Cloning, Bioinformatic Analysis And Expression In Diapauses Of The Dorsal In Delia Antiqua

Diapause is a specific developmental and physiological phenomenon in insects andits related arthropods, during which period the development of orgnisms stagnate and nofood be ate, no moving, metabolism decreased. During diapause, the most genesexpressing in developmental period are closed,instead of that, from the beginning ofdiapause a set of new genes are started and either continued express in the earlydiapause period or express in late diapause period or express althrough the diapauseperiod. Diapause is slow dynamic development process with it seemingly static.Duringdiapause the orgniam's physiological changes is closely related to the expression ofspecific genes in the specific period of time. So the expression study of genes relateddiapause plays an important role in depth understanding of the molecular mechanismsof diapause。Dorsal is one of the members of the proto-oncogene Rel family. Rel familyproteins, including vertebrate proteins NF-κB1, NF-κB2, RelA, RelB, Rel C, andarthropods Dorsal, Dif and Relish (Rel). All the Rel family members contain the RHDhomologous functional areas. The Toll signaling pathways of arthropods andinterleukin-1receptor,(IL-1R)-NF-way of vertebrates is homologous. Ourunderstanding to the functions of Dorsal protein is mainly in Drosophila melagaster.Dorsal is the downstream target gene of the dorsal-ventral signaling pathways whichdetermine the establish of embryonic dorsal-ventral axis. Dorsal also is the downstreamtarget gene of the Toll signaling pathways which regulate the expression ofantimicrobial peptide gene in insect. In Drosophila, before early embryogenesis and celldifferentiation in embryonic development, Dorsal in the nucleus showing thedistribution along the the dorsal-ventral axis with the concentration gradient. Differentgenes are activated by Dorsal protein with different concentrations, as result, cellsdifferentiation and different organizations produced. About the innate immunity ininsect, when the parasites are infected by the Gram positive bacterial or fungal, theDorsal and Dif (Dorsal-related factor) gene are activated by the Toll signal pathway, andthus, the Dorsal and/or Dif protein are synthesized and transported into nucleus, thenthe dimer Dorsal/or Dorsal/Dif play the role of transcription factor and activate the corresponding antimicrobial peptide gene expression, and further the activation ofphagocytic cells, as result the immune response expanding.At present, the reportion of Toll pathway is mainly involved in the innate immunein the early embryo and adult stage, but its function in the innate immunity of the pupalstage, or diapause has not been reported.Many insects have winter diapause or summer diapause, and only the rare fewspecies (such as Mamestra brassicae) has facultative diapause,which means both hassummer diapause and winter diapause, Delia antique is one of this rare insect species.The summer diapause and winter diapause occurre at the same developmental pointafter head eversion of the pupal stage. So onion maggot, Delia antique is one of idealmodel used to diapause study. We cloned two Dorsal gene cDNA of the Delia antiquewith two different sequence through RACE method. The two cDNA sequence aresubmissed to GenBank and get the accession number for JQ790524and JQ804834.Semi-quantitative, real-time quantitative PCR are selected to detect the expression ofDaDorsal gene in the onion maggot diapause period. The results are as follows:①Cloning and sequence analysis Of Dorsal in onion maggot Delia antiquaUsing RACE method,we cloned two Dorsal gene cDNA of Delia antiqua, thefull length of the two sequence are2351bp and2354bp, respectively, and thecorresponding ORF are1974bp (34-2007bp) and1686bp (34-1731bp), encoding657and562amino acids, respectively; the hypothetical protein molecular weight of72.9kDa and62.3KDa and isoelectric point of8.5and8.95, respectively. Respectively,we do comparison of the similarity between the deduced amino acid sequence of twosequences and the congeners in other species from the GenBank,also do thephylogenetic analysis. The results indict Dorsal of the onion maggot Delia antiqua hasthe the maximum similarity with the congener in Drosophila melagaster.②The induction of onion maggot Delia antiqua diapauseAdult feeding: temperature of (23±1.0)℃, photoperiod of16L:8D,50~70%relative humidity conditions, the adults of onion maggot are growing well.Non-diapause induction: temperature (18±1.0)℃, photoperiod of16L:8D,50~70%relative humidity conditions, the non-diapause larvae and pupae are growingwell.Summer diapause induction: the Delia antiqua pupa all enter diapause at (25±0.5)°C, photoperiod of16L:8D,50~70%relative humidity, light intensity of100%.releasing the diapause best at the temperature of16℃. Winter diapause induction: the Delia antiqua pupa all enter the winter diapause attemperature of (15±0.5)°C, photoperiod of12L:12D, relative humidity of50~70%and the weakest light intensity, to open a set of lights (according to the climatic chamberwe have) conditions.③The expression of Dorsal gene in onion maggot,Delia antiqua diapauseUsing Semi-quantitative RT-PCR, real-time quantitative PCR to study the relativeexpression abundance of Dorsal gene in the onion maggot diapause pupae. We selectthe non-diapause1day,2days,2.5days,4days,5day,6days,9.5days,14days of pupaand summer diapause0.5days,1.5days,2days,4days,8days,14days,20days,26days pupa and winter diapause3days,10days,20days,50days,85days,99days pupa,accessing to3pupae as a sample at every piont. The results indicate that: under thewinter diapause and summer diapause condition,The content of Dorsal gene mRNA inonion maggot pre-pupa period is low, but the lowest is at the piont of pupation headeversion before enter diapause; Then, with the emergence and increasing of fat body theexpression of this gene rises gradually and reach the highest when the yellow bodyemergencing and begining to move.With previous scholars reported, Dorsal is the downstream target genes of the Tollpathway in Drosophila and is activated when the parasites are infected by fungi andGram-positive bacterial. Dorsal and Dif format the dimer of homologous orheterologous and then activate the downstream antimicrobial peptide gene, then, theantimicrobial peptide synthesised in the fat body. This indicat that Dorsal genes involvein the regulation of antimicrobial peptide gene expression in the onion maggot diapause.It spent on the safety to the diapause pupae of the onion maggot to adverseenvironmental and plays an important role. However, the study of Dorsal genesinvolved in insect innate immunity is still largely confined to Drosophila melagaster, inother insects there is few reports, or even zero. This study first reported the expressionof the Dorsal gene in the onion maggot, Delia antiqua pupae, it has lay an importantfoundation for the next study of Dorsal participating in the regulation of antimicrobialpeptide gene.