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A novel Lozenge gene in silkworm, Bombyx mori regulates the melanization response of hemolymph
Developmental and Comparative Immunology 53 (2015) 191e198
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Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
A novel Lozenge gene in silkworm, Bombyx mori regulates the melanization response of hemolymph Man Xu a, 1, Xue Wang a, 1, Juan Tan a, Kui Zhang a, Xi Guan a, Laurence H. Patterson b, Hanfei Ding c, Hongjuan Cui a, * a b c
State Key Laboratory of Silkworm Genome Biology (Southwest University), 400715, China Institute of Cancer Therapeutics, University of Bradford, West Yorkshire BD7 1DP, UK Cancer Center, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 February 2015 Received in revised form 10 June 2015 Accepted 1 July 2015 Available online 9 July 2015
Runt-related (RUNX) transcription factors are evolutionarily conserved either in vertebrate or invertebrate. Lozenge (Lz), a members of RUNX family as well as homologue of AML-1, functions as an important transcription factor regulating the hemocytes differentiation. In this paper, we identified and characterized RUNX family especially Lz in silkworm, which is a lepidopteran model insect. The gene expression analysis illustrated that BmLz was highly expressed in hemocytes throughout the whole development period, and reached a peak in glutonous stage. Over-expression of BmLz in silkworm accelerated the melanization process of hemolymph, and led to instantaneously up-regulation of prophenoloxidases (PPOs), which were key enzymes in the melanization process. Further down-regulation of BmLz expression by RNA interference resulted in the significant delay of melanization reaction of hemolymph. These findings suggested that BmLz regulated the melanization process of hemolymph by inducing PPOs expression, and played a critical role in innate immunity defense in silkworm. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Runt-related transcription factor (RUNX) Lozenge Melanization Prophenoloxidases Bombyx mori
1. Introduction Runt-related transcription factors (RUNX) are evolutionarily conserved either in vertebrate or invertebrate (Adewumi et al., 2007; Coffman, 2003; Wildonger et al., 2005). They are characterized by a Runt (or Run) domain, which is highly conserved for DNA binding and proteineprotein interaction and defines a family of heterodimeric transcription factors with essential function in cell development (Coffman, 2003; Kagoshima et al., 1993). There are three RUNX genes in mammals, RUNX1, RUNX2, and RUNX3. They play pivotal roles in normal development and neoplasia (Kim et al.,
Abbreviations: RUNX, Runt-related transcription factor; Lz, Lozenge; AML-1, acute myeloid leukemia factor 1; BmLz, Lozenge gene from Bombyx mori; BmRun, Run gene from Bombyx mori; BmRunt3, Runt3 gene from Bombyx mori; PPOs, prophenoloxidases; DmPPO, PPO gene from Drosophila melanogaster; PCR, polymerase chain reaction; RT-qPCR, real-time quantitative PCR; ORF, open reading frame; UTR, untranslated region; BmPPO, PPO gene from Bombyx mori; PGRP, peptidoglycan recognition proteins; bGRP, b-1 and 3-glucan recognition proteins; PPAE, PPO-activating enzyme. * Corresponding author. E-mail addresses: [email protected], [email protected] (H. Cui). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.dci.2015.07.001 0145-305X/© 2015 Elsevier Ltd. All rights reserved.
2014). In the Dipteral insect Drosophila melanogaster, there are four RUNX members including Lozenge (Lz), Runt (Run), CG42267 and CG34147. CG42267 and CG34147 is less explored, recently a genomewide RNA interference screen identified an isoform of CG42267 which is involved in control of cell survival (Boutros et al., 2004). Deletion of Run in D. melanogaste led to periodic deletions of larval segments and had an effect on sex determination and neurogenesis (Bao and Friedrich, 2008). Lozenge regulates crystal cell lineage commitment by combinatorial interactions with Serpent (Srp) and U-shaped (Ush) during D. melanogaster hematopoiesis (Fossett et al., 2003). When the signals from the NotcheSerrate pathway activated Srp, the expression of Lz is up-regulated, while Ush is down-regulated, which allowing SrpNC to interact with Lz to drive crystal cell lineage commitment. Another possibility is that Ush is activated with Srp, and then directly binds to SrpNC, converting Lz from an activator to a repressor of crystal cell production (Fossett et al., 2003; Lebestky et al., 2000, 2003). In addition, Lz is also in charge of the programmed cell death in D. melanogaster. During this process, Lz directly bind and activate aos and klu through to regulate cell death in cone and pigment cells (Wildonger et al., 2005). It is also reported that RUNX4 in Mosquito is the ortholog gene of Lozenge in D. melanogaster, which regulates prophenoloxidases
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(PPOs) gene expression under the Toll pathway (Zou et al., 2008). Insect PPO is an important innate immune protein due to its involvement in cellular and humoral defense (Lu et al., 2014). It is cleaved to generate the activated PO, which catalyzes the oxidation of phenols into quinones, and subsequently polymerize it into melanin (Binggeli et al., 2014). In D. melanogaster, there are three PPO genes: DmPPO1 (CG5779), DmPPO2 (GC8193) and DmPPO3 (GC2952). DmPPO1 and DmPPO2 are responsible for all the PO activity in the hemolymph, and play important role in the reaction against some Gram-positive bacteria and fungi encapsulation (Binggeli et al., 2014). Two PPOs in silkworm genome were reported, which could be directly cleaved into activated POs by PPOactivating enzyme (PPAE) (Ashida, 1997). In the BmPPOs activation cascade, once peptidoglycan recognition proteins (PGRP),b-1 and 3glucan recognition proteins (bGRP) detected the invading microorganisms, then PPAE would be activated and cleaved. (Ashida, 1997). However, the knowledge of PPOs activation pathway in silkworm is limited. Until now, the RUNX family is not identified in silkworm yet, in this paper, we identified and characterized the RUNX family in silkworm, which were identified as BmRun (Bm008906), BmLz (Bm008908) and BmRunt3 (Bm008970). We conducted the gene expression profile of all of them, and investigated the function of BmLz in melanization reaction in silkworm hemocytes, our result revealed that BmLz increased melanization reaction through regulating BmPPO expression. 