Cloning,Identification And Functional Analysis Of The Genes Encoding The Pattern Recognition Receptor GRP And PGRP In Nilaparvata Lugens

brown planthopper(Nilaparvata lugens)is an important pest of rice in China,which seriously endangers the safety of rice production.Microbial control is the optimal strategy for biological control of brown planthopper,in which entomopathogenic bacteria and symbiotic bacteria are two important microbial resources.However,the effectiveness of microbial control is largely restricted by insect immune defense system.Further study on the immune interaction between BPH and pathogens and symbiotic bacteria,and mining the key immune related genes,will have important application value for the development of new biological control technology of BPH.The immune recognition of insects to microorganisms is the key to start insect immune defense.In this study,six glucan recognition protein genes(NlGRP1、NlGRP2、NlGRP3、NlGRP4、NlGRP5 and NlGRP6)and two peptidoglycan recognition protein genes(Nl PGRP-LB and Nl PGRP-LC)were cloned and identified,and their expression patterns in different developmental stages,tissue parts and different microbial inducements of BPH were analyzed In order to provide scientific basis and gene resources for the development of the technology of "controlling insects with bacteria" of brown planthopper.The results are as follows1.Cloning and sequence analysis of the pattern recognition receptor gene of Nilaparvata lugens: In this study,eight pattern recognition receptor genes NlGRP1、NlGRP2、NlGRP3、NlGRP4、NlGRP5、NlGRP6、Nl PGRP-LB and Nl PGRP-LC were successfully cloned from the brown planthopper genome.Among them,the protein encoded by NlGRP1、NlGRP2、NlGRP3、NlGRP4、NlGRP5 and NlGRP6 belongs to glucan recognition protein family,and its predicted molecular weight ranges from41.9 k Da(NlGRP4)to 63.0 k Da There is a signal peptide at N-terminal and a β-1,3-glucanase like domain at C-terminal between k Da(NlGRP6).In addition,the N-terminal of NlGRP1 、 NlGRP2 and NlGRP3 also contains a β-1,3-glucan recognition domain.The full-length cDNAs of Nl PGRP-LB and Nl PGRP-LC genes are 651 bp and 1176 bp,respectively,encoding a typical insect peptidoglycan recognition protein composed of 216 and 359 amino acids,and each contains a structure with amidase activity at the C-terminus Domain(Amidase).Sequence analysis showed that Nl PGRP-LB had no signal peptide and transmembrane region,while Nl PGRP-LC showed transmembrane region.It was speculated that Nl PGRP-LB existed in the form of cytoplasmic protein,and Nl PGRP-LC functioned as a transmembrane peptidoglycan recognition receptor.Phylogenetic analysis showed that NlGRP1 、 NlGRP2 、 NlGRP3 、 NlGRP4 、 NlGRP5 、 NlGRP6 、 Nl PGRP-LB and Nl PGRP-LC were closely related to the immune recognition proteins of other Hemiptera insects,indicating that they had relatively conservative evolutionary characteristics and similar biological functions in Hemiptera insects.2.Analysis of gene expression patterns of pattern recognition receptors in the brown planthopper:q RT-PCR analysis showed that the expression of 8 immune recognition related genes in brown planthopper had obvious spatiotemporal specificity.Each gene was expressed in different developmental stages of BPH,and the average expression level was in the order of nymph stage > adult stage > egg stage.The expression level of NlGRP4 was significantly higher than that of the other five glucan recognition protein genes.The expression of Nl PGRP-LB in 5th instar nymphs and female adults was relatively low.The expression of NlGRP1、NlGRP2、NlGRP3、NlGRP4、NlGRP5 and NlGRP6 was relatively low in intestinal and ovarian tissues of BPH,but high in fat body and hemolymph of immune tissues.The expression levels of Nl PGRP-LB and Nl PGRP-LC in midgut were significantly higher than those in other tissues.The expression patterns of genes induced by different types of microorganisms are different.The expression levels of NlGRP1 、 NlGRP3 and NlGRP5 increased first and then decreased within 48 h after induction by Metarhizium anisopliae,while the expression levels of NlGRP2 、 NlGRP4 and NlGRP6 increased with the extension of induction time.The pathogenic bacteria Staphylococcus aureus(gram positive)and Escherichia coli(gram negative)could induce the expression of NlGRP1、NlGRP2、NlGRP3、NlGRP4、NlGRP5 and NlGRP6 in varying degrees.The expression of NlGRP1、NlGRP2、NlGRP3、NlGRP4、NlGRP5 and NlGRP6 was also induced by the symbiotic fungus Pichia pastoris at the initial stage(12 h),but was significantly inhibited at the later stage(48 h).The expression levels of Nl PGRP-LB and Nl PGRP-LC did not change significantly under the treatment of pathogenic fungi and symbiotic fungi,but they were significantly induced by Gram-positive bacteria.The immune response characteristics of Nl PGRP-LB and Nl PGRP-LC to gram negative bacteria were different.E.coli could induce the expression of Nl PGRP-LC,but Nl PGRP-LB had no induction effect.3.Biological function analysis of NlGRP1 and NlGRP4 genes: NlGRP1、NlGRP2 and NlGRP3 have β-1,3-glucanase domain and β-1,3-glucan recognition binding domain.Among them,NlGRP1 is expressed at a higher level during the growth and development of the brown planthopper and the gene expression is affected by microorganisms.Significantly induced.NlGRP4 、 NlGRP5 and NlGRP6 haveβ-1,3-glucanase domain but not β-1,3-glucan recognition binding domain.Among them,NlGRP4 has the highest expression during the growth and development of Nilaparvata lugens and its gene expression is affected.There are obvious differences after induction of different microorganisms.Therefore,NlGRP1 and NlGRP4 genes were selected as the research objects,and the NlGRP1 and NlGRP4 genes of Nilaparvata lugens were subjected to RNA interference by microinjection.The results showed that nlgrp1 and NlGRP4 significantly reduced the survival rate of BPH.Compared with the control(injected with dsgfp),the survival rate of BPH decreased by 30% and 47.8% on the fifth day,respectively.Interference of NlGRP1 and NlGRP4 genes will cause the brown planthopper to lay eggs significantly reduced,but the hatching rate of the brown planthopper eggs is not affected.Interference with NlGRP1 and NlGRP4 also significantly affected the expression levels of other genes in the glucan recognition protein family.For example,after 72 hours of interference with NlGRP4 gene,the expression of NlGRP1 and NlGRP3 were significantly up-regulated,while the expression of NlGRP5 was significantly down regulated.The bioassay results showed that after the interference of nlgrp1 and NlGRP4 genes,the defense ability of BPH to the pathogenic fungus Metarhizium anisopliae was significantly reduced,and the cumulative adjusted mortality of BPH on the 10 th day after microinjection of Metarhizium anisopliae was 27.8% and 40% higher than that of the control group,respectively.After the interference of NlGRP1 and NlGRP4 genes,the mortality of Pichia pastoris inoculated by microinjection was slightly higher than that of the control group,but there was no significant difference between the two groups.These results suggest that NlGRP1 and NlGRP4 genes play an important role in the growth,reproduction and immune regulation of BPH.