Annotation And Study On The Function Of Antioxidant Genes In Silkworm, Bombyx Mori

Silkworm, Bombyx mori, is the only highly domesticated economic insect，theindustry formed by silkworm has an important cultural value and commercial value. Asan important economic mode insects and experimental material, Researching in thegenome structure and function of silkworm in-depth is a significance for maintainingsustained and steady development of silk industry. We may encounter many problemsduring sericulture process in our country, such as the popular of silkworm disease anddegradation of breeding ecological environment, it may directly affect the yield andquality of cocoons. When exposed to biological stress, the balance of reactive oxygenmetabolism of organism will lose. Cells, tissues and organs of host will be damaged byreactive oxygen molecules since the reaction of reactive oxygen molecules is notspecificity, so that caused the damage of immune system and physiological functions oforganism. Therefore, the elimination of the excess of reactive oxygen free radicals inorganism will be able to enhance its ability to resist external adverse environment, so asto improve their immunity. All the organisms have evolved a complex antioxidantsystem in the long evolutionary process. The antioxidant system can regulatemetabolism of organisms with the changes of environmental conditions until hereditarywill be changed accordingly. As an important economic insect and the mode oflepidoptera, silkworm is no exception. To breed resistant silkworm varieties, theantioxidant system of silkworm and its gene function have been studied deeply, whichwill also provide important molecular basis for the study of biological control of otherlepidopteran insects and pests.In this study, based on the silkworm9×genome, EST and microarray data, theantioxidant genes of silkworm have been annotated. Evolution and expression of theantioxidant genes have been analysed by the method of bioinformatics. The cloned,expression pattern, enzyme activity and tissue localization of BmPrx4gene have beenstudied and analysied. The obtained results are as follows:1. Based on genome and EST data, the gene number, gene organization,chromosome distribution and subcellular localization of silkworm antioxidant geneshave been identified and analysis, phylogenetic relationships of the antioxidant genebetween the silkworm and the other four major categories of insects (such as A.mellifera,Drosophila, A.gambiae and T.castaneum) have been analysed and compared. The results suggested that these identified genes include nine families, thereinto, superoxidedismutases (SODs), glutathione peroxidases (GPXs), glutaredoxin (GRXs), thioredoxinreductases (TRXRs), thioredoxin peroxidases (TPXs), thioredoxin (TRXs) andmethionine sulphoxide reductases (MSRs) families were highly conserved in terms ofphylogenetic orthologs and the orthologous genes of these conservative subfamilies hadthe same subcellular localization. Therefore, the gene numbers of the five families werealmost the same in the five insects, CAT and HPX families also appeared to belineage-specifically expanded in the silkworm. Moreover, through the phylogeneticanalysis, the signal peptide and transmembrane domains prediction, UTR region andgene structure analysis as well as sequencing analysis, the results suggested thatBmCat4gene was in deed the Cat gene of silkworm rather than bacteria and othermicroorganisms gene mixed in sequencing of the silkworm genome, and its encodedprotein was likely to be a secreted protein, which was not common to containingsecretory CAT protein in insects.2. Based on EST and microarray data, the transcription activity and expressionpatterns of the silkworm antioxidant genes were analyzed, the expression patterns ofantioxidant genes in various tissues were tested by RT-PCR, which cDNA of larvaltissue of fifth instar3day was made as template. The results showed that42of the50antioxidant genes had ESTs, the probes of39antioxidant genes were found and only24of which showed expression signals with values more than400at least in one tissue. Asfor the other eighteen genes without microarray probes, RT-PCR was performed toanalyze their tissue expression patterns in the fifth instar day3larvae. We found thatmost of the antioxidant genes showed tissue-specific expression patterns (for example,the BmHpx genes of lineage-specifically expanded were predominantly expressed in thesilk gland, the function of these genes might be in maintaining cell homeostasis in theprocess of the synthesis of large amounts of silk proteins), only minority genes wereconstitutively expressed.3. Based on the genome data, the candidate specific primers of the BmPrx4(BmTpx2) gene was designed and the full-length CDS of this gene was cloned, sequenceanalysis of BmPrx genes was done by bioinformatics methods. The results suggestedthat the silkworm genome may have five BmPrx genes, only BmPrx4gene OnlyBmPrx4gene contained a signal peptide encoded21amino acids and an open readingframe (ORF) of744bp encoding a protein of248amino acids with a predicted signalpeptide of63bp. The predicted isoelectric point (pI) and molecular weight (MW) were 6.51and27.87Da, respectively. It contained two introns and three exons and theboundary of the exons and introns conformed to the GT-AG rule.4. Based on antioxidant genes BmPrx4may be induced by high temperature, lowtemperature, and secondary metabolites of mulberry leaves, the induced pattern ofBmPrx4gene in fat body and testis tissue of5instar3days larvae was tested byqRT-PCR. The results indicated that BmPrx4gene in fat body and testis tissue of5instar3days larvae could be induced by the high and low temperatures, and the effectof induced in the testis was more obvious. We had also demonstrated the induction ofBmPrx4in silkworm larvae during exposure to quercetin, Moreover, the expression ofBmPrx4mRNA was up-regulated gradually with the rising concentration of quercetin,and induced by short-term treatment more than with long-term treatment.5. The BmPrx4gene was expressed in the Escherichia coli expression system forin vitro expression, target protein was purified using purified fusion proteins columnwith histidine tag and enzymatic assays were tested using purified fusion proteins. Thepurified fusion proteins were then injected into mice to generate the specific antibodies.The results suggested that the peroxidase activity of BmPRX4is thiol-dependent andthe purified recombinant BmPRX4protein with DTT catalysed the removal of H2O2ina concentration-dependent and time-dependent manner. The prepared antibody wasreacted with recombinant protein by western blotting, the results could be found outafter hybridization was a bright and single band, which indicated that the preparedantibodies was specificity and could be used for subsequent experiments.6. The expression patterns of BmPrx4gene in various tissues and at differentdevelopmental stages were studied by RT-PCR and Western blot methods. The resultsfound that BmPrx4gene in various tissues and at different developmental stages was allexpressed both in transcription and translation level, which indicated that the BmPrx4gene of the silkworm might be a housekeeping gene. In addition, the previous studiesshowed that PRX4protein was a secreted protein in mammalian. Similarly, we foundthat BmPrx4gene not only contained a signal peptide, but also had distribution in theserum of silkworm, which showed that BmPRX4proteins in silkworm might also besecreted proteins.7. The tissue localization of BmPrx4in the silkworm head was studied by themethod of immunohistochemistry. The results showed that the expression signals ofBmPrx4was detected in the brain, nerves and the olfactory system. The nervous systemin the body was especially sensitive to free radical damage because of the rich content of easily oxidizable fatty acids and the relatively low content of antioxidants. Theseresults indicated that BmPrx4might play important roles in protecting the brain, nervesand the olfactory system from oxidative damage in silkworm.Through this topic, the number of antioxidant genes in silkworm were identified,and the mutual evolutionary relationship with the antioxidant genes of the other fourmajor categories of insect was studied at the genomic level. The expression patterns invarious organizations of the silkworm were researched at the molecular level, whichprovided theoretical basis and molecular basis to understand evolutionary status andfunctional diversity of antioxidant genes in the silkworm and even insects. At the sametime, the expression patterns, enzymatic assays and tissue localization of the conservedgene (BmPrx4gene) were studied in the silkworm genome, which provide usefulinformation to study function characterization of housekeeping gene in silkworm. Inaddition, it also provides favorable clues for effective prevention and control ofagricultural and forest pests.