2. Materials and methods 2.1. Silkworm strain and cell lines The Chinese silkworm strain Dazao (p50) was from the State Key Laboratory of Silkworm Genome Biology (Southwest University, China). Various larval tissues in different stages were stored in liquid nitrogen until they were needed. The S2 cell line (Schneider, 1972) was cultured at 27 C in Schneider's Drosophila medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). The BmE cell line (Pan et al., 2010) was cultured at 27 C in Grace insect's medium (Invitrogen) supplemented with 10% FBS. 2.2. Full-length cDNA cloning, RT-PCR and qRT-PCR analysis of RUNX factors in B. mori Total RNA was extracted with TRIzol reagent (TaKaRa) according to the manufacturer's protocol. 2 mg RNA was reverse transcribed by M-MLV Reverse Transcriptase (Promega), which was stored at 20 C according to the protocol provided by the manufacturer. The RUNX factors were identified with a bioinformatic method based on silkworm genome, CDS database, 9X genomic sequencing database, EST database, and predicted protein database of Bombyx mori (http://www.silkdb.org/silkdb/) (Xia et al., 2004). The amino acid sequence of the RUNX factors from D. melanogaster, A. aegypti and other insects were obtained from the NCBI GenBank (http:// www.ncbi.nlm.nih.gov/) (Table 1). According to the predicted CDS and EST sequences in the SilkDB, primers were designed, 3’and 5’RNA ligase-mediated rapid amplification of cDNA ends (RLMRACE) was performed to obtain their full-length cDNA by using a GeneRacer™kit (Invitrogen) with the gene-specific primer (The sequence of all primers in Supplemental Table 1). Subsequently, each putative protein was further validated by domain prediction using SMART (http://smart.embl-heidelberg.de/) and Pfam (http:// pfam.sanger.ac.uk/) (Zhang et al., 2014). Quantitative real-time PCR (qRT-PCR) was conducted with SYBR® Premix Ex Taq™ II (TaKaRa) with the ABI 7500 Fast Real-
Time PCR System (Applied Biosystems). 2.3. Expression of recombinant BmLz protein and antibody preparation The truncated coding sequence of BmLz was amplified by PCR using hemocyte cDNA template (The sequence of the primers in Supplemental Table 1). After digested with BamHI and XhoI (TaKaRa), the PCR product was ligated into PET32a vector. Then the recombinant plasmid was transformed into competent E. coli Rosetta (DE3) cells, and the recombinant protein was induced by 0.6 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) for 6 h at 37 C, and purified with the HIS/BIND PURIFICATION KIT (Merck). 200 ug recombinant protein BmLz was used for antibody preparation. Briefly, Kunming strain of mouse was used for immunization. For the first round inoculation, an equal volume of complete Freund's adjuvant (Sigma) was used. For the next three round inoculations, the incomplete Freund's adjuvant (Sigma) was used. The mouse was immunized for every 10 days and for 4 times. All procedures were followed the standard procedures (Ellis et al., 1989). The mouse was euthanatized, and the antiserum of BmLz was collected and prepared. 2.4. Cell transfection and over-expressed BmLz The BmLz full length cDNA was amplified by PCR using Dazao (p50) hemocyte cDNA template, and digested with BamH I and ligated into pSL1180-A4-SV40-EGFP vector (Jin et al., 2014). 2 105 BmE cells and S2 cells were seeded into 24-well plates for 24 h, and transiently transfected with pSL1180-A4-SV40-EGFP-BmLz using XtremeGENE HP DNA Transfection Reagent (Roche). After transfection for 72 h, cells were processed for immunofluorescence and
Table 1 Species, proteins and genbank accession numbers of RUNX used in phylogenetic reconstructions. Species
Name
Accession no.
Aedes aegypti Drosophila melanogaster Tribolium castaneum Bombyx mori Danaus plexippus Nasonia vitripennis Apis mellifera Megachile rotundata] Drosophila melanogaster Aedes aegypti Tribolium castaneum Nasonia vitripennis Apis mellifera Megachile rotundata] Bombyx mori Danaus plexippus Aedes aegypti Drosophila melanogaster Tribolium castaneum Nasonia vitripennis Megachile rotundata] Apis mellifera Apis mellifera Megachile rotundata] Nasonia vitripennis Tribolium castaneum Bombyx mori Danaus plexippus Aedes aegypti Drosophila melanogaster
Aaeg CG34145 Dmel CG34145 Tcas CG34145 Bmor Runt3 Dple CG34145 Nvit CG34145 Amel CG34145 Mrot CG34145 Dmel CG42267 Aaeg CG42267 Tcas CG42267 Nvit CG42267 Amel CG42267 Mrot CG42267 Bmor Lz Dple Lz Aaeg lz Dmel lz Tcas lz Nvit Lz Mrot Lz Amel Lz Amel Run Mrot Run Nvit Run Tcas Run Bmor Run Dple Run Aaeg Run Dmel Run
ACF35310.1 NP_001036285.2 EFA09162.1 EHJ75094.1 XP_008207595.1 XP_394014.2 XP_003702961.1 NP_608399.2 ACF35312.1 EFA09256.1 XP_008207691.1 XP_394013.4 XP_003702821.1 EHJ75091.1 ACF35313.1 NP_511099.2 EFA09195.1 XP_008206481.1 XP_003702823.1 XP_006564924.1 XP_001121886 XP_003702822.1 XP_008206395.1 XP_969277.1 EHJ75093 ACF35311.1 AAC27777.1
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for qRT-PCR. 2.5. Immunofluorescence assay After grown on coverslip for overnight, the cells was washed 1X phosphate balanced solution (PBS) for 5min, fixed with 4% (w/v) paraformaldehyde (PFA) for 30 min, and permeated by 1% TrionX100 (Sigma). Followed with 10% goat serum incubation, the primary antibody against BmLz at 1: 500 for 1 h, and the secondary antibody Alexa Fluor® 595 goat anti-mouse IgG (H þ L) (1:2000, Invitrogen) for 1 h, the cells were counterstained the nuclei with DAPI (Sigma) for 10min, and observed under Olympus FV1000 confocal microscope.
