Identification Of Nosema Bombycis-induced Early Genes And Application For The Control Of Pérbrine In Silkworm

Microsporidia is a group of eukaryotes exclusively composed by obligate intracellular parasites,which has a wide range of hosts and is common in vertebrates and invertebrates.Since the 19th century,the existence of microsporidia has been known.Nowadays,scientists have already discovered about 200 described genera of microsporidia with over 1500 individual species.Among them,Nosema bombycis(N.bombycis)is the first microsporidia to be identified and named.It is the pathogen of Perbrine,the biggest epidemic disease endangering sericulture safety production.At present,the direct economic loss caused by Perbrine in China is up to hundreds of millions yuan per year.The cost of preventing and controlling the Perbrine accounts for about half of the total expenditure of silkworm eggs production in China major silkworm farms.At present,the research on the resistance of silkworms to N.bombycis is mainly through the natural selection and the introduction of endogenous and exogenous resistance elements(such as endogenous and exogenous resistance genes,antibodies,interference fragments,etc.).Although the above methods have improved the resistance of silkworms to N.bombycis to a certain extent,there has been no corresponding application of resistant silkworms in production so far,and there is no effective medicine for N.bombycis,so we urgently need to explore some new targets and new technologies to improve the resistance of silkworms to N.bombycis.In this research,the response of midgut host gene of silkworm in the early stage of N.bombycis infection was analyzed by transcriptome sequencing.It was found that multiple pathways of silkworm were involved.The BmUGT10295,a UDPglycosyltransferase(UGT)gene,was screened and identified,which was only expressed in infected individuals.The induced expression analysis of the gene in the cluster branch of BmUGT10295 in UGTs showed that besides BmUGT10295,BmUGT8453 was the same induced expression gene,and it was found that these two genes could inhibit the proliferation of N.bombycis.Then,the induced expression patterns of the two gene promoters were identified.Further,the proliferation inhibition system for N.bombycis was established based on these promoters and GAL4/UAS binary expression system.Finally,the proliferation inhibition system was evaluated on individuals,and it was found that there was no significant difference in the survival rate and some main economic traits of the corresponding transgenic silkworms,but it had a significant inhibitory effect on microsporidia.However,when Bombyx mori were infected by Nuclear Polyhedrosis Virus(BmNPV)and Beauveria bassiana(B.bassiana),the survival rate of proliferation inhibition system transgenic silkworm was not higher than that of its control group.The main research results are as follows:1.Response analysis of silkworm midgut in the early stage of N.bombycis infectionIn order to analyze the response of silkworm midgut in the early stage of N.bombycis infection,N.bombycis was orally infected to the 5th instar B.mori.At 3,6,12,24 and 48 hours after the infection with N.bombycis,the midgut of normal and infected silkworms were dissected in wax plate for microscopic examination.The results showed that no obvious mature spores were observed at 3-6 hours after infection.There were a few mature spores in the midgut at 12 hours,but there were a large number of mature spores in the midgut at 48 hours after infection.The transcriptome sequencing of the samples at the above time points showed that a total of 13789 gene sequences were finally obtained,including 11279 known genes(81.80%)and 915 newly predicted genes.The differential expression analysis of these genes showed that there were 1729 up-regulated genes and 693 down-regulated genes at 3 hours after infection,1203 up-regulated genes and 588 down-regulated genes at 6 hours after infection.The number of differentially expressed genes at this time point was much higher than that of the samples at 12 hours after infection(296 up-regulated genes and 755 down-regulated genes).At 24 hours after infection,the number of differentially expressed genes increased sharply(2876 up-regulated and 358 downregulated).At 48 hours of infection,the number of differentially expressed genes reached the maximum,with 4310 up-regulated and 197 down-regulated genes.In order to response microsporidia