Application Of Rapid Identification Of Diapause And Elimination Of Diapause Eggs Based On Raman Spectroscopy

Silkworm（Latin name:Bombyx mori Linnaeus）,commonly known as silkworm,is a lepidoptera insect that feeds on mulberry leaves.It has four different developments:egg,larva,pupa and adult.At the stage,each stage of development can be used as a material for biological science research.The silkworms are propagated in the form of eggs,in which the eggs have diapause eggs and the elimination of diapause eggs.How to identify the diapause of silkworm eggs is important in the silk industry.Based on Raman（Raman）spectroscopy,this paper studies the diapause of silkworm at different developmental stages and the identification of silkworm eggs.The diapause and932 varieties of silkworm eggs were used as materials.The samples of silkworm eggs before and after diapause were removed.The samples were randomly taken from 1 to 8 days in different developmental stages.The micro-area Raman spectroscopy was applied at 785 nm.Excitation light excitation,Raman spectral data of silkworm egg samples were collected,and the data of the Raman spectrum peak map was analyzed by partial least squares discriminant analysis（PLS＿DA）method,and the PLS＿DA model was verified to verify the available Raman.The peak data is systematically judged.The results of spectral analysis showed that the spectrum detected by Raman spectroscopy had an amide I band of 1570 cm^-1 and 1622 cm^-1,an amide II band of 1570 cm^-1,an amide III band of 1279 cm^-1,and 1013cm^-1 CH₃ bending vibration,base deformation vibration（CH）and sugar deformation vibration（CH）of 1459 cm^-1,base stretching vibration（ring）of 1521 cm^-1,1455 cm^-1 and1459 cm^-1.The lipid characteristic peak（ester CH₂ shear vibration）,the PLS＿DA model of Raman spectroscopy predicts the ideal effect of Dazao and 932 varieties,and the Raman spectrum can explain the main material components in silkworm eggs.The prediction effect and discriminant effect of the PLS＿DA model based on Raman spectroscopy are significant.The basic difference of these spectra is the marker for the diapause of the silkworm and the932 varieties and the identification of the diapause eggs,which can be used as an early research method.In order to observe in detail the morphological changes and differences of embryos in different developmental stages of diapause and cultivar 1-9 d postpartum,the eggshell phenotype observation and traditional anatomy were used to identify the morphological changes and differences of embryos at different developmental stages.The observation of eggshell phenotype showed that the diapause of Dazao and 932 was slightly different from the surface of the eggshell at the developmental stage of 1-7 d of the diapause,but the naked eye could be identified mainly on the 8th day.The embryo develops to the eucalyptus stage,and there is a small black spot in appearance;the anatomical results of KOH shelling method show that the embryos are successfully dissected and diapause embryos are obtained at different developmental stages,and the morphological map is obtained,and the embryo can be clearly observed.The characteristics of the diapause and the elimination of diapause eggs are clearly identified,and the developmental status of the embryos is known.It is of great significance to identify and identify the developmental patterns of each period.Using the insect circadian clock-associated gene-the silkworm cyclin family gene,the relationship between the gene expression pattern of the diapause eggs and the diapause eggs and the embryonic development period was studied.The cyclin family genes Bm-Cyclin A and Bm-Cyclin B were detected by real-time quantitative PCR（RT-q PCR）using Dazao and932 diapause and the embryos of 1-8 days after the development of silkworm eggs.The expression of 6 genes of Bm-Cyclin B3,Bm-Cyclin E,Bm-Cyclin H and Bm-Cyclin L1 was explored.Molecular biology results showed that the genes of Bm-Cyclin A,Bm-Cyclin B,Bm-Cyclin B3,Bm-Cyclin E,Bm-Cyclin H and Bm-Cyclin L1 were all expressed in the diapause silkworm eggs.Expression,declining trend in the later stage.The expression levels of Bm-Cyclin A,Bm-Cyclin B,Bm-Cyclin B3,Bm-Cyclin E and Bm-Cyclin L1 in diapause eggs basically showed a steady state,which may be related to the mitosis of cells during development.Have a certain relationship.