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2.6. Over expressed BmLz in silkworm 2 mg pSL1180-A4-BmLz-SV40-EGFP in 20 ml medium without FBS and antibiotic with 6 ml X-tremeGENE HP DNA Transfection Reagent (Roche) were mixed and injected into silkworm larva at day 3 of 5th instar after incubating at room temperature for 30 min. The empty vector pSL1180-A4-SV40-EGFP was used as control. After 72 h injection, hemocytes were collected. 2.7. Knockdown of BmLz in silkworm The double strand RNAs (dsRNAs) for BmLz and EGFP were generated by RiboMAX Large Scale RNA Production System-T7 kit
Fig. 1. The genomic locations of the RUNX genes in B. mori and phylogenetic analysis of BmRUNX genes with other insects. (A) The genomic locations of the RUNX genes in B. mori. The red boxes represent the BmRUNX genes localization and transcriptional orientation was indicated by black arrows. (B) Phylogenetic analysis of BmRUNX genes with other insects. The full-length amino acid sequences from B. mori and insect RUNX proteins were aligned using ClustalX. A phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates, and displayed with MEGA 4.0. The BmRUNXs are colored by red. Sequence information was summarized in Table 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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3. Results 3.1. Characteristics and phylogenetic analysis of the B. mori RUNX genes sequence BmRun, BmLz, and BmRunt3 full-length sequences were obtained and identified. They all had a Runt domain, and the sequences and their information were listed in Fig. S1eS3 and Supplemental Table 2, which were all located in the nscaf2930 of chromosome No.3 in silkworm (Fig. 1A). BmRun consisted of five exons and four introns, while BmLz and BmRunt3 consisted of eight exons and seven introns (Fig. S4). The phylogenetic analysis showed that BmRun belonged to Group RUN, BmRunt3 was in the Group CG34145, and BmLz was in the Group Lozenge (Fig. 1B). However, their Runt domain amino acid sequence was highly conservative with that of D. melanogaster and Danaus plexippus (Fig. S5). The homology reached up to more than 70% identity. 3.2. Expression pattern analysis of the B. mori RUNX genes
Fig. 2. The expression patterns of BmRunx genes or only BmLz in B. mori. (A) The expression analysis of BmRunx genes in different larval tissues on day 3 of the fifth instar silkworm larvae by RT-PCR. BmActin3 gene was used as the internal control at each tissue. (BeC) Analysis the relative transcript abundance of the BmLz in different larval tissues on day 3 of the fifth instar larvae and the hemocytes from day 3 of 4th instar to the whole 5th instar of silkworm by qRT-PCR. Abbreviation: Ep, epidermis; He, head; Te, testis; Ov, ovary; Mi, midgut; Ma, Malpighian tube; Si, silk gland; Fa, fat body; Wg, Wing imaginal disc; Ha, hemocytes; Wh, Whole body; M, Molting. BmGAPDH was used as an internal control.
(Promega) (Payungporn et al., 2006). The primers sequence was listed in Supplemental Table 1. 50 mg of dsRNA was injected into silkworm larva at first day of 5th instar to knockdown BmLz. EGFP was used as control. After injected for 3 days, hemocytes were collected.
2.8. PO activity assay Phenoloxidase activities were determined according to Haobo Jiang et al. (Jiang et al., 2003) and Meixian Wang et al. (Wang et al., 2013) methods with slight modification. 40 ml hemolymph immediately mixed with 60 ml cold PBS, then added 200 ml of 10 mM dopamine (Sigma) in 20 mM TriseHCl (pH 7.0). After mixing enough, 200 ml mixture was transferred into microplate wells. After incubating at 37 C for 30 min, the absorbance at 450 nm was assessed using the GloMax-Multi Detection System Photometer (Promega).
The mRNA expression level of RUNX genes on day 3 of 5th (L5D3) instar larvaes was analyzed. As shown in Fig. 2, we collected and compared most of organs in silkworm, including testis, ovary, epidermis, silk gland, malpighian tubule, midgut, head, fat body, wing imaginal disc (adhering hematopoietic organ) and hemocytes. The electrophoresis results showed that BmRun and BmRunt3 were commonly expressed in all organs, although BmRun expression was a little bit low in malpighian and testis, and undetectable in hemocytes. However, only BmLz was specifically expressed in hemocytes and wing imaginal disc (Fig. 2A), which meant that BmLz might play an important role in hemocytes and hematogenesis in silkworm. We confirmed the result by qRT-PCR, and the data showed that BmLz was highly expressed in homocytes comparing to other tissues (Fig. 2B). Further, we collected and analyzed mRNA level from hemocytes in different development stages from day 3 of 4th (L4D3) instar larvae, the whole 5th instar, to the day 2 of prepupa (PP2) larvae. The result showed that BmLz was commonly expressed in hemocytes through the whole development period, and reached a peak in glutonous stage L4D4 and L5D4 (Fig. 2C) which implied that BmLz might involve in homocyte development. 3.3. Localization analysis of BmLz Next, we overexpressed BmLz in BmE cell line, EGFP was used as control. The result showed that the control EGFP signal evenly spread out in cytoplasm and nucleus, in contrast, the BmLz signal only existed in cell nucleus not in cytoplasm (Fig. 3A). Moreover, we validated this result in hemocytes of B. mori by immunofluorescence using BmLz antibody we generated. The result showed that BmLz can be found in two types of hemocytes though observing the cell morphology. Consistent with the result in BmE cell line, BmLz was detected in cell nucleus of spherulocyte, but also could be expressed in the whole cell of oenocytoid (Fig. 3B). So BmLz might play a role in controlling hemocytes differentiation and immune response as a transcription factor. 3.4. Over-expression BmLz up-regulated the PPOs expression in vitro To further examine the role of BmLz in insect immune response, we firstly up-regulated the BmLz expression in S2 cell line (Fig. 4A and B). As shown in Fig. 4C, overexpression of BmLz significantly increased PPO1, PPO2, PPO3 respectively, which are the key enzymes in the melanization process.
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Fig. 3. Localization of BmLz in cell of B. mori. (A) BmLz-EGFP fusion protein localized in the cell nucleus of BmE. (B) Immunofluorescence analysis using BmLz antibodie in the circulating haemocytes of silkworms. BmLz could be detected in oenocytiod and spherulocyte severally. Scale bar ¼ 5 mm.
3.5. Over-expression of BmLz in vivo stimulated melanization process of hemolymph It's known that B. mori hemolymph would blacken when it is exposed to the air, which is attributed to PPOs. After injection of BmLz-EGFP in silkworm for 3 days, the EGFP signal could be detected in circulatory hemocytes. It was similar to the result in BmE cell line, control EGFP signal spread out in cytoplasm and nucleus. In contrast, the BmLz-EGFP signal was detected in cell nucleus, and most of these cells seem to plasmatocyte (Fig. 5A). qRT-PCR analysis was accomplished and showed that BmLz was over expressed comparing to control (Fig. 5B). Overexpression of BmLz dramatically accelerated markedly the melanization process, after exploring to air for 10min, hemolymph started to blacken in overexpressed BmLz group, but not the control group (Fig. 5D), and the PO activity in overexpressed BmLz group was higher than the control group (Fig. 5C). Overexpression of BmLz could significantly increase PPO1 and related melanization enzyme BmPPAE and BmBAEE, especially PPO1 increased at least more than twenty fold (Fig. 5E). Taken together, these findings demonstrated that BmLz could stimulate melanization process in hemolymph of B. mori. Fig. 4. The effect of BmLz was overexpressed by transient transfection in S2 cell line. (A) The expression level of BmLz detection in S2 cell line. (B) qRT-PCR detected the expression level change of BmLz in S2 cell line. (C) The expression level of PPO1, PPO2 and PPO3 detection by qRT-PCR in S2 cell line after overexpression of BmLz. Dmrq49 was used as an internal control. The data were analysed using Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001. Data are shown as the mean ± SE from three independent experiments.
