Molecular Characteristics And Functions Of Transcription Factors LmE75 And LmE93 Genes In Locusta Migratoria

20E(20-Hydroxyecdysone)binds to Ec R(Ecdysteroid receptor)and USP(Ultraspiracle),then reduces a series of downstream "early transcription factors" and “early-late transcription factors” to regulate the expression of downstream genes,which affect the physiological processes of molting,metamorphosis and reproduction of insects.Locusta migratoria is an important agricultural pest.In this paper,the molecular characteristics and biological functions of two transcription factors Lm E75 and Lm E93 genes are studied to provide important molecular targets for pest control.The main results are as follows:1.Analysis of the sequence and expression characteristics of Lm E75 and Lm E93 genesThe c DNA sequences of four Lm E75 genes,Lm E75 A,Lm E75 B,Lm E75 C and Lm E75 D and one Lm E93 gene were obtained based on the transcriptome database of L.migratoria.Bioinformatics analysis shows that four Lm E75 isoforms have only minor differences in the N-terminal A/B domain.Except for Lm E75 D,which does not contain DNA binding domain,Lm E75 A,Lm E75 B and Lm E75 C contain DNA binding domain and ligand binding domain.Using the neighbor-joining(NJ)method of MEGA 6.0 software,it was found that E75 isoforms of different subtypes of insects clustered first.The open reading frame of the Lm E93 gene was 3378 bp,encoding 1125 amino acids,and it contains helix-turn-helix(HTH)conserved domain.Phylogenetic tree analysis showed that Lm E93 clustered with E93 of Schistocerca gregaria.The expression patterns in different tissues and developmental stages of L.migratoria showed that: Lm E75 A,Lm E75 B and Lm E75 C are highest expressed in the integument,and Lm E75 D is highest expressed in the foregut.The four isoforms of Lm E75 were expressed in all developmental stages.The expression of Lm E75 A and Lm E75 B increased first and then decreased at the 4th and 5th instar nymphs.The expression of Lm E75 C increased gradually at both the 4th and 5th instar nymphs,with the highest expression before molting.Lm E75 D was expressed equally in the nymphal stage,and highly expressed in the adult.Lm E93 is highest expressed in ovary,followed by fat body and integument.Lm E93 expressed significantly higher in adult than nymphal stage,and higher expressed in 5th instar nymphs than other nymphal stages.20 E induction experiments in vivo showed that both Lm E75 and Lm E93 are positively regulated by 20 E.2.Functional analysis of Lm E75 gene in L.migratoriaUsing RNA interference(RNAi)technology,the function of Lm E75 during nymph-nymph and nymph-adult development was analyzed.Firstly,ds RNA of the common region of four isoforms of Lm E75 gene were synthesized.After injection of ds Lm E75 into the 4th instar nymphs,the expression of Lm E75 was significantly lower than the control group.When the control group molt into 5th instar nymphs,67.5% of the ds Lm E75-injected nymphs died before molting,and 32.5% of the nymphs arrested development during molting process and died.The HE staining of the integument found that the apolysis occur in the cuticle of ds Lm E75-injected locusts,and old cuticle degradation and new cuticle synthesis was also found.Transmission electron microscopy(TEM)results showed that the thickness of new cuticle synthesis after injection of ds Lm E75 was significantly reduced compared with the control.When ds RNA of common region of Lm E75 isoforms was injected into the 5th instar nymph,it was found that the control group molting into an adult after 7 days,the feeding of ds Lm E75-injected group significantly reduced and activity decreased than the control group after 4 days.Locusts exhibited obvious developmental delay phenotype,and remain in the 5th instar nymph developmental stage as long as 13-18 days.All the locusts could not molt to adults and eventually died.The HE staining of the integument found that the cuticle of ds Lm E75-injected locusts show no apolysis,and the old cuticle did not degrade,and no new epidermis was formed.ds RNA was synthesized corresponding to each specific region of three Lm E75 isoforms.It was found that after injection of ds Lm E75 A into the 4th instar nymphs,delayed developmental phenotype was observed,the mortality was 100%.After injection of ds Lm E75 A into the 5th instar nymphs,there were two phenotypes including died during molting and delayed development.All insects eventually died.But there were no visible phenotypes after the injection of ds Lm E75 B and ds Lm E75 C compared with the control,and they could developed normally to adults.The results indicated that Lm E75 gene plays an important role in the molting process of locusts.3.Functional analysis of Lm E93 gene in L.migratoriaUsing RNAi technology,the function of Lm E93 during nymph-nymph and nymph-adult development was analyzed.The 4th and 5th instar nymphs were injected with ds Lm E93,respectively.The expression of Lm E93 in the wings could be significantly suppressed.After the 4th instar nymphs were injected with ds RNA,compared with the control group,the ds Lm E93-injected group molted in advance.Eventually,11% of the nymphs developed to 7th instar nymphs,which were incapable of migration and reproduction.All ds Lm E93-injected locusts finally died.HE staining of the integument showed that after the 4th instar nymphs injected with ds Lm E93,the apolysis occurred in advance and old cuticle degradation and new cuticle synthesis was also found.There was no significantly different of the structure of cuticle compared with the control group.In addition,the expression of Lm E93 in the ovary was significantly inhibited after ds Lm E93 was injected into the 4th instar nymph.The development of the ovary was severely blocked,the yolk was not formed,and there was no development of ovarian tubule.After 5th instar nymphs were injected with ds Lm E93,there was no obvious in advance molting phenotype.The 6th instar nymphs could be observed,but they did not have the ability to migrate and hold.mandibles,pronotum,fore wings and hind wings were signifantly different with those in the 5th instar nymphs and adults.RT-q PCR showed that the expression of Lm Yorkie and Lm ACP19 genes related to wing development were significantly down-regulated.The results indicated that Lm E93 play a key role in the metamorphosis,reproduction and wing development of locusts.