| The yellow mealworm Tenebrio molitor is an important storage pest.It occurs less in spring and winter,and reaches its peak in summer and autumn.It often breaks out into disasters.It is a completely metamorphosed insect,and undergoes four stages of egg,larva,pupa and adult.T.molitor mostly occurs in warehouses of various agricultural and forestry products.At present,chemical control is mainly used for the control of T.molitor.Chemical control not only seriously pollutes the environment,but also easily makes T.molitor develop resistant to insecticide.Chitin deacetylase(CDAs)can catalyze the hydrolysis of chitin and remove the acetyl group to form chitosan,which is conducive to the formation of dense lamellar structure of chitin and protein in the epidermis,and plays an important role in the growth and development of insects.At present,CDAs have been identified in a variety of insects.For example,Tribolium castaneum contains 9 CDAs,Drosophila melanogaster and Bombyx mori contain 6 CDAs,and Pediculus humanus,Apis mellifera and Anopheles gambiae contain 5 CDAs.Previous studies have shown that CDAs gene play an important role in the maintenance of chitin lamellar structure of insect epidermis,trachea and wings,However,the molecular characteristics and biological functions of CDAs in T.molitor have not been reported.In this paper,the molecular characteristics and biological functions of the TmCDAs were systematically studied.The synthetic dsRNA in vitro was injected into the body cavity of T.molitor to silence the TmCDAs genes.It will provide important theoretical basis for biological control of storage pests.This paper mainly studies on the following four aspects:1.Identification of chitin deacetylase(TmCDAs)Based on the Tenebrio molitor transcriptome database,10 TmCDAs gene sequences were searched,full-length primers were designed,and their full-length c DNA sequences were cloned.Analyze its gene structure,protein functional domain,maximum molecular weight,and isoelectric point characteristics.A phylogenetic tree analysis was conducted on the obtained TmCDAs and other insects,and the results showed that CDAs were divided into 5 subfamilies with high confidence.TmCDAs have conserved sequences in different insects.2.Screening of internal reference genes in quantitative PCR analysis of T.molitorIn this study,the most suitable internal reference genes and the number of internal reference genes were screened in different tissues of pupal and at different developmental stages using ge Norm,Normfinder,and Bestkeeper software.Among them,18 Sr RNA and RPS3 were the most suitable internal reference genes.18 Sr RNA、RPS3、α-tubulin、β-Actin or 18 Sr RNA、RPS3、α-Tubulin was the most suitable internal reference gene combination.3.Molecular characterization of chitin deacetylase(TmCDAs)RT-q PCR was used to detect the expression of TmCDAs genes in different developmental stages and different tissues of T.molitor.The results showed that TmCDA1-TmCDA5 were highly expressed at pupal stage of T.molitor.TmCDA1-TmCDA5 were highly expressed in the body wall,while TmCDA6-TmCDA9 were specifically expressed in the intestine of T.molitor.It was found that TmCDAs were highly expressed before and after molting of T.molitor,and TmCDA1 and TmCDA2 were both highly expressed after molting.It was speculated that the Group I of TmCDAs genes was involved in the process of body wall formation.4.Biological function of chitin deacetylase(TmCDAs)The biological function of TmCDAs was studied using RNAi technology combined with microscopic and ultrastructural observations.Double stranded injection was performed on the second day of pupation to detect the silencing efficiency of dsTmCDA1 and dsTmCDA2,and to observe the phenotype,microstructure,and ultrastructure of T.molitor.After injection of double chain,the molting and death of the yellow mealworm were delayed by one day,with mortality rates reaching 89% and 82%,respectively.The HE staining results showed that there was no significant difference compared to the control group after injection of dsTmCDA1 and dsTmCDA2.The observation results using transmission electron microscopy showed that after injection of dsTmCDA1 and dsTmCDA2,the epidermal lamellar structure disappeared,and the pore structure became deformed and irregular.After injection of dsTmCDA1,the epidermis became thicker,while after injection of dsTmCDA2,the epidermis became thinner.To sum up,this thesis studies the molecular characteristics and biological functions of TmCDAs in T.molitor.The research work would provide an important theoretical basis for the screening of molecular target genes and enrich the theoretical basis for new control methodsbased on green environmental protection. |