3.6. Down-regulation of BmLz expression by RNA interference in vivo delayed the melanization process of hemolymph To examine the role of BmLz in hemocytes of B. mori, BmLz
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dsRNA was designed as in Fig. 6A and B, and injected into silkworm larvae for 3 days. The expression of BmLz was dramatically reduced more than 70% in hemocytes comparing to control (Fig. 6C). As shown in Fig. 6D, the melanization process was delayed distinctly after knockdown BmLz, specifically after exploring to air for 20min. The PO activity analysis showed that PO activity in Lz RNAi group was reduced compare with the control group (Fig. 6E). In addition, immunofluorescence result showed that the expression of BmLz in oenocytoid reduced distinctly (Fig. 6F). 4. Discussion RUNX family has been commonly studied in mammals, as well as in D. melanogaster. They play pivotal roles in several developmental processed including hematopoiesis, neurogenesis, and skeletogenesis (Nah et al., 2014). However, there is no any report about RUNX transcription factors in B. mori. This is the first time we identified and characterized the RUNX family including BmRun, BmLz and BmRunt3 in silkworm. BmRun was clustered into RUN group, BmLz was clustered into Lozenge group, and BmRunt3 clustered into CG34145 group. Their Runt domain sequence was highly conservative with that of D. melanogaster and D. plexippus,
and the identity was up to 70%. BmRun and BmRunt3 were commonly expressed in most of organs, except BmRun was undetectable in hemocytes. However, BmLz was only expressed in hemocytes and imaginal disc, which meant that it might play an important role in hemocyte. The localization analysis of BmLz showed that it expressed in spherulocyte and oenocytoid, which suggested that it might involve in immunoreactions of silkworm. Next, through overexpression and knockdown experiment, we verified that role of BmLz in hemolymph. Overexpressed BmLz could upregulate PPOs expression in vivo and in vitro, which are key enzyme in melaniztion reaction, and accelerate the melanization response of hemolymph of B. mori. Knockdown BmLz downregulated BmPPOs expression, and delay the melanization process of hemolymph. There are four types of hemocyte in D. melanogaster, including prohemocyte, plasmatocyte, crystal cell, and lamellocyte (Minakhina and Steward, 2010). But there are five types of hemocyte in B. mori, including prohemocyte, oenocytoid, plasmatocyte, granulocyte and spherulocyte (Nakahara et al., 2009; Tan et al., 2013). Oenocytoid in B. mori is similar to the crystal cell in D. melanogaster (Strand, 2008). Both of them are active participator in response to microorganisms’ invasion and wound healing
Fig. 5. The effect of BmLz was overexpressed in day 3 of 5th instar silkworm larva hemolymph by transient transfection. (A) BmLz-EGFP fusion protein expressed in the circulating haemocytes though transient transfection of plasmids. (B) The mRNA expression level of BmLz in the hemolymph. (C) PO activity change after BmLz overexpression. (D) Hemolymph was harvested after BmLz overexpression. (E) The mRNA expression level of the melanzation pathway related gens in silkworm detected by qRT-PCR. BmGAPDH was used as an internal control. The data were analysed using Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001. Data are shown as the mean ± SE from three independent experiments.
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Fig. 6. Down-regulation of BmLz by RNA interference in day 1 of 5th instar silkworm larva haemolymph. (A) The sequence of BmLz dsRNA. (B) The synthesis of dsRNA (1, 2 band were EGFP dsRNA; 3, 4 band were BmLz dsRNA). (C) qRT-PCR detected the expression level change of BmLz in hemocytes after RNAi. The data were analysed using Student's t test, **p < 0.01. Data are shown as the mean ± SE from three independent experiments. (D) Survey of haemolymph melanism rate after BmLz was knockdown. (E) PO activity change after RNAi. (F) Immunofluorescence analysis using BmLz antibodie in the circulating haemocytes after RNAi.
(Adewumi et al., 2007; Ashida et al., 1988; Ling et al., 2005). Lz, as a hematopoietic modulation factor, is in control of the fate of crystal cell in D. melanogaster (Lebestky et al., 2000). PPOs, as the enzymes required for melanization response of hemolymph, mainly exist in crystal cell in D. melanogaster, which are cleaved to generate active POs as a result of proteolytic cascade activation and initiate the melanization response (Ferjoux et al., 2007; Gajewski et al., 2007). Mosquito RUNX4, the ortholog gene of D. melanogaster Lz could directly regulate PPO genes expression by binding to the Runt binding motif in the promoter of PPOs to continue the melanization during the maturing process of new crystal cell (Zou et al., 2008). In B. mori, PPOs exist in oenocytoids and plasmatocytes, while PO only exists in oenocytoids (Ashida et al., 1988). These suggest that BmLz promotes the melanization reaction of hemolymph by directly regulating the PPOs in oenocytoids of B. mori. Whereas, the further research and verification are still demanding.
Conflicts of interest The authors have declared that no competing interests exist. Acknowledgment This work was supported by the National Basic Research Program of China (2012CB114603), the Research Fund for the Doctoral Program of Higher Education of China (20130182110003), the Natural Science Foundation of Chongqing (CSTC2013jcyjys0007), and the Fundamental Research Funds for the Central Universities (SWU111014, XDJK2015D021). We are grateful to Prof. Chunfeng Li of the State Key Laboratory of Silkworm Genome Biology for his help in preparation of this manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http://
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mediated sumoylation. Oncogenesis 3, e101. Lebestky, T., Chang, T., Hartenstein, V., Banerjee, U., 2000. Specification of Drosophila hematopoietic lineage by conserved transcription factors. Science 288, 146e149. Lebestky, T., Jung, S.-H., Banerjee, U., 2003. A serrate-expressing signaling center controls Drosophila hematopoiesis. Genes Dev. 17, 348e353. Ling, E., Shirai, K., Kanehatsu, R., Kiguchi, K., 2005. Reexamination of phenoloxidase in larval circulating hemocytes of the silkworm, Bombyx mori. Tissue Cell 37, 101e107. Lu, A., Zhang, Q., Zhang, J., Yang, B., Wu, K., Xie, W., Luan, Y.-X., Ling, E., 2014. Insect prophenoloxidase: the view beyond immunity. Front. Physiol. 5. Minakhina, S., Steward, R., 2010. Hematopoietic stem cells in Drosophila. Development 137, 27e31. Nah, G.S.S., Tay, B.-H., Brenner, S., Osato, M., Venkatesh, B., 2014. Characterization of the Runx gene family in a Jawless vertebrate, the Japanese Lamprey (Lethenteron japonicum). PloS one 9, e113445. Nakahara, Y., Shimura, S., Ueno, C., Kanamori, Y., Mita, K., Kiuchi, M., Kamimura, M., 2009. Purification and characterization of silkworm hemocytes by flow cytometry. Dev. Comp. Immunol. 33, 439e448. Pan, M.-H., Cai, X.-J., Liu, M., Lv, J., Tang, H., Tan, J., Lu, C., 2010. Establishment and characterization of an ovarian cell line of the silkworm, Bombyx mori. Tissue Cell 42, 42e46. Payungporn, S., Chutinimitkul, S., Chaisingh, A., Damrongwantanapokin, S., Buranathai, C., Amonsin, A., Theamboonlers, A., Poovorawan, Y., 2006. Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection. J. Virol. Methods 131, 143e147. Schneider, I., 1972. Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27, 353e365. Strand, M.R., 2008. The insect cellular immune response. Insect Sci. 15, 1e14. Tan, J., Xu, M., Zhang, K., Wang, X., Chen, S., Li, T., Xiang, Z., Cui, H., 2013. Characterization of hemocytes proliferation in larval silkworm, Bombyx mori. J. Insect Physiol. 59, 595e603. Wang, M.-x., Lu, Y., Cai, Z.-z., Liang, S., Niu, Y.-s., Miao, Y.-g., 2013. Phenol oxidase is a necessary enzyme for the silkworm molting which is regulated by molting hormone. Mol. Biol. Reports 40, 3549e3555. Wildonger, J., Sosinsky, A., Honig, B., Mann, R.S., 2005. Lozenge directly activates argos and klumpfuss to regulate programmed cell death. Genes Dev. 19, 1034e1039. Xia, Q., Zhou, Z., Lu, C., Cheng, D., Dai, F., Li, B., Zhao, P., Zha, X., Cheng, T., Chai, C., 2004. A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science 306, 1937e1940. Zhang, K., Xu, M., Su, J., Yu, S., Sun, Z., Li, Y., Zhang, W., Hou, J., Shang, L., Cui, H., 2014. Characterization and identification of the integrin family in silkworm, Bombyx mori. Gene 549, 149e155. Zou, Z., Shin, S.W., Alvarez, K.S., Bian, G., Kokoza, V., Raikhel, A.S., 2008. Mosquito RUNX4 in the immune regulation of PPO gene expression and its effect on avian malaria parasite infection. Proc. Natl. Acad. Sci. 105, 18454e18459.