infection,insects have developed a unique,rapid and efficient immune system.We mainly analyzed the transcription changes of genes related to innate immune pathway,autophagy pathway,apoptosis pathway and detoxification pathway in B.mori.In the Toll receptor pathway,Spz1/Toll9-1/Toll11 are mainly involved in the stress response of N.bombycis.Toll receptor pathway and JAK-STAT pathway were strongly activated in 24-48 hours,while IMD pathway was activated in the whole process of infection.The autophagy pathway was also strongly activated mainly 24-48 hours after infection.In the apoptotic pathway,the apoptotic pathway was inhibited at 3-12 hours after N.bombycis infection.At 24-48 hours after infection,receptor-mediated apoptosis and endoplasmic reticulum stress-induced apoptosis were strongly activated.In addition,we found that the detoxification pathway of B.mori was activated under the microplasma infection.Among that,the UGT genes were strongly activated mainly at 24-48 hours,while the ABCG transporters were significantly activated throughout the infection period.In short,in the stage of microsporidia infection,the stress response of the host to the pathogen is not a simple pathway or a simple change of a limited number of pathways.It is a complex and changeable process involving multiple pathways,which is the result of the mutual game between pathogen and host.2.Screening of N.bombycis-inducible genes in silkwormIn this study,a large number of differentially expressed genes were identified by transcriptome,and it was expected to screen out a number of genes that were induced by infection,but not expressed without infection.Combined with the silkworm gene chip data established by laboratory before,55 differentially expressed genes were screened from the transcriptome for RT-PCR verification.Firstly,the genes with positive amplification bands were removed by RT-PCR using the templats of 5 instar and 3 instar silkworm without infection with N.bombycis.Then the genes with positive amplification bands in the infection group were selected by RT-PCR using the infected materials for 48 hours post infection,and the positive amplification bands of uninfection material were removed.Finally a candidate target,BmUGT10295,was obtained,which was the induced expression gene by N.bombycis,and is used as the target in follow-up studies.3.The identification and function of induced expression BmUGT genesBmUGT 10295 belongs to UDP-glycosyltransferase(UGT)family.Previous evolutionary analysis found that it is located in the cluster I of the UGT gene family.In this study,it was found that,in addition to BmUGT10295,BmUGT8453 was also induced to express in the cluster I of UGT gene family through RT-PCR.And there were no transcripts of BmUGT10295 and BmUGT8453 genes in the various tissues and different periods of the uninfected silkworm.Through RT-qPCR analysis,it was found that BmUGT10295 and BmUGT8453 were mainly transcribed in the midgut and Malpighian tube of the infected silkworm.Furthermore,the full-length cDNA sequences of these two genes were obtained through the 3’RACE and identification of transcription initiation site.The recombinant BmUGT10295 and BmUGT8453 proteins were expressed in Escherichia coli and were prepared for their antibodies.Then Western blotting analysis confirmed that BmUGT 10295 and BmUGT8453 were only expressed in infected silkworms.In order to better investigate the gene function of BmUGT,the transcription of BmUGT10295 and BmUGT8453 in BmN-SWU cells after infection with N.bombycis were detected by RT-PCR.The results showed that BmUGTs were only transcribed in infected cells,and the results were further confirmed by IFA.Next,over-expression of BmUGTs in BmN-SWU cells can significantly reduce the amount of N.bombycis compared with the control.The amount of RNAi BmUGTs in BmN-SWU cells can significantly increase compared with the control.The above results indicate that BmUGT 10295 and BmUGT8453 can inhibit the proliferation of N.bombycis.4.Establishment of proliferation inhibition system and evaluation of pathogeninfectionIn order to establish an proliferation inhibition system,we firstly must determine a promoter that can be induced and activated by N.bombycis.In the above experiments,it has been determined that BmUGT10295 and BmUGT8453 are induced expression genes,and then the upstream sequences of these two genes are intercepted as promoters.Two vectors pSL[P10295-mCherry-SV40]and pSL[P8453-mCherry-SV40]were