| The rice stem borer,Chilo suppressalis（Lepidoptera:Pyralidae,Crambidae）,is one of the most serious rice pests in China,which larvae feed on the stems of rice.Cuticular Protein（CP）of insects is one of the major components of insect cuticles,which plays an important role in the growth and development of insects.Cuticular proteins are cross-linked to chitin which form the major structure of insect cuticles.However,in Lepidoptera,the study of cuticular proteins have been hampered due to low efficiency of RNA interference.Polyamidoamine dendrimer（PAMAM）is a nanocarrier with a unique three-dimensional structure and a high density of amine functional groups on its surface,which has the ability to bind and deliver nucleic acids.Recently,the reports revealed that could form a "fluorine phase" by grafting fluoroalkyls to nanocarriers.A strong interaction chain is formed between the fluorine chains,which reduces the leakage of the drug encapsulated by the nanocarrier.In this paper,the full-length cDNA sequences of CsLCP 16/17 and CsLCP17 genes had been cloned from C.suppressalis,and molecular structure characteristics were analyzed.The fluorinated nanocarrier P2 used in this paper was obtained by fluorination modification.At the cellular level,an efficient delivery system was established on Lepidopteran Sf9 cells by used fluorinated nanocarrier P2.In vivo level,an efficient and convenient transdermal delivery system was established on C.suppressalis by used fluorinated nanocarrier P2.An efficient transdermal interference system for CsLCP 16/17 and CsLCP 17 was established by combining transdermal delivery system and RNAi technology.CsLCP 16/17 and CsLCP17 were efficiently silenced by used this system,and their functions were systematically analyzed.The specific research results are as follows:（1）The full-length cDNA sequences of CsLCP 16/17 and CsLCP 17 were cloned from C.suppressalis by RT-PCR and RACE.The CsLCP16/17 open reading frame is 330 bp long and encodes 109 amino acid residues,while the CsLCP 17 open reading frame is 513 bp long and encodes 170 amino acid residues.The sequence analysis on two cuticular proteins showed that both CsLCP16/17 and CsLCP17 contained the conserved chitin binding domain Chitin Bind 4,which belonged to the RR-1 subfamily of the CPR family.Multiple sequence alignment analysis revealed that CsLCP16/17 was an apparent ortholog to LCP16/17 of Maduca sexta with 74.55%sequence identity,and CsLCP17 was an apparent ortholog to LCP17 of Plutella xylostella with 48.82%sequence identity.Analysis of temporal-spatial expression patterns revealed that the expression level of CsLCP16/17 gradually increased in the larval stage ranging from first instar to fourth instar.After reaching a peak in the fourth instar,the expression level of CsLCP16/17 decreased sharply in the sixth instar.In the pupal and adult stages,CsLCP16/17 was not expressed.The expression level of CsLCP 16/17 was the highest in the cuticles,followed by the foreguts,and the low level in the heads and hindguts.The expression level of CsLCP17 gradually increased in the larval stage ranging from first instar to third instar.After reaching a peak in the third instar,the expression level of CsLCP 17 gradually decreased.In the pupal and adult stages,CsLCP 17 was not expressed.The expression level of CsLCP 17 was the highest in the cuticles,followed by the heads,not expressed in other organizations.These results indicated that the CsLCP 16/17 and CsLCP 1 7 genes of C.suppressalis may play an important role in the growth and development of the larval cuticles and heads.（2）Agarose gel electrophoresis analysis found that the dsRNA band was completely disappeared when the ratio of P2 to dsRNA charge（N/P ratio）was equal to or greater than 1:1.The results indicated that the nanocarrier P2 was able to bind and loaded with dsRNA when the N/P ratio was equal to or greater than 1:1.When the N/P ratio was 32:1 and 64:1,P2/dsRNA complexes showed high transfection efficiency in Sf9 cells examined.RNA interference was performed on the inhibitor of apoptosis protein（IAP）gene of Sf9 cells.The results revealed that treatment with dsRNA against SfIAP using P2 as a vector could trigger potent gene（SfIAP）silencing,and the cells appeared apoptosis.The results indicated that nanocarrier P2 could significantly improve the RNA interference efficiency of lepidoptera cells.Nanocarrier P2 could carry nucleic acid dye-labeled dsRNA through the egg mass and larvae body wall of C.suppressalis into the body,and would not affect the normal growth and development of the egg mass and larvae.The results revealed that nanocarrier P2 carried nucleic acid penetration through the body wall,with safety and low toxicity.（3）This study performed RNA interference to examine the function of CsLCP16/1 7 gene in C.suppressalis.Nanocarrier P2 was used to deliver dsLCP16/17 transdermally into C.suppressalis.Compared with the control group,the expression level of CsLCP 16/17 gene was significantly decreased in the group treated by P2/dsLCP16/17,and the interference efficiency was 80%.The results indicated that the transdermal delivery of dsRNA by nanocarrier P2 could effectively interfere with the target gene,which provided an efficient and convenient transdermal interference system for the study of the gene functions of C.suppressalis.Compared with the control group,the boring behavior of larvae in the treatment group decreased or even fully vanished.Compared with the control group,the larval tracheas in the treatment group was collapsed,and the chitin was discontinuous and broken by fluorescence microscope after staining the cuticle with Fluorescent Brightener 28.The results of the nanoindentation experiment indicated that the elastic modulus of the larvae cuticles in the treatment group was more than twice that of the control group,which proved that the larval cuticles in the treatment group had a higher degree of hardening.These results indicated that CsLCP 16/17 gene played a key role in the growth and development of larval cuticles and tracheas.（4）This study performed RNA interference to examine the function of CsLCP 17 gene in C.suppressalis.Using nanocarrier P2,dsLCP17 was injected into the third instar larvae.Compared with the control group,the expression level of CsLCP17 gene was significantly decreased in the group treated by P2/dsLCP17.The interference efficiency at different time points was above 95%,and the gene silencing was fast and lasted for a long time.The larvae of the treatment group found that abnormal heads development and failed molting.The results indicates that CsLCP 1 7 was indispensable for the larval heads development of C.suppressalis.In this paper,a transdermal delivery system was established by combining nanotechnology and RNAi technology.Two dsRNA that inhibit the growth and development of C.suppressalis were obtained through experiments.The research results will lay foundation for further combining nanotechnology and RNAi technology to develop the RNA pesticides that are green,efficient and target pests’ cuticles. |
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