Cloning And Gene Expression Analysis Of C-Lectin During Diapause Of The Onion Maggot, Deitaantiqua

Diapause is most often observed in insects and is a physiologically dormant statedifferent from other types of dormancy. It allows insects to survive in harsh environ-ments or extend longevity. In general, larval, pupal, or adult non-diapausing insects possessan innate immune system preventing the invasion of microorganisms into their bodies;however, it is unclear whether this system works under the dormant condition of diapause.It isnot clear so far if the diapause insects maintain an active immune response or if they use adifferent strategy from that for the nondiapause counterparts.As a innate immune pattern recognition receptor in insect immunity, C-lectin whichcontains one or more carbohydrate-recognition domains(CRD) takes part in the identificationof the pathogenic microorganism. Ca2+plays an essential role in lectin,so it is called C-typelectin. After recognizing the sugar molecules on the membrane surface of microbial cell,C-lectin will activate the immune system to clear invaded microbes．In this paper,we used Delia antiqua as experimental material．First,we have clonedand characterized the full length cDNA of C-lectin gene by RACE PCR. It has been depositedin GenBank under accession JQ657826. Then we researched the expression level in theimmunization during diapause through the methods of semi-quantitative PCR and Real timeQuantitative PCR．The results are as follows:1. In present study, we cloned a C-lectin gene from the diapause pupa on the basis ofEST sequence screening and combined with RACE cloning technology. The full-lengthcDNA,767bp long, an open reading frame (ORF) of615bp,5′end the translation section(5' UTR)of78bp,3′end the translation section (3' UTR)of74bp, encoding a protein of204amino acids with a calculated molecular weight of2.38kDa and theoretical isolectric point of6.05. According to the further research of C-lectin gene, my experiment offers further supportfor controlling Delia antiqua, and paves the way for the research of immunologic mechanismin insect. 2. The expression of C-lectin in diapause pupa is analysed by semi-quantitative PCRand Real time Quantitative PCR, and the results showed that the final relative mRNA levelsof C-lectin gene is up-regulated in pre-diapause both of summer diapauses (SD) and winterdiapauses (WD),down-regulated in diapause both of SD and WD, and up-regulated again inthe post-diapause both of SD and WD. These results suggested that, C-lectin gene may playan essential role in diapause,during which depressed metabolic and cell proliferating activitiescharacteristic to diapause may admittedly weaken the defense mechanismms involving thesynthesis of damaged cells.