 μL), 1 μL forward and 1 μL reverse primers (0.4 μL each), GoTaq® qPCR Master Mix (10 μL), and Nuclease-Free Water (7.2 μL). The reaction procedure followed the instructions provided by GoTaq® qPCR Master Mix. Based on the expression level of OBPs in male head, the obtained data were analyzed by 2-ΔΔCt (Livak and Schmittgen 2001). The expression of AcinOBP in various tissues and developmental stages was quantified. Each tissue and stage had 3 biological replicates and 3 technical replicates.
Data statistics and analysis
The data analysis was performed using SPSS 20.0 software. Tukey’s honestly significant difference (hsd) was employed to analyze the relative expression of 3 OBPs genes across different tissues and developmental stages. The Least Significant Difference (LSD) T test was used to assess the gene expression within the same tissue but across gender. GraphPad Prism 7.0 software was utilized for generating histograms, presenting mean ± SD, with statistical significance set at P <0.05.
Results
Cloning and sequence analysis of OBPs
According to the transcriptome database, we designed primers targeting sequences of 3 OBPs for amplification: 662 bp, 503 bp, and 441 bp (Fig. 1A–C). By comparing the sequences of the 3 OBPs genes with the transcriptome data, we found that the sequence information remained consistent. We assigned them as AcinOBP8, AcinOBP14, and AcinOBP18. The corresponding GenBank accession numbers are MT999864, MT999870, and MT999874 (Table 2).
Citation: Journal of Entomological Science 59, 4; 10.18474/JES23-86
Citation: Journal of Entomological Science 59, 4; 10.18474/JES23-86
The full-length coding sequences (CDS) of AcinOBP8, AcinOBP14, and AcinOBP18 were 402 bp, 462 bp, and 444 bp in length, respectively (Fig. 2A–C). These CDS encoded proteins consisted of 147, 153, and 133 amino acids. The predicted molecular weights for these proteins were determined to be approximately 16.70 kDa, 16.66 kDa, and 15.02 kDa with isoelectric points of approximately 5.93, 8.40, 9.46, respectively. Furthermore, the presence of signal peptides was detected within the aforementioned genes.
Physicochemical properties
The physicochemical properties of the 3 Minus-C OBPs were predicted using the ExPASy-ProtParam software (Table 3). Amino acid residues for the 3 Minus-C genes were 147, 153, and 133, and molecular weights were determined to be 16.71, 16.66, and 15.03, respectively. Additionally, their predicted theoretical iso-electric points were found to be at pH values of 5.87, 8.63, and 9.12, respectively. The results demonstrated that AcinOBP8 was classified as an acidic protein, whereas AcinOBP14 and AcinOBP18 were categorized as alkaline proteins. The net charge of AcinOBP8 was characterized by a greater number of negative charge residues compared to positive charge residues, whereas the net charge of AcinOBP14 and AcinOBP18 exhibited a reversed trend with fewer negative charge residues than positive charge residues. The instability coefficients of AcinOBP8 and AcinOBP18 were <40, while the instability coefficient of AcinOBP14 >40. This suggests that AcinOBP8 and AcinOBP18 represent stable proteins, in contrast to AcinOBP14, which displays characteristics of an unstable protein. The protein’s thermal stability was indicated by the Aliphatic index, and the results demonstrated that all OBPs were thermally stable proteins. AcinOBP8 and AcinOBP14 had a total average hydrophobicity of −0.020 and 0.016, respectively, indicating that these gene proteins were amphoteric proteins. On the other hand, AcinOBP18 had a total average hydrophobicity of −0.523, suggesting that this gene was hydrophilic (where positive values indicate hydrophobicity while negative values indicate hydrophilicity; the range of −0.5 to 0.5 represents amphoteric proteins).
Sequence alignment and phylogenetic relationship analysis
By comparing the amino acid sequences of the 3 OBPs of the A. cinerarius with those of other Lepidoptera (Figs. 2, 3), it was observed that AcinOBP8 exhibited the highest sequence similarity to Dendrolimus kikuchii Matsumura OBP (32.43%), while displaying lower similarities to Operophtera brumata L., Semiothisa cinerearia Bremer & Grey, and E. obliqua (16.99%, 13.12%, and 8.84%, respectively). AcinOBP14 demonstrated the highest sequence consistency with E. obliqua OBP (72.90%), followed by Streltzoviella insularis Staudinger (59.75%), E. obliqua (21.42%), and O. brumata (11.42%). AcinOBP18 displayed the highest sequence consistency with E. obliqua OBP16 (90.98%), followed by Galleria mellonella L. OBP56a (85.71%), which exceeded 13.10% for S. cinerearia and 8.63% for O. brumata.
