SSH screening on Musca domestica differential genes of defense against infection

WANG Yan，JIN Xiao-bao，ZHU Jia-yong

（1.School of Chemistry and Chemical Engineering，Guangdong Pharmaceutical University，Zhongshan528458，China；2.Guangdong Key Laboratory of Pharmaceutical Bioactive Substances，Guangdong Pharmaceutical University，Guangzhou510006，China）

Fly，a typical arthropod（diptera：cyclorrhapha），is traditionally considered as having the adaptive immunity，which is different from vertebrate.Recently，it has been used to explore the origin and evolution of immune system，and that makes a bridge of the dependability between insects and vertebrate innate immunity.The molecules with common ancestry may have evolved into different molecules for the different functions after their divergence.Fly form its distinct immunologic mechanism after long-term evolutionary process，that’s different from mammalian adaptive immunity，without T cells，B cells，complement system and antibody forming system［1］.When they are invaded by surrounding pathogenic organism，how do molecule immunologic mechanism defense against infection and the express of immunological effector molecule？We designed an experimental approach by infectingMusca domesticawith gram positiveStaphylococcus aureus（S.aureus），negativeEscherichia coli（E.coli）andBeauvoir bassinetbacteria to find out immune-related genes.In this study，technologies of subtracted suppressed hybridized cDNA libraries，and real-time PCR were used.

Materials and methods

Insect and bacterium materials

TheMusca domesticastrain was provided by the Guangdong Center for Disease Control and Prevention.According to the protocol of reference，Musca domesticamaintained in culture was used in the present study.Gram positiveS.aureus，negativeE.coliandBeauvoir bassinetbacteria strains were supplied by Guangdong Institute of Microbiology.

Immune challenge of animals

S.aureus，E.coliandBeauvoir bassinetbacteria were mixed in PBS，then 2μL（108cells/mL）live bacteria PBS suspensions were microinjected into more than 200Musca domestica.About 8 groups were separately collected at 2，4，8，12，16，24，36，and 48hours after the challenge.All tissues were collected in approximately 100mg of ice-cold medium contained in a 1.5mL micro-centrifuge tube.Then the tissues were flash frozen in liquid nitrogen and stored at-80℃ until the RNA was isolated.At all the 8time intervals，about 800 mg were mixed together as tester samples for suppression subtractive hybridization（SSH）analysis.Musca domesticainjected with PBS only was used as driver samples for SSH analysis.

Construction of subtractive cDNA library

For forward subtraction，three different microbial challenged subtractive cDNA libraries were separately constructed between challengedMusca domestica（tester）and unchallengedMusca domestica（driver）using the PCR-Select cDNA Subtraction Kit（CLONTECH，Cat.no.K1804-1）according to the manufacturer＇s protocol.Briefly，forS.aureus-challenged subtractive cDNA library construction，total RNA was prepared using TRIZOL reagent（Invitrogen）from theS.aureus-infected and PBS-treated as the control.Poly（A＋）RNA was extracted using Oligotex mRNA Midi Kit（QIAGEN，Cat.no.70042）.The cDNA was synthesized using the Oligo d（T）primer and purified according to the manufacturer＇s instruction.Tester and driver cDNAs were purified by ethanol precipitation，and then digested with 15units of Rsa I at 37℃overnight to obtain shorter blunt-ended molecules.Two different adaptors，adaptor 1 and adaptor 2R，were then ligated to 5′-end of each strand of tester cDNA.The adaptor-1-ligated and adaptor-2R-ligated cDNAs were separately hybridized at 68℃for 8hours with an excess of driver cDNA after denaturation at 98℃for 90sec.After the first hybridization，the two samples were mixed together without denaturation and hybridized again with freshly heat-denatured driver cDNAs for 20hours at 68℃.The resulting mixture was diluted to 1∶50，and then amplified by 2 rounds of PCR to enrich desired cDNAs containing both adaptors by exponential amplification of these products.The primary PCR was performed with flanking primers against adaptor 1and adaptor 2R，and the amplified products were used as template in the secondary PCR with nested primers，which were provided by the Kit.After the secondary PCR，the same quantities of secondary cDNA products of the subtracted and the unsubtracted libraries were taken，the hybridization efficiency was evaluated by PCR with GAPDH performed on tester（unsubtracted）and subtracted cDNAs for 18，23，28，and 33cycles.For the construction of the bacteria strains andE.coli-infected subtractive cDNA library，the same method was used.

Cloning，sequencing and sequence data analysis

The confirmed subtracted cDNA（secondary PCR product）was ligated into pGEM-T easy vector（Promega，USA）.The ligation was transformed into DH5acompetent cells and plated onto agar plates containing ampicillin， X-gal，and IPTG.White colonies sequences were performed on ABI3730machines （Shanghai Invitrogen Biotech，China）.The sequences were searched in GenBank with BLASTx for comparative analysis.Vector and adaptor sequences were trimmed with the use of VecScreen（http：//www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html）prior to further analysis.Homology searches of all sequences were queried in the GenBank database by using the BLASTn （homology）and BLASTx （translation homology）search at the NCBI network service（http：//www.ncbi.nlm.nih.gov/BLAST）.The cDNA sequence was named according to homologous sequences in the database，and cDNA with BLAST scores＜45bits（no homologous stretch＞50bp）were designated as having no significant similarity.For classification of the genes in those functions，the Arabidopsis database at the MIPS website was searched for each sequence.

