Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 DOI: 10.13102/sociobiology.v60i4.355-361Sociobiology 60(4): 355-361 (2013) Molecular Cloning, Characterization and Expression Analysis of Calreticulin Gene in the Ant Polyrhachis vicina Roger (Hymenoptera: Formicidae) LP Liu, L Dang, GS Xi, F Wang Introduction Calreticulin (CRT) is located both inside and outside of the endoplasmic reticulum, and also distributed on the cell surface (Ostwald et al., 1974). CRT belongs to a multifunc- tional calcium-binding protein, which can regulate calcium balance, assist the protein folding, processing and presenting the antigen. Usually, the gene of CRT composition and the amino acid sequence are highly conservative. Many stud- ies show that the CRT protein may play a critical role in the maintenance of cell functions (Lee et al., 2001; Lynch et al., 2006). CRT was initially purified from hare sarcoplasmic re- ticulum of muscle cells by Ostwald and MacLennan in 1974. However, CRT gene’s cDNA sequence was cloned from the liver of mice by Smith and Koch in 1989. Currently, CRT gene’s cDNA sequence was cloned from vertebrates, inver- tebrates and higher plants. At the same time, the molecular structure and related functions of CRT were studied. In in- sects, CRT was detected as an encapsulative protein. CRT may be relevant to the identification of foreign objects in Abstract Calreticulin (CRT) as a ubiquitous and highly conserved calcium-binding protein that exists in endoplasmic reticulum (ER), which possesses a variety of biological functions in the regulation of cell calcium homeostasis, molecular chaperoning and innate immu- nity. In our research, the calreticulin homologous gene (refered as PvCRT) was cloned from the ant Polyrhachis vicina Roger (Hymenoptera: Formicidae), the full-length cDNA of PvCRT is 1584bp base pairs (bp), contains a 5’-untranslated region of 87bp and a 3’- untranslated region of 246bp. The open reading frame (ORF) of 1251bp encodes 416 amino acids. Using real-time quantitative RT-PCR to study PvCRT mRNA expression levels indicates that this gene was expressed in different developmental stages of castes of P. vicina. The mRNA expression level in both embryos and adults shows that PvCRT gene may play some essential roles in the ant’s development. Sociobiology An international journal on social insects College of Life Sciences, Shaanxi Normal University, Xi’an, China RESEARCH ARTICLE - ANTS Article History Edited by: Kleber Del-Claro - UFU, Brazil Received 07 January 2013 Initial acceptance 04 April 2013 Final acceptance 26 July 2013 Keywords Calreticulin homologous gene, Molecular cloning, Polyrhachis vicina real-time quantitative RT-PCR Corresponding author Gengsi Xi College of Life Sciences Shaanxi Normal University, Xi’an 710062, China E-Mail: xigengsi@snnu.edu.cn the cell defense response, since higher amounts of CRT can be found around the foreign substance. In Drosophila mela- nogaster Meigen CRT named DmCRT acts as a marker for phagocytosis of apoptotic cells by D. melanogaster phago- cytes (Kuraishi et al. 2007). In .addition, DmCRT plays an important role in odor-guided behavior (Stoltzfus et al., 2003) and also is involved in mediating anesthetic sensitivity (Gamo et al., 2003). In Anopheles gambiae Giles AgCRT is known to interact with malaria parasite surface protein (Rodriguez- Mdel et al., 2007). Here, the full-length cDNA sequences of the calreticu- lin gene were cloned from the ant Polyrhachis vicina Roger and the homologs were named PvCRT. A phylogenetic tree was constructed using MEGA 5.0. The results of the phyloge- netic analysis indicated that PvCRT shares high identity with Pteromalus puparum L and Nasonia vitripennis Walker calre- ticulin. The expression pattern of PvCRT mRNAs was studied by using real-time RT-PCR. Our data indicate that this gene is expressed differentially in distinct development stages and different castes. LP Liu, L Dang, GS Xi, F Wang - Calreticulin Gene Investigation in Polyrhachis vicina Roger356 Material and Methods Experimental insects Polyrhachis vicina, which belongs to genus Polyrha- chis (Hymenoptera:Formicidae), is a species of typical euso- cial insect with the characteristic of castes differentiated in females, males and workers. It is a good material for studying the mechanisms of insect development and behavior. P. vicina as a complete metamorphosis insect, is mainly distributed in east and south of China, Burma, Cambodia, Japan, Australia and Papua New Guinea. The colonies of P. vicina were bought from Hongfa Edible Ant Research Center in Nanning City, Guangxi Prov- ince, in People’s Republic of China. Then, the ants were cul- tured in