| Cuticle coveres the whole body of insects,and plays a vital role for insect development.Chitin is the main component of the cuticle,and it is orderly organized into the dedicated cuticle architecture.Since chitin is absent in the human and higher mammal,the cuticle is recognized as a safe target for pest management.Chitin organization is very important for maintaining the integrity of cuticle architecture.The screening and functional analysis of key genes involved in chitin organization will uncover the molecular mechanism of cuticle formation,and provide the theoretical basis for discovering novel molecular target of pest control.In this thesis,we selected the Locusta migratoria as model insect,and studied the biological functions of three kinds of genes potentially responsible for the chitin organization.The main contents are as follows: 1.Identification and molecular characterization of chitin deacetylase(CDAs)The cDNA sequences of CDAs were obtained from the transcriptome of L.migratoria by bioinforamtic analysis,the full-length cDNAs were amplified and sequenced for further confirmation.The domains of CDAs were analyzed and phylogenetic tree was constructed.Four full-length cDNAs of CDAs were obtained,named LmCDA1,LmCDA2,LmCDA4 and LmCDA5,respectively.LmCDA2 and LmCDA5 have two alternative splicing,named LmCDA2 a and LmCDA2 b,LmCDA5a and LmCDA5 b.Reverse transcription-quantitative PCR(RT-qPCR)was applied to reveal the molecular characterization of LmCDAs,tissue expression patterns showed that LmCDA1 and LmCDA4 have the highest expression level in the foregut,and a relatively lower transcripts in the integument and hindgut.LmCDA2 a and LmCDA2 b are higher expressed in the foregut than other tissues.LmCDA5 have the highest expression in the foregut,and higher expression in the integument,hindgut and fat body.Expression patterns from integument of different days during 5th instar nymph showed that LmCDAs highly expressed in the 1st and 2nd day,decreased in following days,then arised before molting.The results suggested that LmCDAs might play an important role during the molting.2.The biological function of chitin deacetylase(CDAs)RNA interference(RNAi)was performed to study the biological function of CDAs.The result showed that the target genes were specifically and efficiently silenced after injection of dsRNA,and insects were difficult to split the old cuticle and finally died when dsLmCDA1,dsLmCDA2 and ds LmCDA2 a were injected into the L.migratoria,respectively.However,locusts could molt to the next stages after injection with dsLmCDA2 b,ds LmCDA4 and ds LmCDA5,respectively.Hematoxylin-eosin(HE)staining after injection of dsLmCDA1 and ds LmCDA2 showed locusts also undergo the process of apolysis,new cuticle synthesis and thickening,old cuticle degradation and thinning,which were similar to the control.Immunohistochemical results showed that the LmCDA2 localized in the apical site of the procuticle,and LmCDA1 localized in both epidermal cell and the exocuticle.Chitosan staining indicated that the contents of chitosan deacetylation degree was significantly decreased when injection of ds LmCDA1.However,compared with the control,the chitosan deacetylation degree was not changed with dsLmCDA2 injection.The results suggested LmCDA1 plays a key role in chitin deacetylation.Transmission electron microscope observation showed that the chitin lamina structures also existed and cuticle became thin after injection of ds LmCDA1,while the chitin laminae disappeared and cuticle became thick with dsLmCDA2 injection.When double silence of LmCDA1 and LmCDA2,chitin laminae were absent and the thickness of cuticle was between single injection of ds LmCDA1 or ds LmCDA2.The results suggested that the LmCDA2 is responsible for cuticle chitin organization.LmCDA1 and LmCDA2 play an important role in maintenance of cuticle thickness.Chitin content detection showed that the chitin content significantly reduced after ds LmCDA1 injection,while there is no influence on chitin content after ds LmCDA2 injection.Double silence of LmCDA1 and LmCDA2,chitin content significantly decreased.The results suggested that the function of LmCDA1 and LmCDA2 are differentiated.3.The molecular characterization and biological function of Knickkopf(Knk)The full-length cDNA of LmKnk was identified after searching from the transcriptome of L.migratoria.Domain analysis showed that the LmKnk contained three conserved functional domains.Phylogenetic tree analysis showed that LmKnk is closely grouped with Knks from other insects,named LmKnk.RT-qPCR was performed to study the molecular characterization of LmKnk.Tissue expression pattern showed that LmKnk has the highest expression level in the foregut,and a higher expression in the integument.Expression pattern from integument of different days during 5th instar nymph showed that LmKnk expressed during the whole stages.The results showed that LmKnk might play an important role during whole 5th instar nymphs.RNAi technology was applied to study biological function of LmKnk.The gene silencing efficiency was detected and phenotype was observed after injection of the dsLmKnk into the L.migratoria.The results showed that ds LmKnk enable to silence the target gene efficiently.Locusts failed to molt and finally died during the nymph-nymph and nymph-adult molting.HE staining showed that locusts go through the apolysis,old cuticle degradation,however,the new synthesized cuticle is significantly thinner than the cuticle of control.Chitin staining was performed to observe the chitin content in the cuticle,results showed that chitin content dropped significantly with ds LmKnk injection.Transmission electron microscope observation showed that when injection of dsLmKnk the chitin laminae disappeared,cuticle became thin and pore canal structure was abnormal.Chitin content detection of cuticle showed that the chitin content decreased after injection of dsLmKnk.Immunohistochemical observation indicated that LmKnk original synthesized in the epidermal cell,and transported into the newly synthesized cuticle when molting,which suggested it might participate in protection of new cuticle.4.The molecular characterization and biological function of Retroactive(Rtv)The full-length cDNA of LmRtv was obtained with validation after sequence searching from the transcriptome of L.migratoria.Expressions of LmRtv were detected in different tissues and integument of different days,and the results showed that LmRtv has the highest expression in the foregut and 1st,2nd days of 5th instar nymph.Locusts were difficult to molt to next instar and finally died after dsLmRtv injection.HE staining showed that the locusts still go through apolysis,old cuticle degradation,but the new produced cuticle significantly decreased.Transmission electron microscope observation showed that chitin laminae in the new cuticle disappeared,pore canal become swelling and abnormal,and the thickness of cuticle became thinner when compared with control.Chitin content detection results showed that chitin content decreased after injection of dsLmRtv.In this thesis,the locusts were difficult to molt and finally died after injection of ds LmCDA1,ds LmCDA2,dsLmKnk and ds LmRtv,and it is difficult to distinguish differences because of similar phenotypes.The microscopic and ultrastructure technologies were performed to extensively explore the reason of death.The results showed that the LmCDA1 localized in the epidermal cell and exocuticle and responsible for the chitin deacetylase and maintain the balance of chitin and chitosan.LmCDA2 localized in the apical site of epicuticle,and was required for the formation of chitin laminae.LmKnk localized in the new cuticle and participated in deposition and organization of chitin content.However,the localization of LmRtv is not clear due to lack of antibody,the function of LmRtv could be similar with that of LmKnk,and responsible for the chitin deposition and chitin lamina maintenance.In this thesis,the biological function of genes involved in chitin organization were investigated systematacially,and the research work would enrich the contents of chitin architecture of insect cuticle.The obtained key genes such as LmCDA1,LmCDA2,LmKnk and LmRtv could provide the potential molecular targets for the pest control. |