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Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
A novel Lozenge gene in silkworm, Bombyx mori regulates the melanization response of hemolymph Man Xu a, 1, Xue Wang a, 1, Juan Tan a, Kui Zhang a, Xi Guan a, Laurence H. Patterson b, Hanfei Ding c, Hongjuan Cui a, * a b c
State Key Laboratory of Silkworm Genome Biology (Southwest University), 400715, China Institute of Cancer Therapeutics, University of Bradford, West Yorkshire BD7 1DP, UK Cancer Center, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 February 2015 Received in revised form 10 June 2015 Accepted 1 July 2015 Available online 9 July 2015
Runt-related (RUNX) transcription factors are evolutionarily conserved either in vertebrate or invertebrate. Lozenge (Lz), a members of RUNX family as well as homologue of AML-1, functions as an important transcription factor regulating the hemocytes differentiation. In this paper, we identified and characterized RUNX family especially Lz in silkworm, which is a lepidopteran model insect. The gene expression analysis illustrated that BmLz was highly expressed in hemocytes throughout the whole development period, and reached a peak in glutonous stage. Over-expression of BmLz in silkworm accelerated the melanization process of hemolymph, and led to instantaneously up-regulation of prophenoloxidases (PPOs), which were key enzymes in the melanization process. Further down-regulation of BmLz expression by RNA interference resulted in the significant delay of melanization reaction of hemolymph. These findings suggested that BmLz regulated the melanization process of hemolymph by inducing PPOs expression, and played a critical role in innate immunity defense in silkworm. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Runt-related transcription factor (RUNX) Lozenge Melanization Prophenoloxidases Bombyx mori
1. Introduction Runt-related transcription factors (RUNX) are evolutionarily conserved either in vertebrate or invertebrate (Adewumi et al., 2007; Coffman, 2003; Wildonger et al., 2005). They are characterized by a Runt (or Run) domain, which is highly conserved for DNA binding and proteineprotein interaction and defines a family of heterodimeric transcription factors with essential function in cell development (Coffman, 2003; Kagoshima et al., 1993). There are three RUNX genes in mammals, RUNX1, RUNX2, and RUNX3. They play pivotal roles in normal development and neoplasia (Kim et al.,
Abbreviations: RUNX, Runt-related transcription factor; Lz, Lozenge; AML-1, acute myeloid leukemia factor 1; BmLz, Lozenge gene from Bombyx mori; BmRun, Run gene from Bombyx mori; BmRunt3, Runt3 gene from Bombyx mori; PPOs, prophenoloxidases; DmPPO, PPO gene from Drosophila melanogaster; PCR, polymerase chain reaction; RT-qPCR, real-time quantitative PCR; ORF, open reading frame; UTR, untranslated region; BmPPO, PPO gene from Bombyx mori; PGRP, peptidoglycan recognition proteins; bGRP, b-1 and 3-glucan recognition proteins; PPAE, PPO-activating enzyme. * Corresponding author. E-mail addresses: [email protected], [email protected] (H. Cui). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.dci.2015.07.001 0145-305X/© 2015 Elsevier Ltd. All rights reserved.