constructed to analyze the induced-patterns of the two gene promoters.The two vectors were transfected into BmN-SWU cells.After induction by different microorganisms,it was found that the promoter of BmUGT10295 gene(P10295)and the promoter of BmUGT8453 gene(P8453)can be activated by Staphylococcus aureus,BmNPV and N.bombycis,but not Escherichia coli.Next,pSL[P10295-GAL4],pSL[P8453-GAL4],pSL[UAS-EGFP]and pSL[UASRicin A]vectors were constructed.When pSL[P10295-GAL4]or pSL[P8453-GAL4]were co-transformed with pSL[UAS-Ricin A]vector,the cell viability of the infected group was significantly lower than that of the uninfected group,which showed that the established infection system can play a role on the cells.Now that the proliferation inhibition system can work on cells,we further constructed and evaluated transgenic silkworms.First,piggyBac[3P3-EGFP+opIE2GAL4],piggyBac[3P3-EGFP+P10295-GAL4],piggyBac[3P3-EGFP+P8453-GAL4],piggyBac[3P3-DsRed+UAS-EGFP]transgenic vectors were constructed,and successfully created the corresponding transgenic silkworm,UAS-Ricin A transgenic silkworm has been established in our previous research.When the opIE2 transgenic silkworm(constitutively expressing GAL4)was hybridized with Ricin A transgenic silkworm,the individual mortality of its offspring reached more than 90%,while there was almost no death in other control groups.P10295-GAL4 and P8453-GAL4 transgenic silkworm were hybridized with UAS-Ricin A transgenic silkworm respectively,and then the survival rate and economic characters(egg number,whole cocoon weight and cocoon layer ratio)of the hybridization strain(P10295×Ricin A and P8453×Ricin A)under normal feeding were investigated.The results showed that there was no significant difference in survival rate and economic characters between the hybridization strain and the control group.Next,the effects of N.bombycis infection were detected.When the 4th instar silkworm was infected with low-dose microsporidia(100 spores per silkworm),the survival rate of the hybridization strain of P10295×Ricin A and P8453×Ricin A transgenic silkworms were much higher than that of the control group,and the loading of microsporidia in the hybrid lines was significantly lower than that of the control group.When the silkworms were infected with high-dose microsporidia(10000 spores per silkworm),compared with the control group,the hybridization strain of P10295×Ricin A and P8453×Ricin A massive dead in a short time after infection with N.bombycis,resulting in a significant reduction in the survival rate,but the survival rate of the hybridization strain was higher than that of the control group after infection 6 to 12 days.And the loading of microplasma in the hybrid line at each time point was significantly lower than that in the control group.In the induction pattern analysis of the above promoters,it was found that promoters P10295 and P8453 could be activated by a variety of pathogens.Therefore,the proliferation inhibition system was used to detect their resistance to BmNPV and Beauveria bassiana.For infection with BmNPV,despite the survival rate of P10295×Ricin A or P8453×Ricin A transgenic silkworm was slightly lower than that of the control group in the early stage,and the survival rate of P10295×Ricin A or P8453×Ricin A transgenic silkworm was slightly higher than that of the control group in the late stage of infection,but there was no significant difference.For B.bassiana infection,the survival rate of P10295×Ricin A or P8453×Ricin A transgenic silkworm was slightly lower than that of the control group in the early stage,and finally reached a consistent level.In conclusion,this study analyzed the host midgut immune response of B.mori in the early stage of N.bombycis infection through the transcriptome data,and screened BmUGT as the induced expression gene of B.mori.Furthermore,overexpression and RNAi BmUGT10295 and BmUGT8453 proved that the expression of this gene could inhibit the proliferation of N.bombycis.Using the promoters of BmUGT10295 and BmUGT8453 genes,a proliferation inhibition system was established through GAL4/UAS binary expression system.Although the system has little effect on the infection of BmNPV and B.bassiana,it can well inhibit the proliferation of N.bombycis.This study provides a new idea for the prevention and control of N.bombycis disease in sericulture production,and also provides a reference for the pathogen prevention and control of other economic insects.