| Cuticle is essential for insects growth and development.As the main component of insect cuticle,chitin and protein interact with each other in the process of cuticle formation and finally make the cuticle appear densely and orderly layered structure.The well arrangement of chitin in cuticle is very important for the integrity of cuticle structure and the maintenance of cuticle function.Changes in cuticle structure and composition may affect the insects sensitivity to environmental stress.There is no chitin in humans and higher animals,insect cuticle is seen as a safe target for pest control.Our research group has been devoted to the identification and functional analysis of key genes involved in chitin metabolism and lamellar arrangement of locust,that is one of the most important agricultural pests.In this article,we chose migratory locust(Locusta migratoria)as experimental materials,to further explore the biological functions of LmKnk2 and LmKnk3 in cuticle formation of locust,the transcriptional regulation of Knickkopf family gene and its role in response of locusts to environmental stress.LmKnk2 and LmKnk3 are two members of Knickkopf family genes which involved in lamellar arrangement of insects cuticle.The results will make great scientific significance for elucidating the molecular mechanism of chitin assembly in locust cuticle and the discovery of new molecular targets for pest control.The main research contents and findings are as follows:1.Validation and molecular characterization analysis of LmKnk2 and LmKnk3LmKnk2 and LmKnk3 were got from several transcriptome databases of Locusta migratoria by bioinformatic method,and there were two selective transcripts of LmKnk3,named as LmKnk3-FL and LmKnk3-5' respectively.First,we cloned the full length cDNA sequences of these 3 genes by PCR,their open reading frames were 2112 bp,4932 bp and 1434 bp,encoding 703,1643 and 477 amino acids,respectively.Then we compared their full-length cDNA sequences with the locust genome to map their gene structure,and analyzed their amino acid sequences by SMART online software.Finally we aligned LmKnk2,LmKnk3-FL,LmKnk3-5' with their homologous amino acid sequences from different insects to construct the phylogenetic tree.The expression of LmKnk2,LmKnk3-FL and LmKnk3-5' were tested in 9different tissues of the fifth-instar nymphs of L.migratoria by RT-qPCR,such as integument,foregut,midgut,hindgut,gastric caeca,Malpighian tubules,fat body,wing pad and trachea.The results showed that LmKnk2 was highly expressed in wing pad,both LmKnk3-FL and LmKnk3-5' were predominantly expressed in the integument.The same method was used to test their expression in different developmental stage of fifth-instar nymphs.The results showed that all the three genes were highly expressed before molting.2.Biological function analysis of LmKnk2 and LmKnk3The biological function analysis of LmKnk2 and LmKnk3 were performed by RNAi combined with phenotype observation,chitin staining and TEM.First,RNAi and phenotype observation were performed.The results showed that the expression of target genes could be significantly silenced at the mRNA level compared with the dsGFP group,However,only the fifth-instar nymphs injected with dsLmKnk3-5' emerged visible phenotype,about ?30%of the nymphs were unable to successfully molt(lethal phenotype),?60% can shed their exuviae successfully and became adults but with wrinkled wings.Then chitin staining,chitin content test and TEM analysis were performed after dsRNA injection,the results showed that there were no significant differences in the cuticle thickness,the helicoidal organization and the chitin content of the nymphs injected with either dsLmKnk2 or dsLmKnk3-FL as compared with those of the control nymphs injected with dsGFP,while after the injection of dsLmKnk3-5',both the cuticle thickness and the chitin content of the nymphs were significantly decreased as compared with the control nymphs,but the helicoidal organization of the newly formed procuticle was the same as that of the dsGFP-injected nymphs.Finally,Bodipy staining were used to study the lipid deposition of cuticle after dsRNA injection.Compared with the control group,the lipidscannot be easily detected in the dsLmKnk3-5'-injected insects.3.Preparation of LmKnk3-5' polyclonal antibodyWe first constructed prokaryotic expression vector and transformed them into E.coli to express LmKnk3-5' recombinant fusion protein and then purified the protein,finally prepared mice-derived polyclonal antibody by using LmKnk3-5' recombinant fusion protein to immunize mice.The results showed that the titer of LmKnk3-5' polyclonal antibody was good for the next experiments.The specificity of LmKnk3-5' polyclonal antibody was well after western blot test and we found that the expression of LmKnk3-5'was low in cuticle.We used immunohistochemical to locate LmKnk3-5'protein in locust and found that it could be located in the epidermal cells and cuticle in L.migratoria.4.Transcriptional regulation research of Knickkopf family gene in L.migratoriaFifth-instar nymphs were used as experimental materials to test whether the expression of Knickkopf family gene in L.migratoria was regulated by20 E.The results showed that 20 E could up regulate the expression of four Knickkopf genes,and after dsLmEcR injection,the expression of the four Knickkopf genes were significantly reduced.5.Effect of Knickkopf family gene on the response of locust to environmental stressThird-instar nymphs were used as experimental materials to test the sensitivity of locust to pesticide,high temperature and dry environment after the expression of Knickkopf family gene was reduced by RNAi.The results showed that after dsLmKnk,dsLmKnk3-5' injection,the sensitivity of locust to environmental stress was significant increased,while after dsLmKnk2,dsLmKnk3-FL injection,the sensitivity of locust to environmental stress was the same as the dsGFP injection group.In a word,in this article,we studied the molecular characteristics and biological function of LmKnk2,LmKnk3-FL and LmKnk3-5',transcriptional regulation of Knickkopf family gene and its effect on the response of locust to environmental stress in L.migratoria.The research not only enriches the research contents of the key genes involved in insect cuticle formation,but also provides a new idea for screening new targets for locust control and green pest control strategies. |
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