Citation: Journal of Entomological Science 59, 4; 10.18474/JES23-86
The phylogenetic analysis revealed that AcinOBP8, AcinOBP14, and AcinOBP18 formed a distinct clade (Fig. 4). Specifically, AcinOBP8 and ObruOBP were grouped together, while AcinOBP14 and ScinOBP clustered in another branch. Furthermore, AcinOBP18 showed close genetic relationship with GmelOBP56a and EoblOBP16. The results showed that AcinOBP8, AcinOBP14, and AcinOBP18 were closely related to the OBPs of O. brumata, S. cinerearia, and E. obliqua, respectively. In addition, AcinOBP18 was also closely related to the OBP of G. mellonella.
Citation: Journal of Entomological Science 59, 4; 10.18474/JES23-86
Expression profiles at different developmental stages
The expression patterns of the 3 OBPs in A. cinerarium at different developmental stages are depicted in Fig. 5 (OBP14 was not expressed in the 4th-instar larvae). OBP8 exhibited an increasing trend initially, reaching its peak during the 4th instar, followed by a decrease. The expression levels were 69.10 and 29.97 times higher than the control (egg stage) for the 3rd and 4th instars, respectively, with a significant difference (P <0.05). No significant differences were found among other developmental periods.
Citation: Journal of Entomological Science 59, 4; 10.18474/JES23-86
The expression of OBP14 showed a gradual increase, reaching the maximum in males, followed by diapausing pupal and adult female stages, which were 240.45, 121.69, and 51.85 times that of the control, respectively, and were significantly different (P <0.05). There was no significant difference in expression levels in other periods of development. The expression of OBP18 was mainly concentrated in non-diapause pupae, which were 84.54 times that of the control and were significantly different (P <0.05). The expression level of OBP18 was extremely low in other periods, with no obvious difference.
Tissue expression profile
The qPCR results revealed the expression of 3 OBPs in all examined tissues of A. cinerarius (Fig. 6). Notably, distinct expression patterns were observed for these genes across different tissues in male and female adults of A. cinerarius. Specifically, the expression level of OBP8 exhibited a gradual increase followed by a decrease in the head, thorax, and abdomen of both male and female adults. Among females, the highest expression level was detected in the abdomen, reaching 13.12 times higher than that of the control group. Conversely, lower expression levels were observed in the legs and wings of males, ranging from only 0.08 to 0.58 times than that of the control.
Citation: Journal of Entomological Science 59, 4; 10.18474/JES23-86
The OBP8 expression showed significant differences only in the abdomen and legs of adult males and females, while no significant differences were observed in other body parts. The expression level of the OBP14 was low in each tissue of female A. cinerarius, with no significant difference detected. In male insects, except for wings, there was a gradual increase in the expression levels across different tissues. Specifically, the expression levels in legs, abdomen, thorax, and head were 81.97 times, 15.27, 12.39, and 5.64 times greater than that of the control group, respectively. Moreover, compared to females, males exhibited significantly higher expression levels of OBP14 in their heads, thorax, abdomens, and legs (P <0.01). The expression pattern of OBP18 in A. cinerarius was the same as that of OBP14, the expression level in each tissue was low and there was no significant difference, while the expression level in male insects was opposite to that of OBP14. It showed a gradual downward trend, and the expression levels in the head and thorax of males were 222.52 and 12.67 times that of the control, respectively. The expression levels in male and female legs were significantly different, while in the head and abdomen reached a significant difference (P <0.01).
Discussion
Odorant binding protein (OBP) is a low molecular weight acidic soluble protein that is widely distributed in the lymph of insect antennae. It typically has a relative molecular weight ranging from 15 to 20 kD and is characterized by the presence of a signal peptide at its N-terminus, consisting of approximately 20 amino acids (Paula et al. 2016, Wang et al. 2022). Generally, OBP contains 6 conserved cysteines, while Minus-C OBPs lack 2 of these conserved cysteines (Gao et al. 2023).
In this study, 3 OBPs gene, namely AcinOBP8, AcinOBP14, and AcinOBP18, were identified for the first time based on transcriptome data from A. cinerarius adults and pupae. Notably, these proteins exhibited a lack of the second and fifth cysteines, suggesting their classification as Minus-C OBPs.