2014). In the Dipteral insect Drosophila melanogaster, there are four RUNX members including Lozenge (Lz), Runt (Run), CG42267 and CG34147. CG42267 and CG34147 is less explored, recently a genomewide RNA interference screen identified an isoform of CG42267 which is involved in control of cell survival (Boutros et al., 2004). Deletion of Run in D. melanogaste led to periodic deletions of larval segments and had an effect on sex determination and neurogenesis (Bao and Friedrich, 2008). Lozenge regulates crystal cell lineage commitment by combinatorial interactions with Serpent (Srp) and U-shaped (Ush) during D. melanogaster hematopoiesis (Fossett et al., 2003). When the signals from the NotcheSerrate pathway activated Srp, the expression of Lz is up-regulated, while Ush is down-regulated, which allowing SrpNC to interact with Lz to drive crystal cell lineage commitment. Another possibility is that Ush is activated with Srp, and then directly binds to SrpNC, converting Lz from an activator to a repressor of crystal cell production (Fossett et al., 2003; Lebestky et al., 2000, 2003). In addition, Lz is also in charge of the programmed cell death in D. melanogaster. During this process, Lz directly bind and activate aos and klu through to regulate cell death in cone and pigment cells (Wildonger et al., 2005). It is also reported that RUNX4 in Mosquito is the ortholog gene of Lozenge in D. melanogaster, which regulates prophenoloxidases
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(PPOs) gene expression under the Toll pathway (Zou et al., 2008). Insect PPO is an important innate immune protein due to its involvement in cellular and humoral defense (Lu et al., 2014). It is cleaved to generate the activated PO, which catalyzes the oxidation of phenols into quinones, and subsequently polymerize it into melanin (Binggeli et al., 2014). In D. melanogaster, there are three PPO genes: DmPPO1 (CG5779), DmPPO2 (GC8193) and DmPPO3 (GC2952). DmPPO1 and DmPPO2 are responsible for all the PO activity in the hemolymph, and play important role in the reaction against some Gram-positive bacteria and fungi encapsulation (Binggeli et al., 2014). Two PPOs in silkworm genome were reported, which could be directly cleaved into activated POs by PPOactivating enzyme (PPAE) (Ashida, 1997). In the BmPPOs activation cascade, once peptidoglycan recognition proteins (PGRP),b-1 and 3glucan recognition proteins (bGRP) detected the invading microorganisms, then PPAE would be activated and cleaved. (Ashida, 1997). However, the knowledge of PPOs activation pathway in silkworm is limited. Until now, the RUNX family is not identified in silkworm yet, in this paper, we identified and characterized the RUNX family in silkworm, which were identified as BmRun (Bm008906), BmLz (Bm008908) and BmRunt3 (Bm008970). We conducted the gene expression profile of all of them, and investigated the function of BmLz in melanization reaction in silkworm hemocytes, our result revealed that BmLz increased melanization reaction through regulating BmPPO expression. 2. Materials and methods 2.1. Silkworm strain and cell lines The Chinese silkworm strain Dazao (p50) was from the State Key Laboratory of Silkworm Genome Biology (Southwest University, China). Various larval tissues in different stages were stored in liquid nitrogen until they were needed. The S2 cell line (Schneider, 1972) was cultured at 27 C in Schneider's Drosophila medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). The BmE cell line (Pan et al., 2010) was cultured at 27 C in Grace insect's medium (Invitrogen) supplemented with 10% FBS. 2.2. Full-length cDNA cloning, RT-PCR and qRT-PCR analysis of RUNX factors in B. mori Total RNA was extracted with TRIzol reagent (TaKaRa) according to the manufacturer's protocol. 2 mg RNA was reverse transcribed by M-MLV Reverse Transcriptase (Promega), which was stored at 20 C according to the protocol provided by the manufacturer. The RUNX factors were identified with a bioinformatic method based on silkworm genome, CDS database, 9X genomic sequencing database, EST database, and predicted protein database of Bombyx mori (http://www.silkdb.org/silkdb/) (Xia et al., 2004). The amino acid sequence of the RUNX factors from D. melanogaster, A. aegypti and other insects were obtained from the NCBI GenBank (http:// www.ncbi.nlm.nih.gov/) (Table 1). According to the predicted CDS and EST sequences in the SilkDB, primers were designed, 3’and 5’RNA ligase-mediated rapid amplification of cDNA ends (RLMRACE) was performed to obtain their full-length cDNA by using a GeneRacer™kit (Invitrogen) with the gene-specific primer (The sequence of all primers in Supplemental Table 1). Subsequently, each putative protein was further validated by domain prediction using SMART (http://smart.embl-heidelberg.de/) and Pfam (http:// pfam.sanger.ac.uk/) (Zhang et al., 2014). Quantitative real-time PCR (qRT-PCR) was conducted with SYBR® Premix Ex Taq™ II (TaKaRa) with the ABI 7500 Fast Real-
Time PCR System (Applied Biosystems). 2.3. Expression of recombinant BmLz protein and antibody preparation The truncated coding sequence of BmLz was amplified by PCR using hemocyte cDNA template (The sequence of the primers in Supplemental Table 1). After digested with BamHI and XhoI (TaKaRa), the PCR product was ligated into PET32a vector. Then the recombinant plasmid was transformed into competent E. coli Rosetta (DE3) cells, and the recombinant protein was induced by 0.6 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) for 6 h at 37 C, and purified with the HIS/BIND PURIFICATION KIT (Merck). 200 ug recombinant protein BmLz was used for antibody preparation. Briefly, Kunming strain of mouse was used for immunization. For the first round inoculation, an equal volume of complete Freund's adjuvant (Sigma) was used. For the next three round inoculations, the incomplete Freund's adjuvant (Sigma) was used. The mouse was immunized for every 10 days and for 4 times. All procedures were followed the standard procedures (Ellis et al., 1989). The mouse was euthanatized, and the antiserum of BmLz was collected and prepared. 2.4. Cell transfection and over-expressed BmLz The BmLz full length cDNA was amplified by PCR using Dazao (p50) hemocyte cDNA template, and digested with BamH I and ligated into pSL1180-A4-SV40-EGFP vector (Jin et al., 2014). 2 105 BmE cells and S2 cells were seeded into 24-well plates for 24 h, and transiently transfected with pSL1180-A4-SV40-EGFP-BmLz using XtremeGENE HP DNA Transfection Reagent (Roche). After transfection for 72 h, cells were processed for immunofluorescence and
Table 1 Species, proteins and genbank accession numbers of RUNX used in phylogenetic reconstructions. Species
Name
Accession no.
Aedes aegypti Drosophila melanogaster Tribolium castaneum Bombyx mori Danaus plexippus Nasonia vitripennis Apis mellifera Megachile rotundata] Drosophila melanogaster Aedes aegypti Tribolium castaneum Nasonia vitripennis Apis mellifera Megachile rotundata] Bombyx mori Danaus plexippus Aedes aegypti Drosophila melanogaster Tribolium castaneum Nasonia vitripennis Megachile rotundata] Apis mellifera Apis mellifera Megachile rotundata] Nasonia vitripennis Tribolium castaneum Bombyx mori Danaus plexippus Aedes aegypti Drosophila melanogaster
Aaeg CG34145 Dmel CG34145 Tcas CG34145 Bmor Runt3 Dple CG34145 Nvit CG34145 Amel CG34145 Mrot CG34145 Dmel CG42267 Aaeg CG42267 Tcas CG42267 Nvit CG42267 Amel CG42267 Mrot CG42267 Bmor Lz Dple Lz Aaeg lz Dmel lz Tcas lz Nvit Lz Mrot Lz Amel Lz Amel Run Mrot Run Nvit Run Tcas Run Bmor Run Dple Run Aaeg Run Dmel Run
ACF35310.1 NP_001036285.2 EFA09162.1 EHJ75094.1 XP_008207595.1 XP_394014.2 XP_003702961.1 NP_608399.2 ACF35312.1 EFA09256.1 XP_008207691.1 XP_394013.4 XP_003702821.1 EHJ75091.1 ACF35313.1 NP_511099.2 EFA09195.1 XP_008206481.1 XP_003702823.1 XP_006564924.1 XP_001121886 XP_003702822.1 XP_008206395.1 XP_969277.1 EHJ75093 ACF35311.1 AAC27777.1
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for qRT-PCR. 2.5. Immunofluorescence assay After grown on coverslip for overnight, the cells was washed 1X phosphate balanced solution (PBS) for 5min, fixed with 4% (w/v) paraformaldehyde (PFA) for 30 min, and permeated by 1% TrionX100 (Sigma). Followed with 10% goat serum incubation, the primary antibody against BmLz at 1: 500 for 1 h, and the secondary antibody Alexa Fluor® 595 goat anti-mouse IgG (H þ L) (1:2000, Invitrogen) for 1 h, the cells were counterstained the nuclei with DAPI (Sigma) for 10min, and observed under Olympus FV1000 confocal microscope.
