| The epidermal degradation of Locusta migratoria is regulated by chitinase.By preparing the paraffin section of 9d-14 d,the prenymphs of the flying locust were found to be formed at 9d and then thickened gradually to the thickest at 11 d.It then began to degrade,and the epidermal cells produced interstitial space,and degraded at 14 d.By immunofluorescence staining,LmCht5-1 was found to be expressed in epidermal cells at the beginning and transported to the cell and epidermal space,and finally to the epidermis with chitin.Using dsLmCht5-1 to interfere with the expression of LmCht5-1 in pre-nymphal stage,it was found that the protein expression was decreased during the whole pretreatment of the skin of the pre-nymphs,and the degradation of the old epidermis was blocked,which caused the death of pre-nymph dysplasia.Further FB28 staining showed that the degradation of chitin in pre-nymphal epidermis was significantly lower than that in the control group.Therefore,we believe that LmCht5-1 is responsible for the degradation of chitin in the epidermis of prenymphs during the pre-nymphal period.Insect chitinase is a big family,during the process of chitin degradation the chitinase(Cht5s)play an important role,it can be catalytic by beta 1,4glycosidic bond connected of N-acetyl glucosamine acetamide based off of acetyl,forming acetyl chitin(chitosan).Group early identified that chitinase5 has the phenomenon of gene replication in the Locusta migratoria,the chitinase 5 of Locusta migratoria contain two genes named LmCht5-1 and LmCht5-2,in which the gene of LmCht5-1 play an important role during the development from 5 instar nymphy to adult period that had been shed,while the function of gene of LmCht5-2 is unclear.However,the function of LmCht5-1 and LmCht5-2 in the embryonic stage remains to be studied,and the functional differentiation of LmCht5-1 and LmCht5-2 also needs to be further studied.The lack of antibody has limited the research on gene function.Further through gene expression profile of the study found that thetwo genes during embryonic development period of the Locusta migratoria with different mode of epidermis may participate in the process of different epidermal growth,its development model is not clear.Based on the research basis of early research,this study took the gene of LmCht5-1 as the research object,carried on the prokaryotic expression,prepared LmCht5-1 specific polyclonal antibody successfully,and studied the genes in embryonic development of Locusta migratoria.The study provides important basic datais for exploring gene about chitin metabolism during the process of development,and for new targets for the screening of pesticide,which is environmental and friendly,and provides the theoretical basis of chitosan production methods on the chitin of agricultural pests named Locusta migratoria as substrate.This paper mainly studies from the following three aspects:The preparation technology of paraffin section was improved by optimizing the pretreatment,washing,dehydration and transparency of Locusta migratoria embryos.The modified paraffin section technique was used to obtain the complete and clear histological tissue section of the epidermal structure,and HE staining was used to study the epidermal development of the Locusta migratoria embryo.The results showed:(1)under the condition of 30 ?,migratory locust embryonic cuticle was full at 5d,and formed in the embryonic development,and began to degrade at 8 d.(2)under the condition of 30 ?,migratory locust embryonic pre-nymph cuticle was full at 11 d,and formed in the embryonic development and began to degrade at 12 d,and degraded completely at 14 d,and embryo of Locusta migratoria formed new cuticle.This study optimized the preparation steps of the paraffin section of migratory locusts and revealed the epidermal development model of the locust serous membrane and provided the basic information for the study of insect embryo epidermal metabolism.2.Prokaryotic expression,purification and antibody preparation and verification of locust LmCht5-1.Select LmCht5-1 antigen structure area,construction of plasmid pET32a-OVA-LmCht5-1,will be builded a successful restructuring of plasmid into E.coli induced expression of target protein,the success of protein expression using Ni-NTA affinity chromatography column for protein purification,purification of components using 12% sds-page method to detect the purification result,then the determination of the purified protein concentration,to reach 10 mg concentration of antibody preparation of purified protein to the biotechnology company.After antibody synthesis,the Western blot technique was used to verify the antibody.The specificity of antibody titer and specificity was detected,and the specificity of antibody was detected by immunohistochemistry,which provided important experimental materials and platform conditions for the in-depth analysis of the function of LmCht5-1.3.LmCht5-1 gene was used to organize localization and function analysis during the development of migratory locust embryos.The localization and changes of LmCht5-1 gene were detected by immunohistochemistry.The effects of LmCht5-1 gene on the development of migratory locust embryos were studied by means of RNAi interference and transmission electron microscopy.Immunohistochemical results showed that LmCht5-1 was located in the cytoplasm.The results showed that LmCht5-1affected the last molting of migratory locust embryos,which affected the metamorphosis of the migratory locust embryo into nymphs. |
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