Molecular Characterization And Functional Analysis Of The PIWI Subfamily And Prmt5 Genes In Locusta Migratoria

Locusta migratoria is a worldwide agricultural pest.Currently,the chemical control methods used have brought a series of safety issues.Sterile insect technology has the advantages of species specificity and environmental friendliness,and has broad application prospects in pest control.Therefore,the study of insect fertility has important theoretical and practical significance.PIWI subfamily proteins is conserved in animals and associated with pi RNA in gonads to regulate transposon silencing,maintain genome integrity,and preserve normal gametogenesis and reproduction.In this pathway,PRMT5 is required to N-terminal arginine methylation of PIWI subfamily proteins to ensure the normal function of PIWI subfamily proteins.In this paper,the molecular characteristics,expression characteristics and functions of Lm PIWI genes and their methylation gene Lmprmt5 in Locusta migratoria were studied.The main results are as follows:1.Molecular characterization of Lm PIWI subfamily genesBased on the Locusta migratoria transcriptome databases,we identified and characterized the PIWI subfamily genes,Lmpiwi1,Lmpiwi2 and Lmago3.Lm Piwi1 and Lm Piwi2 belong to the Piwi/Aub clade,and Lm Ago3 belongs to the Ago3 clade.Gene structure analysis showed that Lmpiwi1,Lmpiwi2 and Lmago3 contained 16,15 and 17 exons,respectively.Analysis of the protein conserved domains showed that all three genes contain all typical PIWI subfamily proteins conserved domains: N-terminal domain,PAZ domain,MID domain and PIWI domain.In eukaryotes,the last leucine(L)at the C-terminus of the PIWI subfamily proteins is conserved.It is located in the channel formed by the MID and PIWI domains where it might associate a Magnesium ion(Mg2+)for 5' end of pi RNA recognition and binding.2.Expression analysis of Lm PIWI subfamilyThe expression characteristics of PIWI subfamily genes in Locusta migratoria were analyzed.At different developmental stages,Lmpiwi1 expressed the highest in eggs and fifth instar nymphs,Lmpiwi2 reached the highest expression in fifth instar nymphs and adults,and Lmago3 expressed at equivalent levels in each development stage.In different tissues,Lmpiwi1 was highest expressed in testes and ovaries,Lmpiwi2 was highest expressed in testes,and Lmago3 was highest expressed in ovaries.The expression of Lm Piwi1 protein was basically consistent with the m RNA expression trend,with the highest expression in the testis.3.Functional analysis of Lm PIWI subfamily genesThe RNAi technology was used to study the function of the Lm PIWI genes.Detection of the silencing efficiency showed that Lmpiwi1,Lmpiwi2 and Lmago3 could not be silenced in the ovary,while the silencing efficiency in the testis was 92.72%,86.16% and 90.85%.However,these ds Lm PIWI-treated locusts did not have any obvious defects during spermatogenesis,and the testicular morphology,sperm cell morphology,and hatchability were normal.We found that a lower survival rate in the ds Lmpiwi1-treated nymphs,and the mechanism is still unclear.4.Molecular characterization and functional analysis of Lmprmt5Sequence analysis showed that the N-terminus of Lm PIWI subfamily proteins has a motif that can be catalyzed by PRMT5.In this study,the molecular characteristics of Lmprmt5 gene were identified and analyzed.Analysis of expression characteristics showed that Lmprmt5 was expressed at high levels in both testis and ovary,and the most highly expressed stages was the 5th instar nymph.Using RNAi technology to study the function of Lmprmt5,it was found that there was no abnormality in spermatogenesis of knock down Lmprmt5.In summary,here we analyzed the molecular characteristics and expression characteristics of the PIWI subfamily genes and prmt5 gene in Locusta migratoria,and used RNAi technology to study the functions of these genes.These genes can't be silenced in the ovary,but are significantly silenced in the testis.There is no abnormality in testis development and spermatogenesis.However,Lmpiwi1 plays a role in maintaining the longevity or survival of locusts.These data provide basic information for subsequent studies on PIWI/pi RNA pathway functions and molecular mechanisms in Locusta migratoria.To provide a reference for the biological significance of RNAi mechanism in reproductive system.