Functional Analysis Of DEAD-box Family Genes In Locusta Migratoria

DEAD-box proteins are ATP-dependent RNA helicases,which exist ubiquitously in bacteria,fungi,animals and plants,and are involved in almost every step of RNA metabolism.DEAD-box proteins play an indispensable role in the growth and development of organisms.However,except in Drosophila,it has not been much studied on the functions of these proteins in insects.In this study,we analyzed the expression patterns of the DEAD-box genes at development stages and in various tissues of Locusta migratoria.We also explored the functions of these DEAD-box genes in the growth and reproductive development of L.migratoria using RNAi method.The results from this study lay a foundation for further comprehensive functional analysis of the DEAD-box genes in L.migratoria,and provide also new target genes for pest biological control.Here we focus on the expressions and biological functions of the DEAD-box genes(DDXs)in L.migratoria and the main results are as follows:1.Molecular characterization of 32 LmDDXsAccording to the DDXs sequences from human and fly,we obtained 32 LmDDXs by blasting transcriptome databases.LmDDXs were named based on their orthologs in human.The sizes of LmDDXs sequences vary from 840 to 2502 bp.26 LmDDXs own full-length CDS sequences,which contained 9 specific conserved motifs of DDXs.Most of LmDDXs,except genes with partial sequences,contained DEXDc and HELICc domains,and some LmDDXs with specific domains at their N-,C-terminal regions,like DUF4217,Zn?C2HC,KH and DBP10 CT domains.The gene structures of LmDDXs vary with different exon numbers.For instance,there is only one exon in LmDDX23 and LmDDX41,respectively,however,fifteen in LmDDX27.Phylogenetic tree showed that almost all LmDDXs have orthologous genes in other examined insects,suggesting that DDXs are highly conserved in insects.Furthermore,most LmDDXs own orthologs in human as well,implying that DDXs are conserved in animal evolution.2.mRNA expression and functional analysis of LmDDXsWe performed RT-qPCR at various developmental stages and in different tissues of locust to examine the transcription activities of 32 LmDDXs.The results showed the expression patterns of LmDDXs during whole developmental processes from egg to adult.Most of LmDDXs exhibited a lower expression in egg stage and a relative higher level at second(2nd)and third(3rd)instar nymphs.Some LmDDXs,like LmDDX4,LmDDX49,LmDDX52,were expressed at relatively constant level at all stages.The tissue-specific expression patterns showed that most LmDDXs were highly expressed in both testis and ovary with a relative lower expression in other tissues,including integument,fat body,foregut,midgut,gastric caecum and malpighian tubule.We further analyzed the biological functions of 32 LmDDXs members by RNAi technology.Silence efficiency of these LmDDXs varies from 52% to 98% at 3rd nymph locusts.RNAi analysis informed that seven genes were essential for locust survival,among which LmDDX18,LmDDX39,LmDDX41,LmDDX47 and Lme IF4 A played an important role in maintaining normal gut and gastric caecum.These data demonstrate that LmDDXs are essential for gut development in L.migratoria.3.Effects of LmDDX47 on gut development of L.migratoriaTo understand the effects of LmDDXs on gut development of the locust,we chose LmDDX47 as a case.To verify whether LmDDX47 function is stage specific,we performed RNAi at 5th nymph and adult locust.The experiment results showed that LmDDX47 is indispensable for maintenance of the normal midgut and gastric caecum at all these developmental stages.Furthermore,using H&E staining it was revealed that the number of midgut epithelial cells and intestinal stem cells was significantly reduced after knockdown of LmDDX47.It was also found that knockdown of LmDDX47 increased apoptosis of the intestinal epithelial cells and reduced the proliferation ability of intestinal stem cells,most mitochondria were enlarged and distributed throughout the cell in the gut.To better understand the mechanism of LmDDX47 in gut development and maintenance,we constructed p IEx4-LmDDX47-GFP plasmid and transfected S2 cells.The results showed that LmDDX47 was located in the nucleolus,suggesting that LmDDX47 is involved in the synthesis of ribosomes.This is consistent with reduced synthesis of 18 S r RNA in the LmDDX47 knockdown locust.In addition,knockdown of LmDDX47 enhanced the levels of acetylation and crotonylation of some proteins in the midgut.4.Effects of LmDDXs on the reproductive development of L.migratoriaOur experimental data showed that LmDDXs had high expression in testis and ovary,implying their functions in the reproductive development of locust.To our surprise,we found no obvious defects in testis development of LmDDXs knockdowns.We thus focus on the effects of these LmDDXs in locust ovary development.By RNAi analysis,we found that LmDDX3,LmDDX6,LmDDX18,LmDDX39,LmDDX41,LmDDX47 and Lme IF4 A are required for ovary development and oocyte maturation.LmDDX3 and LmDDX6 were further investigated,and it was revealed that both LmDDX3 and LmDDX6 knockdowns decreased the expression of LmVg and JH signal pathway genes,and resulted in impaired ovary development and oocyte maturation.However,there are still some differences between the functions of LmDDX3 and LmDDX6.For example,down-regulation of LmDDX3 increased the expression of LmVg R,and while down-regulation of LmDDX6 reduced the transcripts of LmMet and LmGrp78-2.This suggests that LmDDXs have different functional mechanisms in the ovary deveoplemnt of L.migratoria.In summary,we obtained 32 DEAD-box family genes from the genome of L.migratoria by bioinformatics and analyzed their gene and protein structures.RT-qPCR analysis revealed the expression patterns of these LmDDXs in different developmental stages and tissues.Seven LmDDXs genes were found to be essential for the growth of locust by RNAi method.Furthermore,we explored the effects of LmDDX47 on the gut development,LmDDX3 and LmDDX6 on ovary development in details.Taken together,these data have thus revealed the biological functions of some LmDDXs in the growth and development of L.migratoria,laying a foundation for further investigation of other LmDDXs in locust,and providing new targets for pest biological control as well.