Differential screening using reverse northern dot blot

DIG High Prime DNA Labeling and Detection Starter Kit I（Roche，Germany）was used.The PCR products（1uL）from each clone of the subtractive library were printed onto Hybond-N＋nylon membranes（Roche，Germany）.After air drying，membranes were denatured in 0.6MNaOH for 15min，neutralized in 0.5MTris-HCl（pH 7.5）for 2min，and rinsed in distilled water for 30 sec.Samples were cross-linked to membranes by baking for 30min at 120℃and then were stored at-20℃.GAPDH cDNA was also printed on each membrane as an internal control.The PCR-amplified cDNA was RsaI digested to remove the adaptor sequences.Digests were purified with the QIA quick PCR purification kit（QIAGEN，Holland）.Digoxigenin-labeled probes of the SSH tester and SSH driver were synthesized with the use of the DIG DNA labeling kit（Roche，Switzerland）ac-cording to the manufacturer’s instructions.The membranes were prehybridized for 3hours at 42℃and then hybridized overnight at 40℃with probes of the SSH tester and SSH driver.

Real-time PCR analysis of the patterns of gene expression

Total RNA was prepared using TRIZOL reagent（Invitrogen）from the independently infectedMusca domesticaat 2，4，8，12，16，24，36and 48 hours after the microbial injection and PBS-treatedMusca domesticaas the control.After digested with DNase I（RNase free，Takara）to eliminate the genomic contamination，the cDNA was synthesized with the Superscript III reverse transcriptase（Invitrogen）using the Oligo d（T）primer.Realtime PCR was performed with a MJ Mini Option instrument（Bio-Rad）.SYBR Premix Ex TapTM（TaKaRa）was used for PCR reaction.Reaction mixtures were incubated for 1.5sec at 95℃，followed by 40cycles of 5sec at 95℃，20sec at 51℃，and finally 20sec at 72℃，melting curve from 70.0℃to 95.0℃，read every 0.3℃，held for 10sec.Primer sequences and the amplicon sizes are listed in Table 1.Musca domesticaGAPDH was used as control to normalize the starting quantity of RNA.Standard curves were constructed for target genes and GAPDH with two-fold serial dilutions of cDNA.The PCR products were analyzed in the 1.2%agarose gel electrophoresis.For calculations of significance，the logs of the REVs for each gene were analyzed by ANOVA（Analysis of Variance）and a Waller-Duncan multiple range test（SPSS Institute Inc.SPSS User＇s Guide，Version 15.0）.All data were analyzed to determine differences between groups（P≤0.05）.

Tab.1 Primer sequences for quantitative real-time PCR

Results

Infection of Muscadomestica

After infecting 1st-instar larvae ofMusca domesticawithS.aureus，E.coliandBeauvoir bassinetbacteria，the changing of character and survival rate were observed to confirm the LC50，about 1010cells/mL，109cells/mL and OD600 1.98.

Effectiveness of SSH

To evaluate the success of the subtraction，expressions of the GAPDH gene were compared between subtracted and unsubtracted cDNA by PCR.In subtracted cDNA，PCR products were detected firstly at 23cycles.In contrast，the amplification products of unsubtracted cDNA were observed firstly at 18cycles.The results indicated that existence of the GAPDH gene was reduced by up to 25-fold by subtractive hybridization，which showed that the subtraction procedure was successful，seen in Figure 1.

PCR was performed on the unsubtracted or subtracted nested PCR product with the attacin defensin cecropin 5＇and 3＇primers included in the Table 1.The three genes at different levels have been enriched after subtraction，seen in Figure 2.

A gene profile in response to bacterial challenge

A total of 252positive clones were obtained，and the identification of the inserted cDNA fragments in subtractive library was done by PCR.The results showed that there were inserted fragments of 250-750bp，which would provide useful baseline for the screening and cloning of specific anti-infection genes of immunity inMusca domestica.The search for sequence homology in the GenBank nr and EST database by BLASTn revealed the distribution of EST clusters for high homology genes.The high homology genes were grouped into clusters based on the best known function of there protein products：signaling transduction/communication，cell structure/mobility，transporters，apoptosis，metabolism，translation/expression regulation，cell/body defense，and the cluster，whose function was unknown or without an ontological theme.The profile of gene transcription by SSH was reported in this paper.Compared with mammals，Musca domesticahas the same acute immune defense genes（proteases inhibitor，lysozymes，respiratory burst，and antimicrobial peptide，etc.），and a similar pattern and level of temporal gene expression，while C-reactive protein，serum amyloid P and ser-um amyloid A provided a further evidence for the use ofMusca domesticato study the evolution of the immune system.Further functional analyses on these newly identified genes with or without homolog invertebrates assisted immunologists to study the origin and phylogeny of the vertebrate immune system，seen in Table 2.

