Function Analysis Of Nuclear Receptor HR3 And ?FTZ-F1 In The Molting Of Locusta Migratoria

Locusta migratoria belongs to Insecta,Orthoptera,Acrididae.The scope of its distribution is extremely wide,involving Eurasia and Oceania,and the coastal provinces of North China and East China.At the same time,the locusts have a strong ability to migrate and jump,and their main hazards display in some cash crops such as wheat,corn,sorghum and rice etc.Therefore,the locusts are a kind of widely-spreading and extremely destructive agricultural pest in the world,which seriously threaten agricultural production and affect people's normal life.The locust is a kind of hemimetabolous insects.The metamorphosis of insects is accompanied by the occurrence of molting.Insect molting is regulated by ecdysone(20-Hydroxyecdysone,20E)and juvenile hormone(JH),which act through downstream transcription factors,mainly nuclear receptors(NR).Nuclear receptors are a large family and are widely involved in insect molting,reproduction,immunity and morphogenesis.Therefore,nuclear receptors have always been a research hotspot,and they are also new potential targets for pest control.In this paper,two nuclear receptors,HR3 and ?FTZ-F1,were obtained based on the locust transcriptome and genome database,and their roles were explored in the development of epidermis and wing.The main results are as follows:1.Sequence and Expression Characteristics of HR3 and ?FTZ-F1The c DNA sequences of Lm HR3 and Lm?FTZ-F1 were cloned and sequenced based on the transcriptome and genomic database of the Locusta migratoria.Their lengths were 1626 bp and 2196 bp,respectively,encoding 455 and 731 amino acids,and they all had typical nuclear receptor conserved domains: DBD(DNA Binding Domain)and(Ligand Binding Domain).Cluster analysis results showed that the two were clustered with Blattaria.The expression results of different tissues and stages of 5th instar nymphs showed that Lm HR3 was expressed in all tested tissues,with the highest expression level in the cuticle and reached peak on the 6th day.The expression of Lm?FTZ-F1 was high in all tissues and the expression of period showed that it reached peak on the 7th day.The expression pattern of both were consistent with the 20 E titer,suggesting that they may be induced by 20 E.Subsequently,results about 20 E induction experiments and interfering 20 E receptor gene Ec R in vivo indicated that they were positively regulated by 20 E.2.Functional analysis of Lm HR3In order to investigate the function of Lm HR3 in locusts,firstly,10 ?g ds Lm HR3 was injected into the 5th instar nymph for RNA interference,and the same amount of ds GFP of green fluorescent protein gene was injected as control,feeding normaly and observing phenotype.The results showed that all the 5th instar nymphs were unable to molt and die after silencing expression of Lm HR3.Compared with the control group,H&E and chitin staining results showed that the nymphs epidermis of the treatment group were able to undergo apolysis,but the new epidermis layer was significantly thinner.The results with transmission electron microscopy showed that the new epidermis could hardly be formed and the old could not be degraded.RT-q PCR analysis of chitin metabolism-related genes revealed that chitin synthesis(Lm UAP1 and Lm CHS1)and degradation(Lm Cht5 and Lm Cht10)genes' expression were significantly reduced compared to the control group,indicating that Lm HR3 was involved in the regulation of chitin metabolism to control the transformation of nymphs to adults.In order to further investigate the role of Lm HR3 in nymph-to-nymph transformation,silencing of Lm HR3 in early nymphs(3rd instar and 4th instar)using the same method above led to failure of normal eclosion and eventually die.Similar to the 5th instar to the adult,the expression of chitin metabolism-related genes in early nymphs was significantly down-regulated in the treatment group.The results indicated that Lm HR3 could both control the transition of nymphs to nymphs and nymphs to adult by adjusting the expression of chitin-related genes.3.Functional analysis of Lm?FTZ-F1Using the RNAi method described above,the ds RNA of Lm?FTZ-F1 was injected into the body cavity of 5th instar nymphs to knock down its expression.Compared with the control group,the results showed that the 5th instar nymphs of treatment group exhibited a measure of delayed development.Although most of them eventually became adults,the adults body presented wrinkled shape and wing.The results also revealed that delayed apolysis of the treatment group's wing pads by observing the development of 5th instar nymph wing pads with H&E staining.Through observation of transmission electron microscopy we could find that the cell structure in the dermal cell layer was destroyed in both the epidermis and the fore wings of the treatment insects.Similarly,the expression of Lm?FTZ-F1 was silenced in early nymphs(4th instar nymph)and the phenotype of treatment insects being adults similar to those in the fifth instar nymphs.To illustrate the mechanism of Lm?FTZ-F1 in locusts,the expression of Lm?FTZ-F1 was slienced in the second day of the 5th instar nymphs and the total RNA was extracted from the wing pads of the control and treatment groups after 48 h for transcriptome sequencing analysis.A total of 19142 unigenes with 177 differentially expressed genes were obtained,of which 96 were up-regulated and 81 were down-regulated.Further analysis found that chitin metabolism-related genes,cuticle protein genes of wing-specific and carboxypeptidase metabolism-related genes(related to apolysis)were included.The results of RT-q PCR displayed that the expression of carboxypeptidase metabolism-related genes was significantly down-regulated compared to the control group.Hence it was speculated that Lm?FTZ-F1 may be involved in the regulation of carboxypeptidase metabolism to control the occurrence of ecdysis in Locusta migratoria.