Research On The Function And Mechanism Of The Epidermal Protein Genes TcCP65, TcCPR69 And TcCPR22 In The Red Rice Thief

The insect cuticle plays an important role in the growth and development of insects and the defense against the outside world,and the insect cuticle is mainly composed of epicuticle,exocuticle,endocuticle and epidermis.One of the main components of the exocuticle and endocuticle is cuticle protein,indicating that cuticle protein plays an important role in the formation of insect cuticle.At present,the functional studies of cuticular proteins mainly focus on the order Lepidoptera and Orthoptera,such as silkworm,migratory locust and brown planthopper etc.However,there are few studies on Coleoptera.Among them,the research on the cuticle protein mainly focuses on the CPR-RR2 family and its effect on elytra in Tribolium castaneum.while little is known about the functions of other family members.T.castaneum is one of the most harmful grain storage pests in the world,and it has been used as a good material for studying gene function because of its highly efficient systematic RNA interference mechanism.Therefore,this experiment takes the T.castaneum as the research object,in order to better understand the cuticle protein function of coleopteran insects,and then provide a theoretical basis for the biological control of such pests.In order to comprehensively understand the conservation of the various family members of the T.castaneum,we performed a cluster analysis on all the cuticle proteins identified by the T.castaneum,and the results showed that most of the family members of the cuticle protein are relatively conservative.Therefore,three cuticular proteins,TcCP65 gene from the unclassified family,and TcCPR69 and TcCPR22 gene from the CPR-RR1 family,which have high expression in the hindwings of T.castaneum were selected as the target genes of the study,in order to explore the biological functions of different epidermal proteins in different families of Epilepto raectorum.Firstly,the ORF sequences of TcCP65,TcCPR69 and TcCPR22 were obtained,and the structure and protein sequences of TcCP65,TcCPR69 and TcCPR22 were analyzed.Protein sequence analysis showed that TcCP65 did not contain chitin binding domains,while TcCPR69 and TcCPR22 contained chitin binding domains,suggesting that the biological functions of these three cuticle proteins were different to some degree.Then,in order to explore the biological functions of the three cuticle proteins,by analysis of expression patterns in different periods and tissues showed that TcCP65,TcCPR69 and TcCPR22 were all highly expressed in late pupae,and tissue expression analysis of the three genes were all highly expressed on the hidwing.But it also showed some differential expression,TcCP65 was expressed in the ovary at a high level,and TcCPR69 was also expressed at a high level in the gut.This suggests that TcCP65,a member of the unclassified family,is involved in reproduction,while TcCPR69,a member of the CPR-RR1 family,is involved in gut formation.Finally,in order to further explore the biological effect of TcCP65,TcCPR69 and TcCPR22,RNAi interference showed that TcCPR69 not only affected the pupation and eclosion of the beetles,but also affected the peritrophic membrane formation in the midgut of the adults.TcCPR22 mainly affect the normal eclosion of T.castaneum in pupal stage,while TcCP65 had no effect on the abnormal development of the beetle.Although the three cuticular proteins were highly expressed in the hindwings of adults,they had no effect on the hindwing morphology of adults.To further investigate whether TcCP65,TcCPR69 and TcCPR22 are involved in the formation of epidermis and their related mechanisms.we performed HE paraffin section observation on the pupal stage samples.The results showed that ds TcCP65 had no effect on the thickness of new epidermis in late pupa,but ds TcCPR69 and ds TcCPR22 group compared with the control group,the new abdomen cuticle in the late pupal stage was significantly thinner.Moreover,DAPI was used to stain the new epidermal cells,and it was found that the nucleus of the new epidermal cells in the experimental group was significantly abnormal compared with the control group.In addition,through the chitin binding experiment,this article further analyzed whether TcCPR69 and TcCPR22 participate in the process of epidermis formation by regulating the chitin metabolism pathway.First,three cuticular proteins prokaryotic proteins were expressed in vitro.Taking TcCPR69 as an example,the experimental results verified that proteins containing the conserved functional domain of chitin could bind to chitin.Meanwhile,interference with TcCPR69 and TcCPR22 significantly down-regulated the expression of key genes in the chitin pathway,leading to a significant decrease in the content of chitin in cuticle.Ecdysis hormone is very important for regulating the metamorphosis and ecdysis of insects.Cuticle proteins are one of the main components of the cuticle.Are the three cuticle proteins regulated by 20E? The results showed that TcCP65 was not regulated by the 20 E pathway,while TcCPR69 and TcCPR22 were positively regulated by the 20 E pathway.According to the above research results,epidermal proteins TcCPR69 and TcCPR22 are regulated by 20 E,and the interference will affect the expression of key genes in the chitin metabolism pathway and the formation of chitin,thus affecting the new cuticle formation of late pupae,leading to the failure of emergence of.According to the above research results,cuticle proteins TcCPR69 and TcCPR22 are regulated by 20 E,and the interference will affect the expression of key genes in the chitin metabolism pathway and the formation of chitin,thus affecting the new cuticle formation of late pupae,leading to the failure of emergence of T.castaneum.Although the TcCP65 did not affect the metaplasia development of,but it’s could affect the development of the female ovary and embryonic of T.castaneum.Compared with the control group,the number of female eggs was reduced and the eggs cannot be hatched into larvae.The expression of TcCP65 in embryo indicated that the expression of TcCP65 was high express during the 8-24 and the 34-72 h.DAPI staining is used to observe the embryonic development at various stages.TcCP65 mainly plays an important role in the appendage formation stage of embryonic development.