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2.6. Over expressed BmLz in silkworm 2 mg pSL1180-A4-BmLz-SV40-EGFP in 20 ml medium without FBS and antibiotic with 6 ml X-tremeGENE HP DNA Transfection Reagent (Roche) were mixed and injected into silkworm larva at day 3 of 5th instar after incubating at room temperature for 30 min. The empty vector pSL1180-A4-SV40-EGFP was used as control. After 72 h injection, hemocytes were collected. 2.7. Knockdown of BmLz in silkworm The double strand RNAs (dsRNAs) for BmLz and EGFP were generated by RiboMAX Large Scale RNA Production System-T7 kit
Fig. 1. The genomic locations of the RUNX genes in B. mori and phylogenetic analysis of BmRUNX genes with other insects. (A) The genomic locations of the RUNX genes in B. mori. The red boxes represent the BmRUNX genes localization and transcriptional orientation was indicated by black arrows. (B) Phylogenetic analysis of BmRUNX genes with other insects. The full-length amino acid sequences from B. mori and insect RUNX proteins were aligned using ClustalX. A phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates, and displayed with MEGA 4.0. The BmRUNXs are colored by red. Sequence information was summarized in Table 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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3. Results 3.1. Characteristics and phylogenetic analysis of the B. mori RUNX genes sequence BmRun, BmLz, and BmRunt3 full-length sequences were obtained and identified. They all had a Runt domain, and the sequences and their information were listed in Fig. S1eS3 and Supplemental Table 2, which were all located in the nscaf2930 of chromosome No.3 in silkworm (Fig. 1A). BmRun consisted of five exons and four introns, while BmLz and BmRunt3 consisted of eight exons and seven introns (Fig. S4). The phylogenetic analysis showed that BmRun belonged to Group RUN, BmRunt3 was in the Group CG34145, and BmLz was in the Group Lozenge (Fig. 1B). However, their Runt domain amino acid sequence was highly conservative with that of D. melanogaster and Danaus plexippus (Fig. S5). The homology reached up to more than 70% identity. 3.2. Expression pattern analysis of the B. mori RUNX genes
Fig. 2. The expression patterns of BmRunx genes or only BmLz in B. mori. (A) The expression analysis of BmRunx genes in different larval tissues on day 3 of the fifth instar silkworm larvae by RT-PCR. BmActin3 gene was used as the internal control at each tissue. (BeC) Analysis the relative transcript abundance of the BmLz in different larval tissues on day 3 of the fifth instar larvae and the hemocytes from day 3 of 4th instar to the whole 5th instar of silkworm by qRT-PCR. Abbreviation: Ep, epidermis; He, head; Te, testis; Ov, ovary; Mi, midgut; Ma, Malpighian tube; Si, silk gland; Fa, fat body; Wg, Wing imaginal disc; Ha, hemocytes; Wh, Whole body; M, Molting. BmGAPDH was used as an internal control.
(Promega) (Payungporn et al., 2006). The primers sequence was listed in Supplemental Table 1. 50 mg of dsRNA was injected into silkworm larva at first day of 5th instar to knockdown BmLz. EGFP was used as control. After injected for 3 days, hemocytes were collected.
2.8. PO activity assay Phenoloxidase activities were determined according to Haobo Jiang et al. (Jiang et al., 2003) and Meixian Wang et al. (Wang et al., 2013) methods with slight modification. 40 ml hemolymph immediately mixed with 60 ml cold PBS, then added 200 ml of 10 mM dopamine (Sigma) in 20 mM TriseHCl (pH 7.0). After mixing enough, 200 ml mixture was transferred into microplate wells. After incubating at 37 C for 30 min, the absorbance at 450 nm was assessed using the GloMax-Multi Detection System Photometer (Promega).
The mRNA expression level of RUNX genes on day 3 of 5th (L5D3) instar larvaes was analyzed. As shown in Fig. 2, we collected and compared most of organs in silkworm, including testis, ovary, epidermis, silk gland, malpighian tubule, midgut, head, fat body, wing imaginal disc (adhering hematopoietic organ) and hemocytes. The electrophoresis results showed that BmRun and BmRunt3 were commonly expressed in all organs, although BmRun expression was a little bit low in malpighian and testis, and undetectable in hemocytes. However, only BmLz was specifically expressed in hemocytes and wing imaginal disc (Fig. 2A), which meant that BmLz might play an important role in hemocytes and hematogenesis in silkworm. We confirmed the result by qRT-PCR, and the data showed that BmLz was highly expressed in homocytes comparing to other tissues (Fig. 2B). Further, we collected and analyzed mRNA level from hemocytes in different development stages from day 3 of 4th (L4D3) instar larvae, the whole 5th instar, to the day 2 of prepupa (PP2) larvae. The result showed that BmLz was commonly expressed in hemocytes through the whole development period, and reached a peak in glutonous stage L4D4 and L5D4 (Fig. 2C) which implied that BmLz might involve in homocyte development. 3.3. Localization analysis of BmLz Next, we overexpressed BmLz in BmE cell line, EGFP was used as control. The result showed that the control EGFP signal evenly spread out in cytoplasm and nucleus, in contrast, the BmLz signal only existed in cell nucleus not in cytoplasm (Fig. 3A). Moreover, we validated this result in hemocytes of B. mori by immunofluorescence using BmLz antibody we generated. The result showed that BmLz can be found in two types of hemocytes though observing the cell morphology. Consistent with the result in BmE cell line, BmLz was detected in cell nucleus of spherulocyte, but also could be expressed in the whole cell of oenocytoid (Fig. 3B). So BmLz might play a role in controlling hemocytes differentiation and immune response as a transcription factor. 3.4. Over-expression BmLz up-regulated the PPOs expression in vitro To further examine the role of BmLz in insect immune response, we firstly up-regulated the BmLz expression in S2 cell line (Fig. 4A and B). As shown in Fig. 4C, overexpression of BmLz significantly increased PPO1, PPO2, PPO3 respectively, which are the key enzymes in the melanization process.
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Fig. 3. Localization of BmLz in cell of B. mori. (A) BmLz-EGFP fusion protein localized in the cell nucleus of BmE. (B) Immunofluorescence analysis using BmLz antibodie in the circulating haemocytes of silkworms. BmLz could be detected in oenocytiod and spherulocyte severally. Scale bar ¼ 5 mm.