Furthermore, the presence of signal peptides in the 3 OBPs genes examined in this study suggests their classification as secretory proteins. However, it should be noted that certain OBP proteins such as SfruOBP3,31,35,41,55-56,59-61, and Spodoptera frugiperda SfruOBP1,7,9,14,17-18,20,23-24 and 26-27 (Jia et al. 2023) in A. glabripennis lack a signal peptide at the N-terminus. Conversely, MaltOBP1 from Monochamus alternatus Hope (Zhang et al. 2020), AglaOBP (Li et al. 2018), and YsigOBP1-YsigOBP10 from Yemma signatus Hsiao (Song et al. 2021) exhibit signal peptides at their N-terminus. These findings indicate that the presence or absence of signal peptides does not serve as an identifying feature for insect odorant binding proteins.
In this study, the identity of AcinOBP8 with O. brumata and S. cmerearia was lower than that of D. kikuchii (32.43%), with the lowest identity being only 8.84% for E. obliqua. On the other hand, AcinOBP14 showed a higher identity with the OBP sequence of S. cmerearia at 72.90%, while its similarity to E. obliqua was lower. Finally, AcinOBP18 had the highest identity with OBP16 sequence of E. obliqua at 90.98%, but its similarities to both S. cmerearia and O. brumata were only 13.10% and 8.63%, respectively. Similar findings were also observed in other insects. For instance, HtheOBP3 exhibited the highest homology with PmicOBP4; however, their sequence similarity was only 55.56% (Zhang et al. 2023). Conversely, the sequence identity between Bactrocera minax Enderlein BminOBP25 and Dacus dorsalis Hendel BdorOBP was determined to be 88% (Si et al. 2018), indicating varying degrees of differentiation among different OBPs within and across insect species.
In general, homologous sequence consistency is generally higher, however, it is important to note that two sequences with low consistency may still be considered homologous. In this study, the identity between AcinOBP8 and the OBP sequence of D. kikuchii was found to be 32.43%, which is higher than that of O. brumata (16.99%). Nevertheless, in the phylogenetic analysis, AcinOBP8 exhibited a closer genetic relationship to O. brumata despite its lower sequence identity. Similar findings were also reported in a study of Helopeltis theivora Waterhouse (Zhang et al. 2023).
The distribution range of OBPs in insects is widely believed to be closely associated with their functional roles, and gene expression profiling serves as a crucial approach for investigating gene function. In this study, qPCR was applied to examine the expression patterns of OBP8 during different developmental stages and in various tissues of male and female adults. Our findings revealed that AcinOBP8 exhibited consistent expression throughout the developmental stages, with significantly higher levels observed in the 3rd and 4th larval instars. Notably, the highest expression level was detected in 4th-instar larvae. SzeaOBP1 and SzeaOBP28 were expressed in both larval and adult stages of S. zeamais, but the expression levels differed (Tang et al. 2019). TcasOBP7G is expressed at all developmental stages and is highly expressed in late larvae and late adults (Gao et al. 2023). This may be related to the feeding activity of larvae and adults, because OBPs are related to the dissolution of fatty acids in food (Ishida et al. 2013) and the transport of nutrients (Ribeiro et al. 2014). AcinOBP8 exhibited high expression exclusively in the 3rd-4th instar larvae due to comparative food ingestion during the egg and adult stages of the A. cinerarius. The dietary intake gradually increased from the 1st- to 4th-instar larvae, reaching its peak in the 4th instar, followed by a decline after the 5th instar. In the pupal stage, no feeding occurred, thus its high expression in the 3rd-4th instar larval stage. AcinOBP14 is highly expressed during the pupal diapause and adult stages, potentially playing a crucial role in olfactory recognition of external environmental cues for both pupae and adults. Given that A. cinerarius diapausing pupae experience extreme temperature variations throughout hot summers and cold winters, this OBP may aid A. cinerarius perception of adverse factors in their external environment.
Furthermore, the absence of AcinOBP14 in the 4th-instar larvae may be attributed to its low expression levels, which were below the detection limit in the selected tissues and developmental stages during qPCR analysis. Additionally, among the 39 OBPs identified in P. xylostella, 12 were not detected (Cai et al. 2021). Notably, AcinOBP18 exhibited significantly higher expression levels during non-diapause periods compared to other times (P <0.05), suggesting its potential involvement in physiological processes beyond olfactory recognition.
