Molecular Characteristics And Functions Of Key Genes Involved In Molting In Locusta Migratoria

The ecdysis and metamorphosis are jointly regulated by 20E(20-hydroxyecdysone)and JH(Juvenile hormone)in insects.20 E binds to ecdysteroid receptor and Ultraspiracle protein(USP in holometabolous insects),which is called RXR(Retinoid X receptor)in hemimetabolous insects.Furthermore,it regulates the expression of a series of downstream transcription factors,thus regulating physiological processes such as ecdysis and metamorphosis.Nucleation of Ec R-B1,a subtype of Ec R,requires the involvement of the GTPase Ran and Nuclear Transport Factor 2(NTF2).Phosphoacetylglucosamine mutase(PAGM)is one of the key enzymes in the synthesis of chitin.In this paper,the molecular characteristics and biological functions of four genes(Lm RXR,Lm Ran,Lm NTF2 and Lm PAGM)in ecdysis and metamorphosis were studied in Locusta migratoria.The research results are as follows:1)The full-length ORF c DNA sequences of Lm Ran and Lm NTF2 genes were obtained by searching the L.migratoria transcriptome database,which encode 215 and 130 amino acids,respectively.RT-q PCR was used to analyze the m RNA expression of these two genes in different tissues and different developmental stages.The results showed that Lm Ran highly expressed in foregut,wing,testisand ovary,and was expressed in different stages.Lm NTF2 was most highly expressed on the first day of the fifth nymph of L.migratoria,and was highly expressed in gastric caecum,midgut and hindgut.The functions of these two genes were studied with RNAi technology.The result showed that after ds Lm Ran injection at 4th and 5th stages,the expression of Lm Ran in ds Lm Ran-injected locusts was significantly decreased compared with that in the control group,and all died before molting,indicating that Lm Ran gene played an important role in the metamorphosis of L.migratoria nymph to nymph molting and nymph to adult molting.After the silencing of Lm NTF2,both the experimental group and the control group were able to develop into adults.Further section histological analysis of the integument was carried out.H&E staining results showed that there was no degradation of old cuticle or formation of new cuticle after silencing Lm Ran.In addition,after silencing Lm Ran,the weight of L.migratoria was significantly lower than that of the control group,and anatomy showed that the gastriccaecum of L.migratoria was severely atrophic in the ds Lm Ran group.RT-q PCR was used to analyze the expressions of CYP family genes,20 E signaling pathway genes and key genes involved in chitin metabolism,and it was found that after silencing Lm Ran for 48 hours,the expressions of Lm CYP302a1,Lm CYP315a1 and Lm CYP314a1 of CYP family genes involved in 20 E synthesis and Lm Gfat and Lm CHT10 of key genes involved in chitin metabolism were significantly down regulated.2)According to the Lm RXR-L and Lm RXR-S c DNA sequences published in NCBI(Gen Bank number: AAQ55293.1 and AAF00981.1),specific PCR amplification primers were designed for Lm RXR-L and Lm RXR-S.RT-q PCR was used to study m RNA expression characteristics,and the results show that the Lm RXR-L and Lm RXR-S are highly expressed in testis and ovary,followed by integument,foregut,midgut,gastric caecum,hidgut,fat body,malpighian tube and wing.Both Lm RXR-L and Lm RXR-S were highly expressed at the second and third day of the fifth stages.The function of Lm RXR gene was studied by RNAi technology,and the results showed that after ds Lm RXR injection at the fourth and fifth stages,the expression of Lm RXR in the experimental group was significantly decreased compared with that in the control group,and 90% of the locusts were finally unable to successfully molting and died,and the remaining 10% of the locusts died before molting without developmental delay.However,there is no remarkably difference in the histological structure of cuticle between ds Lm RXR injected nymphs and control.The results of transmission electron microscope and chitin contents showed that there was no significant change in the chitin lamellar structure and chitin contents of the old cuticle and the new cuticle of the experimental group compared with that of the control group.The weight of ds Lm RXR-injected group was lower than the control group,and the gastric caecum was severely atrophic.RT-q PCR was used to analyze the expression of CYP family genes,20 E signaling pathway genes and key genes of chitin metabolism.The results showed that after silencing Lm RXR for 48 hours,the expression of all genes was not affected except for Lm CYP302a1,which is down regulated and Lm FTZ-F1,which is up regulated.3)The full-length ORF c DNA sequence of Lm PAGM was obtained by analyzingthe transcriptome data of L.migratoria,which encode 497 amino acids.RT-q PCR was used to analyze the expression of Lm PAGM in different tissues and different days of the fifth stages.The results showed that Lm PAGM was expressed in integument,foregut,midgut,hindgut,gastric caecum,malpighian tube,fat body and wing,and the expression increased significantly on the eighth day of the fifth stage.The biological function of Lm PAGM was explored by RNAi.The expression of ds Lm PAGM-injected group was significantly down regulated compared with the control group,and about30% of locust could not successfully molt into adults.The results of this study lay a foundation for further understanding the molecular regulatory mechanism of migratory locust molt development,and provide new molecular targets for locust control.