| The brown planthopper(BPH),Nilaparvata lugens(St(?)l),is an important pest of rice in China and Southeast Asia.It has a short life cycle and strong reproductive capacity,which is likely to cause large-scale outbreaks and bring huge losses to rice production.The main influencing factors of BPH reproductive capacity are the external environment,reproductive related genes and non-coding RNA.Among the non-coding RNAs,piRNA is a kind of non-coding small molecular RNA with a length of 24-32 nt,which mainly exists in animal germ cells.In the process of animal germ cell formation,piRNA forms a silencing complex with PIWI subfamily members,which has an inhibitory effect on transposon and plays an important role in protecting the integrity of germ line genome.There are several cofactors that play an important role in piRNA’s generation and silencing,except members of the PIWI subfamily.Mutations or deletions of these genes will have an impact on the reproductive system of animal.Therefore,it is of great significance to study the PIWI subfamily members and cofactor genes of BPH.It also provides new ideas for the prevention and control of BPH.The annotated genome information of BPH showed that the members of PIWI subfamily contained three genes(Ago3,Piwi and Aub)which the specific functions were still unclear.To this end,this study used two member genes of the PIWI subfamily of BPH(named Nl Ago3 and Nl Siwi)and a cofactor gene(named Nl YC)as the research object.We cloned these three key genes and analyzed their gene expression patterns in the development process and different tissues,and studied the function of the genes with RNAi technology.The role of these three genes in the reproduction of BPH was discussed.The aim is to lay a foundation for the study of piRNA pathway in BPH,and to form a theoretical basis for the study of new insect resistance strategies.In this study,we chose three key genes Nl Ago3,Nl Siwi and Nl YC from BPH genome and transcriptome.The open reading frame(ORF)sequences were obtained respectively: The ORF length of Nl Ago3 is 2657 bp,which is potential to encode a predicted 100.16 k Da protein containing 885 amino acid residues.the theoretical isoelectric point of Nl Ago3 protein is 9.47.The ORF length of Nl Siwi is 3248 bp,encoding a protein of 1082 amino acids with a expected molecular weight of 121.83 k Da and a theoretical isoelectric point of 9.84.The ORF of Nl YC is 2744 bp and encodes 914 amino acids.The predicted molecular weight is 103.07 k Da and the theoretical isoelectric point is 4.89.Protein prediction results indicate that both Nl Ago3 and Nl Siwi proteins contain conserved PAZ and piwi domains.Phylogenetic tree analysis showed that these two proteins were clustered with PIWI subfamilies of other animals.The Nl YC gene contains three Tudor domains and belongs to the TUDOR protein family.First,the spatiotemporal expression pattern analysis showed that the three genes were expressed in all developmental stages of BPH,and the expression level of nymphs at 1-5 instars was lower than that in the adult stage.These three genes were also expressed in various tissues of BPH.The highest expression of Nl Ago3 was found in antennae,fat bodies and ovaries,followed by foot and salivary glands,and the lowest in testis.Nl Siwi was also the highest expression level in the antennae,fat bodies and ovaries,followed by testis and foot,and the expression level of salivary gland was the lowest;Nl YC was different from both of them.Nl YC has the highest expression in the antennae,followed by fat bodies,and the expression levels in the four tissues of the foot,salivary glands,testis and ovaries are not much different.Secondly,the functions of Nl Ago3,Nl Siwi and Nl YC genes were verified by microinjection.Within the first three days after injection,the survival rate of BPH in the treatment group and the control group decreased sharply,then the survival rate gradually stabilized and finally reached more than 60%.The expression level of Nl Ago3 was significantly decreased on the third day after silencing,which was 25 times higher than that of the control group.The single-pair mating test showed that the rate of nymph emergence was 50%in the control group,and no nymph hatched after Nl Ago3 silence.After the silencing of Nl Siwi,the expression level was significantly reduced,22.6 times as much as that of the control group on the first day.On the third day,the expression level increased,but it was still 6.3 times lower than the control group.After mating,the emergence rate of the control group was 40%,and Nl Siwi was incubated without nymphs.After silencing of Nl YC,the expression level was 5.6 times that of the control group,and the insect emergence rate was 30% in the control group.No nymph hatched in the treatment group.Above all,the silencing of Nl Ago3,Nl Siwi and Nl YC genes affects the reproductive capacity of BPH,which indicates that members of the PIWI subfamily and cofactors may affect the piRNA production pathway,thus affecting the formation of reproductive cells,piRNA pathway affects the gamete formation of BPH,which can also be used as a potential target gene for the control of BPH. |
