| Locusta migratoria is a typical explosive agricultural pest around the world.The outbreak of locust plague limits the stable development of agriculture.For a long time,the control measures of locusts are mainly based on the use of chemical pesticides,but the long-term and extensive use of chemical pesticides has caused serious "3R problems".Therefore,it is urgent to develop new green methods for pests.Metarhizium anisopliae is one of broad-spectrum entomopathogenic fungus,which has been widely used in the control of agricultural and forestry pests,but its slow effect hinders its industrialization as an important resource for biological control.The reason for the low insecticidal efficiency of entomogenous fungi is that when they are infected,they are resisted by the innate immune system.Therefore,it is of great significance to improve the efficiency of biological control by carrying out the research on the immune system of insect pests,mining the key genes of immune entomogenous fungi,and taking this as the target to develop new,safe and efficient biological insecticidal agents.Based on this,the main research contents of this paper are as follows: on the basis of obtaining the key gene Toll-likereceptor(TLR)of Locusta migratoria against Metarhizium anisopliae,LmTLR was cloned and its structure was analyzed,and the phylogenetic tree was constructed;the expression pattern of key immune gene LmTLR and its induced expression pattern induced by Metarhizium anisopliae were analyzed;the function of LmTLR in immune Metarhizium anisopliae was studied.The recombinant Metarhizium anisopliae expressing ds TLR with hairpin structure was constructed and its virulence was determined.The main results are as follows:1.On the basis of partial TLR sequence of Locusta migratoria obtained from transcriptome database,the full-length cDNA sequence of TLR gene was obtained by RACE combined with RT-PCR technique,in which ORF is 3387 bp,its molecular weight(MW)is about 127.8 KDa,and isoelectric point(PI)is 5.88.Through bioinformatics analysis,it is found that LmTLR is a type I transmembrane receptor,which can be divided into three parts:extramembrane region,transmembrane region and intramembrane region.The extracellular domain,the N-terminal domain,is composed of repetitive sequences rich in leucine,and the TIR domain in the cytoplasm is highly homologous to interleukin-1 receptor(Interleukin-1receptor,IL-1R).The spatio-temporal expression pattern LmTLR was analyzed by qRT-PCR.The results showed that the expression of LmTLR was the highest in the epidermis,and the expression of LmTLR was the highest at 3th,4th and 5th instar.Through phylogenetic analysis,it was found that LmTLR was highly homologous to the TLR of Gryllus bimaculatus.2.The expression patterns of LmTLR induced by fungi and bacteria were detected by qRT-PCR.The results showed that both bacteria and fungi could induce the expression of LmTLR.In order to further clarify the function of LmTLR in immune response,RNA interference technique was used to silence LmTLR.It was found that LmTLR could be silenced significantly after injecting ds RNA after 24h.After interfering with LmTLR,the antibacterial activity of migratory locusts decreased significantly,and the expression of Toll signal key genes LmMyD88 and LmPelle were also down-regulated,indicating that LmTLR regulates immune response by affecting the expression of two key genes LmMyD88 and LmPelle in Toll pathway.In addition,we also found that LmTLR could also affect the expression of Lmtransferrin.3.The regulation mode of LmTLR expression was studied by inducing ecdysone and interfering with ecdysone receptor Ec R.The results showed that both endogenous and exogenous 20E could induce the expression of LmTLR,and the change of LmTLR expression at 5th instar was consistent with the titer of ecdysone.After silencing EcR,both endogenous and exogenous ecdysone could not induce the expression of LmTLR.This suggests that 20E regulates the expression of LmTLR by binding to LmEc R.4.In order to improve the pathogenic efficiency of Metarhizium anisopliae,the vector pSilent-1-TLR1-TLR2 was constructed and successfully introduced into Metarhizium anisopliae protoplasts to express ds LmTLR.The results showed that transgenic Metarhizium anisopliae could express and secrete ds LmTLR.Through qRT-PCR detection,it was found that transgenic Metarhizium anisopliae could significantly reduce the expression level of LmTLR(decreased by about 81%).The virulence level was significantly higher than that of the original strain,and the whole death time was about 2 days ahead of schedule,which greatly increased the insecticidal efficiency of Metarhizium anisopliae.In this study,the function of LmTLR in immunity to Metarhizium anisopliae was analyzed by molecular biological techniques,the hormone regulation mode of LmTLR was revealed,and the highly pathogenic Metarhizium anisopliae engineering strain expressing ds LmTLR was successfully constructed.The results are of great significance for improving the efficiency of biological control and promoting the industrialization of entomogenous fungi as high-efficiency green insecticides. |