boxes at the laboratory, under a controled temperature of 28ºC, relative humidity of 40% and natural light-dark pe- riods. The ants were usually reared on fruits, meat and hon- eydew. Egg, larvae, pupae and adults (workers, winged males and winged females ) were collected from the two colonies, 6 individuals were used respectively for each caste, immersed immediately in liquid nitrogen, stored at -80°C for RNA ex- traction (Lv et al., 2008; Guo et al., 2010). RNA preparation and cDNA synthesis Total RNA was extracted from pooled samples of 12 frozen larvae selected randomly with RNAiso Plus (Takara Bio Inc., Shiga, Japan), and then immediately reverse-transcribed in order to generate cDNA using the First-Strand cDNA Sythesis Kit with oligo (dT) primer (Fermentas Life Sci- ences, Burlington, Ontario; http://www.fermentas.com/). All steps were followed through the manufacturer’s instructions. Molecular cloning and sequencing of PvCRT The full-length cDNA of CRT from P. vicina named PvCRT was cloned based on the scheme shown in Table 1. First, a partial cDNA fragment of PvCRT was gained using degenerated primers (S1 and A4), according to the above fragment, primers P1 and L4 were designed and used these pair primers, and then Seq2 fragment was obtained. The four primers were designed on the basis of the conserved motifs of published PvCRT of other insect species (Pteromalus pupar- um L., Bombyx mori L, Culex quinquefasciatus (Say), Naso- nia vitripennis Walker, Galleria mellonella L. PCR products were purified from agarose gel using Gel Extraction Kit (Qia- gen, Germany http://www.qiagen.com/) and purified products were cloned into PMD19-T Vector (Takara Bio Inc.), then PCR positive clones were obtained for sequencing. After that, using 3’ and 5’ rapid amplification of cDNA ends (RACE) to get the full length of PvCRT, the specific primers of RACE are shown in table 1. All the primers were designed using PrimerPremier 5.0 and Oligo 6.0. Structural and phylogenetic analysis of PvCRT The ORF of PvCRT was obtained using the National Center for Biotechnology Information ORF Finder (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html). Signal peptide predic- tion was completed using the SingalP program (http://www. cbs.dtu.dk/services/SignalP/ Bendtsen J.D. et al. 2004; Center for Biological Sequence Analysis;). The secondary structure of the PvCRT protein was predicted usingSOPM (http://npsa- pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_hnn. html). The tertiary structure of P. vicina CRT was also pre- Table1. Oligonucleotide primers used for cDNA cloning and real-time quantitative RT-PCR. Note: Y: C or T; V: A or G or C; M: A or C; W: A or T; D: A or G or T; R: A or G; H: A or C or T; Tm: Annealing temperature. Target Name Primer Sequence 5′-3′ Expected size(bp) Tm(°C) CRT Fragment S1 A4 P1 L4 GAYTCVTGGGAAMACMATTGG CTTGCCAYAAATCVAGWCCDAC TGAGGATAAAAAACCAGAGGAC CATCRTGCTCTTCAATHTCAGG 873 606 52 52 3’RACE 3outer F1 3inner Q4 3’oligo(dT) GCTGTCAACGATACGCTACGTAACG CATTTGTGGCCCAGGAATTAGG CGCTACGTAACGGCATGACAGTG TACCCGATAGCGACGATGAT GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T)18 310 60 5’RACE 5outer H9 5inner M1 CATGGCTACATGCTGACAGCCTA TCGCCATCCATTTCGTCATCC CGCGGATCCACAGCCTACTGATGATCAGTCGATG ATCATTTTCTGGGTCATTCC 300 56 Real time E13 G13 CGACAACGAGGAAGTAGAGT ATTCGCCATCCATTTCGTCATC 224 60 Β-actin B1 B2 TGAACAGGAGATGGCTACAG CCGATGGTGATGACTTGAC 80 60 Sociobiology 60(4): 355-361 (2013) 357 dicted using Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/ html/page.cgi?id=index). Potential functional motifs were analyzed using PROSITE database (Expert Protein Analy- sis System, Swiss Institute of Bioinformatics, Basel; http:// myhits.isb-sib.ch/cgi-bin/motif_scan). Sequence alignments based on the amino acid sequences of published CRT genes were performed with Clustal X 2 and DNAman. From align- ments, a phylogenetic tree of PvCRT was generated by MEGA 5.0, based on the neighbor-joining method with a bootstrap test calculated with 500 replicates and a Poisson correction model. Real-time quantitative RT-PCR Real-time quantitative RT-PCR was used to quantify the mRNA expression levels of PvCRT from two colonies at different development stages and different castes including egg, wholly body of one to four instars larvae’s, pupae and wholly body of caste adult ants (ergate, winged male ant and winged female ant) of P. vicina were used in this experiment. For each of these developmental stages, samples were taken from 6 individuals. Reactions volume, reactions temperature are 25.5ul and 60ºC, respectively. Such reactions were performed in ABI Stepone with a SYBR Premix Ex Taq Kit (Takara Bio Inc). β-actin was