Cloning And Expression Of The DaARF Gene In The Winter And Summer Diapauses Of The Onion Maggot, Delia Antiqua

Diapause is a physiological phenomenom of insects when they are overcoming the bad environment, such as drought,food shortage, low temperature and high temperature. It is also a survival strategy of insects in the progress of exploiting seasonal resources. During diapauses, insects will appear some physiological phenomenon, such as reduce metabolism., stop growing. Such behaviors are closely related to diapause specific gene expression on which our study is very limited. So that the further study of diapause gene expression is very helpful for us to understand the molecular mechanism of diapause.Delia antiqua is a worldwide agricultural pest belonging to the genus Delia, family Anthomyiidae of order Diptera, which is widely distributed in Asia, Europe and North America and mainly damages Liliaceae vegetables in temperate regions of Northern Hemisphere. Delia antiqua larvae bore into the bulbs and feed, which made the vegetables rot and eventually wilted to death. Delia antiqua is a model species for comparatively studying on the molecular mechanisms of winter diapause(WD) and summer diapause(SD) of insects.We got differentlly expressed fragment of ADP-ribosylatin factors (ARF) from the construction of a subtractive cDNA library. ARF is the key component of transport channel of Eukaryotic cell vesicles, which involved in cell transport and signal transduction. During diapauses, the generation, transduction and material transport of diapause signal are very significant. The purpose of the research is whether ARF is related to the progress of diapause.In the research, we firstly cloned and characterized the full length cDNA of ARF from Delia antiqua, DaARF, by Semiquntified PCR, Real-time quantified PCR and sequence analysis. The results are as follows.①The cloning and sequence analysis of ARFThe full-length cDNA,858bp long, and an open reading frame (ORF) of540bp (234-773), encoding a protein of180amino acids with a calculated molecular weight of20.9kDa and theoretical isolectric point of6.42. By multiple sequence analysis, the gene and other Diptera Insect ARF have more than96%similarity. The conserved GTP domain was identified to range a.a.24to129, which is characteristic feature of ARF. ②ARF mRAN levels in different periodsWe reaserch ARF mRAN levels in different periods by Semiquantified PCR and Real-time quantified PCR., which shows ARF mRNA levels were a little different to those of non-diapause. ARF transcription is down-regulated in both summer diapauses and winter diapauses, while being increased when diapause was terminated. These results suggested that ARF is down-regulated in both SD and WD, and it may play an essential role in the signal transduction and material transport of cell during diapause.