Cloning And Expression Of The Sex-determining Gene Tra-1 In Ovomermis Sinensis

The sex of Ovomermis sinensis,an entomopathogenic nematode,is determined by the nutritional competitive pressure in parasitic period.It is a kind of environmentdependent sex determination(ESD),but its molecular mechanism is poorly understood. To study the molecular mechanism of sex determination in O.sinensis,the sequence and expression profile of Ovo-tra-1 were investigated.This was the first time that zinc-finger domain-containing transcription factors were isolated from a parasitic nematode.The main results were summarized as follows:(1)Tra-1 gene was partially cloned from O.sinensis by using RT-PCR and 3'-RACE.The 5' truncated cDNA was 800bp in length and encoded a putative protein of 195 amino acids,which contained three consecutive C₂H₂ zinc finger motifs,and the first 105 amino acids were in zinc-finger 2-5 domain.The deduced amino acids sequence had high homology with GLI3,CiD,CeI-TRA-1A,Cbr-TRA-1A and Ppa-TRA-1 in the zinc-finger domain.The identity were 70%,71%,73%,77%,71%respectively,while outside this domain the amino acid identity was only 6%.The study showed that Ovo-tra-1 encoded a transcription factor of the C₂H₂ zinc-finger class and belonged to the Gli/CiD family of proteins,the conservation was limited to the zinc-finger domain.(2)The transcript levels of tra-1 in different stage of O.sinensis(embryos, pre-parasites,parasitic juveniles,post-parasites,exuviated female adults,exuviated male adults,mated female adults),and parasites of different ratio of host to nematodes(1:40, 1:10)were analyzed by real-time fluorescent quantitative PCR.The results showed that the tra-1 transcript presented high levels in the exuviated female adults(P＜0.01),The expression dropped observably during the stages of embryos and mated female adults, but it remained significantly higher than the level of other groups(P＜0.05).However,the expressions among pre-parasites,parasitic juveniles,post-parasites,had no remarkable differences(P＞0.05).Comparing with the observed results of morphology,it showed that the high expression of tra-1 prompted the gonad differentiating into female gonad,while the low level expression of tra-1 led to the male gonad genesis during the core stage of gonad differentiation.This is to say that as for the O.sinensis,TRA-1A took part in the control of the gonad development and differentiation.Studying on the basis of quantitative results,the author presumed the TRA-1A had a share in the control of the embryogenesis and embryonic development of the O.sinensis.To sum up,the previous researches illustrated the function similarity of TRA-1A in the O.sinensis and other free-living nematodes,and further proved its comparable conservation of TRA-1A on its function.These results showed that the sequences of tra-1 in zinc-finger domain and the function of the gene was conserved,which suggested that mechanisms to control sexual development are similar in parasitic and free-living nematodes.Therefore,it further paved the way for the research of the sex-determination and differentiation mechanism of mermithids.