used as an endogenous control to research the gene expression. Primers (B1, B2) of β-actin and primers (E13, G13) of PvCRT were used in real-time RT-PCR (Table 1). The amplified fragments of two pairs of primers are 80bp and 224bp, respectively. The detailed protocol was as follows: 95ºC for 10min, 40 cycles of 95ºC for 15s and 60ºC for 1min, followed by a melting-curve program from 59 to 95ºC with a heating rate of 0.3ºC every step and continuous-fluorescence acquisition. All RT-PCR reactions were completed in tripli- cate. The relative expression quantification of PvCRT was determined by the formula F=10∆Ct,t/At-∆Ct,r/Ar (Zhang et al., 2005). One of cDNA samples was chose to construct stan- dard curve of PvCRT gene. In this study, we chose the third instar as calibrator to analyze PvCRT expression at different development stages and different castes. The relative expres- sion levels of PvCRT were analyzed using one way analysis of variance (ANOVA) with Dunnett’s multiple comparision test. Results Cloning and characterization of PvCRT cDNA The full-length cDNA of PvCRT was obtained by RT- PCR and RACE. The full-length cDNA of PvCRT is 1584bp. The PvCRT cDNA includes a 5’-untranslated region of 87bp, an ORF of 1251bp and a 3’-untranslated region of 246bp (Fig.1). A putative polyadenylation signal (AATAAA) was found upstream from the 18-nucleotide poly (A) tail, which Fig. 1. The nucleotide and deduced amino acid from the PvCRT. The full sequence of PvCRT is 1584bp, which encodes 416 amino acid. The start codon ATG and the stop codon TAA are underlined and shaded, and the polyadenylation signal (AATAAA) is underlined. A lot of important functional motifs of PvCRT protein are marked. Six casein kinase II phosphorylation sites at 67-70 (TSQD), 84-87 (SNKD), 223-226 (SIDD), 247- 250 (TEPE), 324-327 (TIFD), and 401-404 (SDDD); one N-myristoylation site at 64-69 (GIQTSQ); four protein kinase C phosphorylation sites at 77-79 (SKK), 84-86 (SNK), 95-97 (TVK), and 180-182 (TYK); two tyrosine kinase phosphorylation sites at 161- 167 (RCKDDIY), and 277-284 (KQIDNPNY); calreticulin family signature 1 at 97-112 (KHEQNIDCGGGYLKVF); calreticulin family signature 2 at 129-137 (IMFGPDICG); calreticulin family repeated motif signature at 224-236 (IDDPEDKKPEDWD); endo- plasmic reticulum targeting sequence at 413-416 (HDEL). LP Liu, L Dang, GS Xi, F Wang - Calreticulin Gene Investigation in Polyrhachis vicina Roger358 coincides with the fact that the polyadenylation signal is most often present 11-30 nucleotide upstream from the poly (A) tail (Fitzgerald et al., 1981). The deduced proteins of PvCRT have a calculated molecular mass of 48.5kDa and an isoelectric point (PI) of 4.37. Nucleotide sequence of PvCRT has been deposited into the GenBank database and obtained the acces- sion number of JQ783054. Protein structure prediction The ORF of PvCRT encodes 416 amino acid, and the predicted protein shares 99% similarity with N. vitripennis and 88% with Plutella xylostella L. Analysis with the SignalP program showed that there was N-terminal signal sequence, and the cleavage site between 18 and 19 amino acid, so the protein was determined secreted protein. A series of pre- dicted functional motifs were found by PROSITE program, including six casein kinase II phosphorylation sites at 67-70 (TSQD), 84-87 (SNKD), 223-226 (SIDD), 247-250 (TEPE), 324-327 (TIFD), and 401-404 (SDDD); one N-myristoylation site at 64-69 (GIQTSQ); four protein kinase C phosphoryla- tion sites at 77-79 (SKK), 84-86 (SNK), 95-97 (TVK), and 180-182 (TYK); two tyrosine kinase phosphorylation sites at 161-167 (RCKDDIY), and 277-284 (KQIDNPNY); calreticu- lin family signature 1 at 97-112 (KHEQNIDCGGGYLKVF); calreticulin family signature 2 at 129-137 (IMFGPDICG); calreticulin family repeated motif signature at 224-236 (ID- DPEDKKPEDWD); endoplasmic reticulum targeting se- quence at 413-416 (HDEL). The predicted secondary structure of the PvCRT pro- tein contains 13.70% alpha helix, 14.66% extended strand and 71.63% random coil (Fig. 2). The prediction of tertiary struc- ture is shown in Fig.4. Alignment analysis and phylogenetic-tree construction in in- sects A multiple alignment of the deduced amino acid se- quence of PvCRT with other known calreticulin homologues was performed by Clustal X 2 and DNAman (Fig.3). A phy- logenetic tree was constructed using MEGA 5.0 based on the neighbor-joining method (Tumura et al., 2007) in Fig. 5. The results of the phylogenetic analysis indicated that PvCRT shares high identity with P. puparum and N. vitripennis cal- reticulin (Fig.5). Expression analysis of PvCRT mRNA The expression level of PvCRT mRNA at different developmental