Cloning, Expression and Analysis of a Novel Defense Gene from Antheraea pernyi.

Byline: Cen Qian, Xiao-Juan Zhang, Bao-Jian Zhu, Lei Wang, Guo-Qing Wei, Yu Sun and Chao-Liang Liu

Abstact

In this study, we first reported the defense gene (ApDef) in lepidoptera insect, the Chinese oak silkworm Antheraea pernyi (Lepidoptera: Saturniidae). It consists of 677 bp with a putative open reading frame (ORF) of 492 bp encoding 163 amino acids residues. A signal peptide with 18 amino acids and a typical reeler domain was found in ApDef protein. Phylogenetic analysis indicated that ApDef gene had the closest genetic distance with Antheraea mylitta Defense. The results of real-time quantitative PCR (qRT-PCR) showed that this gene was highly expressed in fat body, low expressed in hemocytes and midgut, and not expressed in epidermis. After being challenged by three types of pathogens (Escherichia coli, Micrococcus luteus and Beauveria bassiana), the expression levels of ApDef were up-regulated significantly compared with the control, and reached the maximum at 12 h post infection. These data indicated that ApDef may play an important role in innate immune responses of A. pernyi.

Key words: Defense gene, Reeler domain, Ap Def Gene, Antheraea pernyi, qRT-PCR, Innate immune, Chinese Oak silkworm.

INTRODUCTION

Immune response is usually divided into two branches, innate immunity and acquired immunity. Unlike vertebrates, invertebrates don't have complex and memory property acquired immune system, but only rely on the innate immune system to resist the invasion of pathogens and infection (Kimbrell and Beutler, 2001; Jiravanichpaisal et al., 2006). Insects, as the largest groups in invertebrates, living in a complex environment, have the quite perfect innate immune system, which can mediate immune process quickly and efficiently through specific proteins (Gillespie et al., 1997; Lavine and Strand, 2002; Cherry and Silverman, 2006). Pathogen-associated molecular patterns, PAMP, were widely existed on the surface of pathogenic microorganisms, such as lipopolysaccharide, LPS (Gram negative bacteria); peptidoglycan, PGN (Gram negative bacteria) andAY-1,3-glucan (Fungi) (Medzhitov and Janeway, 2002).

Insects can identify the PAMP effectively through their membrane receptors and conservative or induced specific proteins, then regulate and stimulate the humoral and cellular immune response through activating a series of signal pathways, such as Toll, IMD, JAK-STAT, JNK, Ras/Rho, NO and so on (Williams et al., 2005, 2006; Dijkers and O' Farrell, 2007; Lamprou et al., 2007; Sackton et al., 2007).

A type of immune-related defense gene, with an typical reeler domain, have been identified in lepidopteran insects, such as Samia ricini, Antheraea mylitta, Bombyx mori, Lonomia oblique, Hyphantria cunea and Manduca sexta (Shin et al., 1998; Bao et al., 2003; Zhu et al., 2003; Veiga et al., 2005; Gandhe et al., 2007; Bao et al., 2011). The reeler domain was initially identified from a secreted glycol protein in the mouse (D'Arcangelo et al., 1995). In mammals, it plays an important role in the development of the central nervous system (Quattrocchi et al., 2002). In insects, it can be induced by pathogenic microorganism challenge and participated in both cellular (nodule formation) and humoral (melanization cascade) immune responses (Bao et al., 2011). So we speculated that AsDef protein in insects may be involved in various signaling pathways for different types of microbial infection.

Antheraea pernyi (Lepidoptera: Saturniidae) is an economically valuable silk-producing insect and commercially cultivated mainly in China, India and Korea. It contained a large number of bioactive peptides/proteins, which have an obvious inhibitory effect against bacteria, fungi, virus and cancer cells (Kavanagh and Reeves, 2004; Strand, 2008; Liu, et al., 2010). Here, we cloned and characterized a defense gene with a typical reeler domain from A. pernyi, and described its expression profiles after the different pathogens challenge. This will provide the foundation for further functional research on insects reeler domain Defense proteins.