Fig.1 Reduction of GAPDH abundance by PCR-select subtraction

Fig.2 Reduction of attacin defensin cecropin abundance by PCR-Select subtraction

Reverse northern dot blot

The efficiency of the SSH was further retested by reverse northern dot blot.PCR amplification of bacterial suspension was taken on 35cloned Broth.After purification，the PCR products were performed for point membrane and reverse northern dot blot.Marking the two probes，the sixth spot was clearly visible when the amount of dilution to 0.1pg/μL.It was indicated that the probe labeling reached the desired results of the calculated amount of labeled probe 70pg/μL seen in Figure 3.The results showed that there were 32of those appeared differences in gene expression afterMusca domesticainfection，and the positive rate was 91.5%，seen in Figure 4.These genes expressed up-regulatively in the infectiousMusca domesticato some extent，which indicated that the SSH was apowerful technique to enable researchers to identify differential genes.

qRT-PCR

The cecropin，defensin and attacin mRNA levels fromMusca domesticawere clearly up-regulated after challenge.Cecropin and defensin gradual-ly increased at 4hours and peaked at 16and 12 hours challenged and then sharply decreased，seen in Figure 5.The attacin showed a similar pattern of expression.Attacin mRNA levels were low at the beginning，increased gradually and peaked at 2 hours，slightly peaking at 16hours，seen in Figure 5C.The antibacterial peptide genes were detected at different development stages of Musca domestica.The qRT－PCR data suggested that the greatest levels of mRNA for all three genes were in the 3rdinstars and adults.Levels of mRNA were relatively lower in eggs，1st－instar larvae and pupae，seen in Figure 5D，E and F.

Tab.2 Genes up-regulated after bacterial challenge

Fig.3 Determination of labeling efficiency

Fig.4 Result of differential screening with reverse northern dot blot

Discussion

Those microorganisms invading the general body cavity were countered by both humoral and cellular reactions［2］.Thus，an experiment was designed by infectingMusca domesticawithS.aureus，E.coliandBeauvoir bassinetbacteria，and the SSH was used to find out the immune-related genes.Compared with the other two bacteria，S.aureushad a strong resistance to infection，which induced the expression of genes at most.From the SSH library，many of genes were the homologs of mammalian acute immune defense genes，including ribosomal protein，metalloproteinase and proteases inhibitor，lysozymes and antimicrobial peptide，genes involved in the complement system，and genes responsible for respiratory burst.

Antibacterial peptide was a significant part of humoral immunity effector molecule inMusca domestica.Following infection or septic injury，insect antimicrobial peptides were rapidly synthesized and cleaved by signal peptidases.They become functionally active，and were then rapidly secreted into the hemolymph［3］.The antibacterial peptide genes expression ofMusca domesticawas constitutive.They expressed at different development stages ofMusca domestica，but the expressing levels were evidently different.During all the three development stages ofMusca domestica，antibacterial genes had varied transcript patterns.They showed the highest level of mRNA in 3rd-instars and adults.In contrast，the transcripts were less abundant in the egg，1st-instar larvae，and pupae.Larvae of bothA.gambiaeandD.melanogasterhave been reported to have high levels of antimicrobial mRNA，which were most likely involved in response to bacterial challenge encountered during larval development［4］.The high mRNA levels observed in the present study for cecropin，defensin and attacin in theMusca domestica3rd-instars could be related to increased microbial challenge and perfect of immune defense mechanism gradually during these stages in development.The low levels of antibacterial mRNA in 1st-instar larvae might be due to lack of microbe consumption［5］.Meanwhile，it indicated that antibacterial peptide was a significant part during larval development.In 1st-instar larvae lack of antibacterial peptide genes expression might be female source transcripts，thus it had low levels of antibacterial mRNA［6］；the insect of holometabolous development had power of defense against infection surrounding in pupal stage.The data showed that levels of antibacterial mRNA are low in pupae，but relative greater in larval，which suggested antibacterial peptide was an significant immunity effector molecule［7］.Thus，the mature of immune system，increased microbial challenge，and adaptive evolution of each area might explain the slight differences observed in adults［8－9］.Following infection or septic injury，transcript of antibacterial genes in-creased in few hours，kept for 24hours and then sharply decreased.Antibacterial peptides defended against infection temporarily.

Fig.5 Cecropin，defensin and attacin mRNA expression at different development stages and challenged respectively of Musca domestica

In summary，the acute immune response to microbial challenge inMusca domesticashared a certain similarity to that in mammals，which provided a further evidence for the use ofMusca domesticato study the evolution of immune system.At the same time，theMusca domesticaalso showed certain specific recognitions for different microbes and might apply different strategies to defend those microbial intrusions.Furthermore，some novel acute immune response genes were also discovered.Further functional analyses on these newly identified genes may assist immunologists to further study the immune system ofMusca domestica.

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