| The diamondback moth, Plutella xylostella(L.), is a worldwide and destructive pest that damagecruciferous crops.Because of the abuse ofchemical pesticides,diamondback moth(DBM) has developed high resistance to them, even including the Bt micro-pesticides, and caused huge economic losses to vegetables cultivation every year. So it is very urgent for researchers to explorenewstrategies and microbial insecticideto biological control DBM. In this study, the transcriptomes of DBM infected by entomopathogenic fungus, Isaria fumosorosea were sequenced and the immunity-related genes with the different expression were analyzed and further validated by q RT-PCR.RNAi technology was used to study the function of Toll families to fungal infection in DBM. The results of this study will provide a new idea to explore new fungal pesticides targeted the immunity system to biological control DBM.In order to clarify the dynamic mechanism of immunity of DBM to I. fumosorosea, 3rd instar larva infected by I. fumosorosea spore(107CFU/ml)at 12 h,18h, 24 h and 36 h were collected respectively and DGE were sequenced by using RNA-seq technologies. Data analysis showed showed there were 119, 117, 635 and 117 genes were up-regulated, and meanwhile 747, 869, 665, 869 genes were down-regulated at different infection time. In these genes, there are 78 genes related to Toll and IMD signalpathwaysof DBM immune system, including 11 pattern recognition receptors(PRRs), 43 pathway components, 9antimicrobial peptides(AMPs). The results also showed the expression of 15 toll geneswhich as core components of Toll pathways were up/down-regulated significantly.These results suggested that the immune pathway of P. xylostella, especially Toll signal pathways were induced by I. fumosorosea and activated the expression of AMPs against fungal infection.On the basis of DGE data and DBM genome data, the full length cDNA sequence encoding Toll 2, Toll 5 and Toll11 were obtained by using RT-PCR and RACE technology.Sequence analysis showed that the full length of cDNA sequence of Toll 2 was 3008 bpwithopen reading frame of 2010 bp, which encoding 669 amino acids, the transmembrane domain was 462-486 amino acids, with the molecular weight of 76.7 k Da,isoelectric point of 6.31. The full length cDNA sequence of Toll 5 was 3497 bp, open reading frame of 2052 bp, encoding 683 amino acids, the transmembrane domain was434-456 aa, with tethemolecular weight of 75.2 k Da, isoelectric point of 5.30. The full length cDNAsequenceof Toll 11 was 4904 bp with open reading frame of 4710 bp, which encoding 1389 amino acids, the transmembrane domain was 1321-1343 amino acids, with the molecular weight of 159.7 k Da, isoelectric point of 5.89.Sequence alignment and phylogenetic tree analysis showed that Toll 2 belonging to a family of Toll-like receptor6,Toll 5 belonging to Toll- like receptor 8 and Toll 5 belonging to Toll- like receptor 8.Using the body wall dipped method, 3rdinstar larvae of DMB were treated by Beauveria bassiana and Metarhizium anisopliae, three tissues including epidermis, fat body,and hemocyte were collected. q RT-PCR results showed that dynamic expressions of Toll 2,Toll 5 and Toll 11 induced by fungus were complex. In different tissues, the expressions of Toll 2, Toll 5 and Toll 11 induced by I. fumosorosea, B. bassiana and M. anisopliae were different, in comparing, the peak time of expression of Toll 2, Toll 5 and Toll 11 in fat body was earlier than other two tissues. Above results showed that three Toll genes, as the internal components of Toll signal pathways, were up and down regulated by fungal infection at different times, which will further indicate the complexity of expression of signal pathway factors triggered by fungus in DBM.In order to investigate the functions of Toll 2, Toll 5 and Toll 11, using filamentous fungi silencing vector p Silent-1 plasmid, RNAi vectors targeted Toll genes were constructed successfully, and transferred into protoplast of I. fumosorosea If B01 by using PEG400 mediator.The genetic stable recombinant strain, ds Toll 2 was obtained and testified by using 600ug/ml of hygromycin B for preliminary screening and using PCR for final screening. The recombinant strain was used to prepare the spore suspensionfor concentration of 10~4, 10~5, 10~6, 10~7 CFU/ml and infected the 3rdinstar larvae using wallimmersion method, results showed that the survival rate of larvae infected by the recombinant strains with the concentration of 10~4CFU/ml, 10~5CFU/ml and10~6CFU/mlwas decreased to 0%at the 6thday.The survival rate of 3rdinstar larvae infected by the recombinant strains with the concentration of 10~7 spores / ml down to 0% was the 5th day.The survival rate of the DBM infected by the unmodified I. fumosorosea with concentration of 104CFU/ml, 105CFU/ml, 106CFU/ml and 10~7CFU/ml is 54.67%, 52% and29.33% and 22.67% at 6thday. The concentration of unmodified I. fumosorosea to keep the0% survival rate of DBM is 10~9CFU/ml. The above results indicated that therecombinant strains, ds Toll 2 has greater impact on the survival rate of DBM and can significantly shorten the death time of DBM.In order to further explore the structure and function of Toll protein in P.xylostella.The expression plasmid p GEX4T-Toll-2 was constructed, and transformed into the competent DE3 cell. Toll proteins were expressed with the induction of IPTG. SDS-PAGE analysis showed that p GEX4T-Toll-2 can be expressed with the molecular weight(Mr) 60.0k Da. The recombinant protein of Toll2 was purified by GST affinity chromatography, and the antiserum of anti-Toll 2 was prepared in rabbit. Western Blot results confirmed that theanti-Toll2 serum can bind purified protein positively with highly quality. The above results provided rich proteinresource to study on the structure and function of Toll 2 protein in future.In conclusion, the transcriptomes of DBM infected by entomopathogenic fungus,Isaria fumosorosea were sequenced and the immunity-related genes with the different expression were analyzed and further validated by q RT-PCR. RNAi technology was used to study the function of Toll families to fungal infection in DBM. The results of this study will provide a new idea to explore new fungal pesticides targeted the immunity system to biological control DBM. |
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