3.5. Over-expression of BmLz in vivo stimulated melanization process of hemolymph It's known that B. mori hemolymph would blacken when it is exposed to the air, which is attributed to PPOs. After injection of BmLz-EGFP in silkworm for 3 days, the EGFP signal could be detected in circulatory hemocytes. It was similar to the result in BmE cell line, control EGFP signal spread out in cytoplasm and nucleus. In contrast, the BmLz-EGFP signal was detected in cell nucleus, and most of these cells seem to plasmatocyte (Fig. 5A). qRT-PCR analysis was accomplished and showed that BmLz was over expressed comparing to control (Fig. 5B). Overexpression of BmLz dramatically accelerated markedly the melanization process, after exploring to air for 10min, hemolymph started to blacken in overexpressed BmLz group, but not the control group (Fig. 5D), and the PO activity in overexpressed BmLz group was higher than the control group (Fig. 5C). Overexpression of BmLz could significantly increase PPO1 and related melanization enzyme BmPPAE and BmBAEE, especially PPO1 increased at least more than twenty fold (Fig. 5E). Taken together, these findings demonstrated that BmLz could stimulate melanization process in hemolymph of B. mori. Fig. 4. The effect of BmLz was overexpressed by transient transfection in S2 cell line. (A) The expression level of BmLz detection in S2 cell line. (B) qRT-PCR detected the expression level change of BmLz in S2 cell line. (C) The expression level of PPO1, PPO2 and PPO3 detection by qRT-PCR in S2 cell line after overexpression of BmLz. Dmrq49 was used as an internal control. The data were analysed using Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001. Data are shown as the mean ± SE from three independent experiments.
3.6. Down-regulation of BmLz expression by RNA interference in vivo delayed the melanization process of hemolymph To examine the role of BmLz in hemocytes of B. mori, BmLz
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dsRNA was designed as in Fig. 6A and B, and injected into silkworm larvae for 3 days. The expression of BmLz was dramatically reduced more than 70% in hemocytes comparing to control (Fig. 6C). As shown in Fig. 6D, the melanization process was delayed distinctly after knockdown BmLz, specifically after exploring to air for 20min. The PO activity analysis showed that PO activity in Lz RNAi group was reduced compare with the control group (Fig. 6E). In addition, immunofluorescence result showed that the expression of BmLz in oenocytoid reduced distinctly (Fig. 6F). 4. Discussion RUNX family has been commonly studied in mammals, as well as in D. melanogaster. They play pivotal roles in several developmental processed including hematopoiesis, neurogenesis, and skeletogenesis (Nah et al., 2014). However, there is no any report about RUNX transcription factors in B. mori. This is the first time we identified and characterized the RUNX family including BmRun, BmLz and BmRunt3 in silkworm. BmRun was clustered into RUN group, BmLz was clustered into Lozenge group, and BmRunt3 clustered into CG34145 group. Their Runt domain sequence was highly conservative with that of D. melanogaster and D. plexippus,
and the identity was up to 70%. BmRun and BmRunt3 were commonly expressed in most of organs, except BmRun was undetectable in hemocytes. However, BmLz was only expressed in hemocytes and imaginal disc, which meant that it might play an important role in hemocyte. The localization analysis of BmLz showed that it expressed in spherulocyte and oenocytoid, which suggested that it might involve in immunoreactions of silkworm. Next, through overexpression and knockdown experiment, we verified that role of BmLz in hemolymph. Overexpressed BmLz could upregulate PPOs expression in vivo and in vitro, which are key enzyme in melaniztion reaction, and accelerate the melanization response of hemolymph of B. mori. Knockdown BmLz downregulated BmPPOs expression, and delay the melanization process of hemolymph. There are four types of hemocyte in D. melanogaster, including prohemocyte, plasmatocyte, crystal cell, and lamellocyte (Minakhina and Steward, 2010). But there are five types of hemocyte in B. mori, including prohemocyte, oenocytoid, plasmatocyte, granulocyte and spherulocyte (Nakahara et al., 2009; Tan et al., 2013). Oenocytoid in B. mori is similar to the crystal cell in D. melanogaster (Strand, 2008). Both of them are active participator in response to microorganisms’ invasion and wound healing
Fig. 5. The effect of BmLz was overexpressed in day 3 of 5th instar silkworm larva hemolymph by transient transfection. (A) BmLz-EGFP fusion protein expressed in the circulating haemocytes though transient transfection of plasmids. (B) The mRNA expression level of BmLz in the hemolymph. (C) PO activity change after BmLz overexpression. (D) Hemolymph was harvested after BmLz overexpression. (E) The mRNA expression level of the melanzation pathway related gens in silkworm detected by qRT-PCR. BmGAPDH was used as an internal control. The data were analysed using Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001. Data are shown as the mean ± SE from three independent experiments.
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Fig. 6. Down-regulation of BmLz by RNA interference in day 1 of 5th instar silkworm larva haemolymph. (A) The sequence of BmLz dsRNA. (B) The synthesis of dsRNA (1, 2 band were EGFP dsRNA; 3, 4 band were BmLz dsRNA). (C) qRT-PCR detected the expression level change of BmLz in hemocytes after RNAi. The data were analysed using Student's t test, **p < 0.01. Data are shown as the mean ± SE from three independent experiments. (D) Survey of haemolymph melanism rate after BmLz was knockdown. (E) PO activity change after RNAi. (F) Immunofluorescence analysis using BmLz antibodie in the circulating haemocytes after RNAi.
(Adewumi et al., 2007; Ashida et al., 1988; Ling et al., 2005). Lz, as a hematopoietic modulation factor, is in control of the fate of crystal cell in D. melanogaster (Lebestky et al., 2000). PPOs, as the enzymes required for melanization response of hemolymph, mainly exist in crystal cell in D. melanogaster, which are cleaved to generate active POs as a result of proteolytic cascade activation and initiate the melanization response (Ferjoux et al., 2007; Gajewski et al., 2007). Mosquito RUNX4, the ortholog gene of D. melanogaster Lz could directly regulate PPO genes expression by binding to the Runt binding motif in the promoter of PPOs to continue the melanization during the maturing process of new crystal cell (Zou et al., 2008). In B. mori, PPOs exist in oenocytoids and plasmatocytes, while PO only exists in oenocytoids (Ashida et al., 1988). These suggest that BmLz promotes the melanization reaction of hemolymph by directly regulating the PPOs in oenocytoids of B. mori. Whereas, the further research and verification are still demanding.
Conflicts of interest The authors have declared that no competing interests exist. Acknowledgment This work was supported by the National Basic Research Program of China (2012CB114603), the Research Fund for the Doctoral Program of Higher Education of China (20130182110003), the Natural Science Foundation of Chongqing (CSTC2013jcyjys0007), and the Fundamental Research Funds for the Central Universities (SWU111014, XDJK2015D021). We are grateful to Prof. Chunfeng Li of the State Key Laboratory of Silkworm Genome Biology for his help in preparation of this manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http://
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