MATERIALS AND METHODS

Experimental animals

The A. pernyi larvae were kindly provided by the Sericultural Research Institute of Henan and reared on the leaves of oak until pupation (Wei et al., 2011). The heat-inactived gram-negative bacterium (Escherichia coli 5 L, 109 cfu/mL), gram-positive bacterium (Micrococcus luteus 5 L, 0.5 mg/mL) and fungi (Beauveria bassiana 5 L, 109 cfu/mL) were used for injecting into the abdomen of A. pernyi larvae (Wang et al., 2014). All the microorganism pellets were diluted in the sterilized PBS. The larvae injected with sterilized PBS were used as negative control. Six larvae from each group were dissected to collect fat body, midgut, hemocytes, malpighian tubule and epidermis at different time points after injection, respectively. These tissues were frozen immediately in liquid nitrogen and stored at -80C.

Total RNA extraction and cloning of the ApDefgene

Total RNA was extracted with TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. The purity and quantity of the extracted RNA were quantified by the ratio of OD260/OD280 by a NanoDrop 1000 spectrophotometer (NanoDrop Technologies Wilmington, DE). Using 500 ng of total RNA per sample to generate first strand cDNA with PrimeScriptTM One Step RT-PCR Kit Ver.2 (Takara, Japan) following the manufacture's instruction. Oligonucleotide primers (Table I) were designed by Primer Premier 5.0 software based on the reported defense sequences from A. mylitta. PCR conditions were denaturation at 94C for 5 min, followed by 30 cycles of 94C for 30 s, annealing at 55 C for 40 s and extension at 72 C for 1 min. 5'- and 3'-RACE were performed according to the manufacture's introduction.

The PCR products were separated on 1% agarose gel and purified with Gel

Extraction Mini Kit (Aidlab, China). Purified PCR products were then ligated into the pMD19-T simple cloning vector (Takara, Japan) and transformed into competent cells of E. coli strain DH5a. Positive clones were sequenced at Invitrogen, Shanghai.

Table I.- Primers used in this study.

Primers###Primers sequence

number

DF1###5'-GGCGGATCCATGTTAAAATTGTCATTTG-3'

DR1###5'-CAGAAGCTTTTATTTTACATTGACATTG-3'

DF2###5'-CGGACTTCAGATATCGTG-3'

DR2###5'-GACGGCGTTATTAGGTTC-3'

5' RACE-G1###5'-CGGGAGCAGTCCAGAGGTATGAGA-3'

5' RACE-G2###5'-GACCAGGATGTGGTTGTGGTTCAG-3'

3' RACE###5'-TGAAGTAACCATCAGCGGCAATA-3'

Sequence analysis

The molecular weight (MW) and the isoelectric point (pI) of the deduced amino acid sequences were calculated by ExPASy (http://web.expasy.org/compute_pi/);hydrophobicity was analyzed by Protscale (http://web.expasy.org/ protscale/); transmembrane structure was analyzed online (http://www.ch.embnet.org/software/ TMPRED_form.html); signal peptide was predicted by SignalP (http://www.cbs.dtu.dk/services/SignalP- 2.0/); other species sequences were downloaded from NCBI public databases (http://www.ncbi.nlm.nih.gov/); phylogenetic tree was constructed by MEGA version 5.0 using the neighbor-joining (NJ) method with bootstrap test of 1000 replications.

Protein expression, purification and antibody preparation

The PCR products were ligated to pET-28 (a+) after digesting with corresponding restriction enzymes. The recombinant plasmids pET-28a- defense were identified by sequencing, then transformed into competent E. coil BL21 (DE3) cells for protein expression induced by different concentrations of IPTG. The recombinant proteins were analyzed with 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and western-blotting. nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography (Qiagen, Germany) was used to purify the recombinant proteins according to the instructions. The purified fusion proteins that were homogenized in complete Freund's adjuvant were used to immunize male New Zealand rabbits for three times at two-week intervals. A boost injection in incomplete Freund's adjuvant was given for another week. Rabbit serum was collected seven days after the last immunization. Antiserum was prepared according to the method described (Harlow and Lane, 1999).