The physiological functions of genes are usually related to their expressed tissues (Hua et al. 2012). The physiological functionality of a gene is typically associated with the tissue in which it is expressed (Hua et al. 2012). Most OBP genes are specifically or highly expressed in insect antennae, which is related to the involvement of OBPs as carriers in transporting fat-soluble odor molecules through antennal lymph (Benton et al. 2007). The expression of OBP18 was found to be significantly up-regulated in the male head (including the antennae), as revealed by our study. In this study, a significant level of OBP18 expression was observed in the male head. The expression levels of AmalOBP3 and AmalOBP8 in Agrilus mali Matsumura are not only highly expressed in the antennae, but also detected in the abdomen and wings (Cui et al. 2018). The expression level of AcinOBP8 was highest in the abdomen of female insects, indicating its potential involvement in oviposition site selection or sex pheromone production. Conversely, AcinOBP14 exhibited the highest expression level in the legs of male insects, suggesting its role in mate searching or transportation of non-volatile odorants (Jia et al. 2020). Some OBPs of Culex pipiens L. (Pelletier and Leal 2009), Anopheles gambiae Giles (Li et al. 2004), Apis mellifera L. (Foret and Maleszka 2006), and Bemisia tabaci Gennadius (Wang et al. 2017) are also highly expressed in various other parts of insects, suggesting that these OBPs may perform other physiological functions beyond their role in olfactory recognition.
The present study focused on the cloning of 3 Minus-C OBPs genes and investigated their expression profiles across various tissues, developmental stages, and gender in the A. cinerarius. Our findings provide information regarding the sequence, physicochemical properties, and expression pattern map of these 3 Minus-C OBPs in this species, thereby providing a foundation for future investigations into the functional roles of Minus-C OBPs.
Cloning of full length CDS of AcinOBP in A. cinerarius.
Nucleotide and encoded deduced amino acid sequence of AcinOBP8 (A), AcinOBP14 (B) and AcinOBP18 (C) in A. cinerarius. The shadow part is the signal peptide. Boxes: four cysteines.
Multiple amino acid sequence alignment of OBPs from A. cinerarius and other insects.
Note: Source species of OBPs proteins and their GenBank accession numbers. Apocheima cinerarius (AcinOBP8: MT999864; AcinOBP14: MT999870; AcinOBP18: MT999874); Operophtera brumata (ObruOBP: KOB71255.1); Dendrolimus punctatus (DpunOBP24: ARO70183.1); Dendrolimus kikuchii (DkikOBP: AII01002.1); Semiothisa cinerearia (ScinOBP: QRF70927.1); Streltzoviella insularis (SinOBP11: QLI62014.1); Dendrolimus punctatus (DpunOBP20: ARO70179.1); Ectropis obliqua (EobliquaOBP16: ALS03864.1); Galleria mellonella (GmelOBP56a: XP_026763748.1); Eogystia hippophaecolus (EhipOBP: AOG12864.1).
Phylogenetic tree based on amino acid sequence of OBPs from A. cinerarium and other insects.
Note: The tree was constructed by MEGA 6.0 using the Neighbor-Joining (NJ) method, the OBPs protein of A. cinerarium was marked with filled circle. Source species of OBPs proteins and their GenBank accession numbers. Apocheima cinerarius (AcinOBP8: MT999864; AcinOBP14: MT999870; AcinOBP18: MT999874); Operophtera brumata (ObruOBP: KOB71255.1); Dendrolimus punctatus (DpunOBP24: ARO70183.1); Dendrolimus kikuchii (DkikOBP: AII01002.1); Semiothisa cinerearia (ScinOBP: QRF70927.1); Streltzoviella insularis (SinsOBP11: QLI62014.1); Dendrolimus punctatus (DpunOBP20: ARO70179.1); Ectropis obliqua (EoblOBP16: ALS03864.1); Galleria mellonella (GmelOBP56a: XP_026763748.1); Eogystia hippophaecolus (EhipOBP: AOG12864.1); Dendrolimus punctatus (DpunOBP24; ARO70183.1); Spodoptera litura (SlitPBP1: AIS72935.1; SlitPBP2:QYF65652.1; SlitPBP3: AIS72934.1; SlitGOBP1: XP_022816701.1;SlitGOBP2: AKI87961.1;SlitOBP3: XP_022827633.1; SlitOBP4: XP_022832058.1; SlitOBP5: AKI87966.1); Helicoverpa armigera (HarmOBP1: AEB54580.1; HarmOBP2: AEB54586.1; HarmOBP3: AEB54582.1; HarmOBP4: AEB54584.1; HarmOBP5: AEB54581.1); Semiothisa cinerearia (ScinOBP1: QRF70921.1; ScinOBP2: QRF70922.1; ScinOBP3: QRF70923.1 ScinOBP4: QRF70924.1; ScinOBP5:QRF70925.1).
Expression levels of AcinOBP in different developmental stages of A. cinerarius.
Expression levels of AcinOBP in different tissues of A. cinerarius adults.
Contributor Notes