Development Of SSR Molecular Markers And Expression Analysis Of Clathrin In Vitellogenesis Of Calliptamus Italicus (L.)

Calliptamus italicus(L.)is one of the pests inhabiting the desert and semidesert in the north of Xinjiang Tianshan Mountains,which has seriously threatened the development of local agriculture and animal husbandry.Microsatellite polymorphism has high resolution,which is of great significance for biomolecular markers and population studies.As a key protein of endocytosis,clathrin plays an important role in the growth,development and reproduction of organisms.Therefore,on the basis of transcriptome sequencing,this study excavated,analyzed and identified the SSR loci of Calliptamus italicus(L.).The full length sequences of the heavy chain of Calliptamus italicus(L.)clathrin were obtained to investigate the spatiotemporal expression of Calliptamus italicus(L.)clathrin1.Analysis of SSR loci and development of molecular markers of Calliptamus italicus(L.)A total of 156 500 effective SSR loci were found in the 718 872 Unigenes of Calliptamus italicus transcriptome database,which were distributed in 126 369 Unigenes,and the occurrence frequency was 17.58%.Among them,the single nucleotide repetition was 60.88%,mainly A/T(44.38%).Primers were designed based on the selected SSR loci,and 24 pairs of primers were randomly selected to conduct PCR amplification on DNA samples of adult Calliptamus italicus(L.)from 10 different geographical populations.A total of 6 pairs of primers were successfully amplified.2.Full length cloning and bioinformatics analysis of CICHCThe full-length sequence of CiCHC was 6 345 bp,with an open reading frame of 5 031 bp.It was estimated to encode 1 677 amino acids and the molecular weight of the encoded protein was 191.85 k Da.CiCHC has seven typical clathrin heavy chain repeats.The phylogenetic tree construction showed that Calliptamus italicus CHC was the closest to Locusta migratoria manilensis,and the sequence consistency of the two was 100%.These results laid a foundation for further elucidation of the regulatory role of Calliptamus italicus in vitellogenesis.3.Dynamic analysis of CiCHC expressionThe dynamic expression of CiCHC transcriptional level is closely related to vitellogenesis.The expression level of CiCHC was increased at Previtellogenesis(1-7days after emergence),and reached the peak at 7 days after emergence(P<0.05).In thevitellogenesis stage(8-13 days after emergence)and in the Posvitellogeny stage(14-25 days after emergence),the expression level was low.The expression trend of CiCHC protein level is similar to that of transcription level,but there is a delay.The expression analysis at the cell level showed that CiCHC was uniformly distributed in oocytes at Previtellogenesis.During vitellogenesis,clathrin mainly accumulates around the oocyte membrane and co-locates with vitelloprotein.In this study,transcriptome data were used to excavate the SSR loci of locust,laying a foundation for the study of molecular markers,population genetics and functional genes of Calliptamus italicus.The full-length sequence of CiCHC was obtained,and the expression dynamics of CiCHC at transcription level,protein level and cell level at different stages of vitellogenesis were analyzed,which provided theoretical basis and data reference for the research of reproductive physiology of Calliptamus italicus,and provided a new idea for the green pest control strategy of Calliptamus italicus.