Western blotting

Proteins that prepared for SDS-PAGE were subjected to SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane by an electrophoretic transfer system (Bio-Rad). Membranes were blocked with 5% non-fat milk in PBST (PBS contained 0.1% Tween-20) for 2 h at room temperature, then washed with PBST five times at 5 min interval and subsequently incubated with primary antibodies for 2 h at room temperature. After washing with PBST, membranes were incubated with horseradish peroxidase (HRP)- conjugated sheep antirabbit IgG antibody (Sigma) for 1 h at room temperature. The final detection was performed with a HRP-DAB Detection Kit (Tiangen, China).

qRT-PCR analysis

Total RNA of each tissue was extracted with TRIzol reagent (Takara, Japan) according to the manufacturer's instructions. Then, RNA samples were treated with RQ1 RNase-free DNase (Promega, USA) to remove genomic DNA contamination. 5g of total RNA was reverse transcribed into cDNA with M-MLV reverse transcriptase (Invitrogen, USA). The gene-specic primers (Table I) were designed by Primer Premier 5.0 software. qRT-PCR was carried out to measure the expression levels of ApDef in various tissues and different treatments.

The PCR reaction was performed in a total volume of 25 L containing 12.5 L 2 A- SYBR Premix Ex TaqII (Tli RNase Plus) (Takara, Japan), 1 L of each primer, 1.5 L of 1:8 diluted cDNA templates and 9 L RNase-free H2O. QRT-PCR was performed using an CFX96TM real-time detection system (Bio- Rad, USA) using the following procedure: initial denaturation at 95 C for 30 s, followed by 40 cycles of denaturation at 95 C for 5 s, annealing at 58 C for 30 s, and a final extension at 72 C for 25 s. The relative quantitative method (2-CT) was used to determine the expression level changes (Livak and Schmittgen, 2008). All data were represented as meanSD and analyzed by Student's t-test, differences were considered statistically significant when p-values less than 0.05.

RESULTS

Sequence analysis of ApDef

A cDNA fragment of 677 bp was obtained by RT-PCR and RACE-PCR (Fig. 1A). Nucleotide sequence analysis revealed that the ApDef contained a putative ORF of 492 bp encoding 163 amino acids residues. A signal peptide with 18 amino acids and a typical reeler domain was found in ApDef protein. Sequence alignment showed that ApDef had 49.7%, 50.9%, 70.2%, 64.6% and 88.2% identity with M.sexta, B. mori, S. ricini, L. obliqua and A.mylitt ApDefs, respectively (Fig. 1B). Phylogenetic analysis indicated that ApDef gene had the closest genetic distance with A. mylitta defense (Fig. 1B).

Prokaryotic expression, protein purification and polyclonal antibody preparation

A recombinant protein with a molecular weight of about 18 kDa from E. coli BL21 (DE3) was detected by SDS-PAGE, and its expression level was not influenced by different IPTG concentrations (Fig. 2A). Western blotting of recombinant proteins using the anti-His-tag antibody suggested that a consensus protein of 18 kDa was detected (Fig. 2B). All of these indicated that the recombinant protein obtained stable expression in E. coli cells. Soluble fusion proteins were successfully purified and detected by SDS- PAGE showing a single band in the gel (Fig. 2C).

Expression of ApDef in various tissues qRT-PCR and immunoblot were carried out to determine the expression levels of ApDef in different tissues including the fat body, midgut, hemocytes, malpighian tubule and epidermis of A. pernyi. Actin gene was used for normalization. Interestingly, the results for qRT-PCR and western blot analysis were just consistent. They both showed especially high expression levels of ApDef in fat body, while low in midgut, hemocytes, malpighian tubule and epidermis tissues (Fig. 3).