stages and in different castes was analyzed by means of real-time quantative RT-PCR. Previous stud- ies revealed that the analyzed β-actin mRNA levels remain constant in tissues of insects regardless of their developmen- tal and physiological condition (Simonet et al., 2004). The analyzed results indicated that PvCRT was expressed in all templates (Fig.6). During the developmental stages, the high- est expression level was found in the third instars and the lowest expression level was found in embryos. Among the three adult’s castes, the PvCRT gene expression was highest in workers and lowest in males. Discussion In this study, the full-length cDNA of calreticulin gene was cloned and characterized from P. vicina. The cDNA se- quence of PvCRT is highly conserved with calreticulin ho- mologs found from other insects, which indicated that this sequence is indeed calreticulin of P. vicina. The results of pre- dicted functional motifs found that in PvCRT protein exists an ER targeting sequence HDEL, which is closely related to the cellular localization of CRT in ER. This result may shows that the PvCRT possesses function of calregulin in ER mainly in- volved in directing proper conformation of the proteins, con- trolling calcium level, and participating in immune responses (Wang et al., 2012; Byung-Jae Park et al, 2001;) including: regulation of intracellular Ca2+ homeostasis chaperone activ- ity, steroid-mediated gene regulation, and cell adhesion (Me- saeli et al., 1999). The phylogenetic tree (Fig.5) was analyzed and found that PvCRT is most closely related to that of P. puparum and N. vitripennis. Usually, P. civica is similar to P. puparum and N. vitripennis in classification status. All of them belong to hymenoptera insects, which may indicate that the calregu- lin possesses conservative properties. Khanna et al. (1987) demonstrated that“the physicochemical and structural prop- erties of calregulin from a single tissue have been highly conserved during vertebrate evolution after comparison of calregulins from bovine, rabbit and chicken livers”Luana et al. (2007) cloned the full length cDNA of calreticulin from Fenneropenaeus chinensis Osbeck (FcCRT). disply that Cal- reticulin belong to a highly conserved calcium-binding pro- tein. The deduced amino acid sequence of FcCRT showed high identity with those of B. mori (88%), Drosophila mela- nogaster Meigen (83%), Mus musculus L. (82%) and Homo sapiens L.(82%). In this paper the analysis of real-time RT-PCR indicated that PvCRT gene is expressed in each template, but the expres- Fig. 2. Predicted secondary structure of the PvCRT protein, where c is random coil; h is alpha helix; e is extended strand; t is beta turn. Sociobiology 60(4): 355-361 (2013) 359 Fig.3. Amino acid sequence alignments of PvCRT with the homologues sequences of other insects and one crustacean. Species and Gen- Bank accession numbers for alignmental and phylogenetic analysis are as follows: N. vitripennis (Nvi) NM_001161679; P. puparum (Ppu) FJ882064; P. vicina (Pvi) JQ783054; B. mori (Bmo) FJ360528; Pieris rapae L. (Pra) EU826537; Plutela xylostella L. (Pxy) HM240516; Cotesia rubecula (Marshall) (Cru) AY150370; D. melanogaster (Dme) NM_079569; Anopheles stephensi Liston (Ast) JN559458; Culex quinquefasciatus (Say) (Cqu) XM_001848772; Litopenaeus vannamei Boone (Lva) (Crustacea) JQ682618. Similarity of 100%, (>=75%), (>=50%) in all CRTs are marked in black, dark green and gray, respectively. LP Liu, L Dang, GS Xi, F Wang - Calreticulin Gene Investigation in Polyrhachis vicina Roger360 sion levels are different. During the developmental stages, the highest expression level was found in the third instars and the lowest expression level was found in embryos. According to those results, we guess that third instars’s nervous system and sense organs may reach an development peak due to calregu- lin can promote the development of nerve and Sense organs (Silerová et al., 2007; Gamo et al.,1998). In the three adult’s castes, the PvCRT gene expression was highest in workers and lowest in males. This result coincide with Stoltzfus and Horton’s conclusion (2003) “the calregulin plays a key role in olfactory system function, possibly by establishing its overall sensitivity to odorants”. Among P. civica adult’s castes, the worker olfactory system is the most developed especially in antenna and chest foot aspects because of the main responsi- bilities related to building and defending nests, feeding the young ants and queens. The main task of male ants is only to mate with the queen, so its olfactory system function may be the weakest among the castes. 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