Induced expression profiles of ApDef

To further understand the induced expression profiles of s ApDef gene in response to a microbial infection, qRT-PCR was performed by fat body. Fat body was prepared after 0, 1.5, 3, 6, 12, 24 and 48 h challenge by three types of pathogens (E. coli, M. luteus and B. bassiana), respectively. Results revealed that the expression levels of ApDef were obviously increased from 1.5 to 48 h after infection and reached the maximum at 12 h (Figs. 4A, 4B and 4C). These results suggested that ApDef played an important role in defending against many types of pathogenes infection.

DISCUSSION

In this study, we reported for the first time the cloning, characterization and identification of a defense gene from A. pernyi. Based on homologous alignment and phylogenetic analysis, the defense protein from A. pernyi was highly homologous to that of A. mylitta. ApDef protein has an obvious signal peptide sequence and a reeler domain, which are similar with other insect defense protein. This will provide effective data to analyze the biological functions of A. pernyi defense.

Compared with the other examined tissues, the expression levels of ApDef were highest in fat body. To investigate the role of ApDef in response to microbial infection, the expression of ApDef was determined after being challenged by three different types of pathogens. The transcriptional expression profiles of ApDef indicated that its expression level was significantly up-regulated in fat body post bacterial infection. Also, among all types of pathogens used in experimental treatments, we found the transcriptional level of ApDef reached the highest post M. luteus injection, Therefore, we considered the ApDef showed stronger bactericidal activity in response to the Gram-positive bacterium than to the Gram-nagative bacterium and fungi. All the results suggested that ApDef played an important role in defending against the pathogens infection. Although the characterization and expression of ApDef were investigated, their physiological functions still need further study.

ACKNOWLEDGMENTS

This work was supported by the earmarked fund for Modern Agro-industry Technology Research System (CARS-22-SYZ10), Sericulture Biotechnology Innovation Team (2013xkdt-05), and Ph.D. programs in Biochemistry and Molecular Biology (xk2013042).

REFERENCES

BAO, Y., MEGA, K., YAMANO, Y. AND MORISHIMA, I., 2003. cDNA cloning and expression of bacteria-induced Hdd11 gene from eri-silkworm, Samia Cynthia ricini. Comp. Biochem. Physiol. C., 136: 337-342.

BAO, Y.Y., XUE, J., WU, W.J., WANG, Y., LV, Z.Y. AND ZHANG, C.X., 2011. An immune-induced reeler protein is involved in the Bombyx mori melanization cascade. Insect Biochem. mol. Biol., 41: 696-706.

CHERRY, S. AND SILVERMAN, N., 2006. Host-pathogen interactions in drosophila: new tricks from an old friend. Nat. Immunol., 7: 911-917.

D'ARCANGELO, G., MIAO, G.G., CHEN, S.C., SOARES, H.D., MORGAN, J.I. AND CURRAN, T., 1995. A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature, 374: 719-723.

DIJKERS, P.F. AND O'FARRELL, P.H., 2007. Drosophila calcineurin promotes induction of innate immune responses. Curr. Biol., 17: 2087-2093.

GANDHE, A.S., JOHN, S.H. AND NAGARAJU, J., 2007. Noduler, a novel immune up-regulated protein mediates nodulation response in insects. J. Immunol., 179: 6943- 6951.

GILLESPIE, J.P., KANOST, M.R. AND TRENCZEK, T., 1997. Biological mediators of insect immunity. Annu. Rev. Ent., 42: 611-643.

HARLOW, E. AND LANE, D., 1999. Using antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. JIRAVANICHPAISAL, P., LEE, B.L. AND SAlDERHALL, K., 2006. Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization. Immunobiology, 211: 213-236.

KAVANAGH, K., AND REEVES, E.P., 2004. Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens. FEMS Microbiol. Rev., 28:101- 112.

KIMBRELL, D.A. AND BEUTLER, B., 2001. The evolution and genetics of innate immunity. Nat. Rev. Genet, 2: 256-267.

LAMPTOU, I., MAMALI, I., DALLAS, K., FERTAKIS, V., LAMPROPOULOU, M. AND MARMARAS, V.J., 2007. Distinct signalling pathways promote phagocytosis of bacteria, latex beads and lipopolysaccharide in medfly haemocytes. Immunology, 121: 314-327.

LAVINE, M.D. AND STRAND, M.R., 2002. Insect hemocytes and their role in immunity. Insect Biochem. mol. Biol., 32: 1295-1309.

LIU, Y.Q., LI, Y.P., LI, X.S. AND QIN, L., 2010. The origin and dispersal of domesticate Chinese oak silkworm Antheraea pernyi in China: are construction based on ancient texts. J. Insect Sci., 10: 180.

LIU, Q.N., ZHU, B.J., WANG, L., WEI, G.Q., DAI, L.S., LIN, K.Z., SUN, Y., QIU, J.F., FU, W.W. AND LIU, C.L.,2013. Identification of immune response-related genes in the Chinese oak silkworm, Antheraea pernyi by suppression subtractive hybridization. J. Insect Physiol.,114: 313-323.

LIVAK, K.J. AND SCHMITTGEN, T.D., 2002. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Methods, 25: 402-408.

MEDZHITOV, R. AND JANEWAY, CA J.R., 2002. Decoding the patterns of self and nonself by the innate immune system. Science, 296: 298-300. QUATTROCCHI, C.C., WANNENES, F., PERSICO, A.M., CIAFRE, S.A., D'ARCANGELO, G., FARACE, M.G. AND KELLER, F., 2002. Reelin is a serine protease of the extracellular matrix. J. biol. Chem., 277: 303-309.

SACKTON, T.B., LAZZARO, B.P., SCHLENKA, T.A., EVANS, J.D., HULTMARK, D. AND CLARK, A.G., 2007. Dynamic evolution of the innate immune system in Drosophila. Nat. Genet., 39: 1461-1468.

SHIN, S.W., PARK, S.S., PARK, D.S., KIM, M.G., KIM, S.C., BREY, P.T. AND PARK, H.Y., 1998. Isolation and characterization of immune-related genes from the fall webworm, Hyphantria cunea, using PCR-based differential display and subtractive cloning. Insect Biochem. mol. Biol., 28: 827-837.

STRAND, M.R., 2008. The insect cellular immune response. Insect Sci., 15: 1-14.

VEIGA, A.B., RIBEIRO, J.M., GUIMARAES, J.A. AND FRANCISCHETTI, I.M., 2005. A catalog for the transcripts from the venomous structures of the caterpillar Lonomia obliqua: identification of the proteins potentially involved in the coagulation disorder and hemorrhagic syndrome. Gene, 355: 11-27.

WANG, L., LIU, D.R., YANG, L., ZHU, B.J., AND LIU, C.L., 2014. Expression patterns of calreticulin from bombyx mori after immune challenge. Pakistan J. Zool., 46: 1731-1737.

WEI, Z.J., YU, M., TANG, S.M., YI, Y.Z., HONG, G.Y. AND JIANG, S.T. 2011. Transcriptional regulation of the gene for prothoracicotropic hormone in the silkworm, Bombyx mori. Mol. Biol. Rep., 38: 1121-1127.

WILLIAMS, M.J., ANDO, I. AND HULTMAK, D., 2005. Drosophila melanogaster Rac2 is necessary for a proper cellular immune response. Genes Cells, 10: 813-823.

WILLIAMS, M.J., WIKLUND, M.L., WIKMAN, S. AND HULTMARK, D., 2006. Rac1 signaling in the Drosophila larval cellular immune response. J. Cell Sci., 119: 2015-2024.

ZHU, Y., JOHNSON, T.J., MYERS, A.A. AND KANOST, M.R., 2003. Identification by subtractive suppression hybridization of bacteria-induced genes expressed in Manduca sexta fat body. Insect Biochem. mol. Biol., 33: 541-559.

Article Details

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Author:	Qian, Cen; Zhang, Xiao-Juan; Zhu, Bao-Jian; Wang, Lei; Wei, Guo-Qing; Sun, Yu; Liu, Chao-Liang
Publication:	Pakistan Journal of Zoology
Article Type:	Report
Geographic Code:	9CHIN
Date:	Apr 30, 2015
Words:	2935
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