| Saprolegnia is a kind of filamentous fungi and belong to the Oomycetes, theSaprolegniales and Saprolegnia. As animal pathogenic Oomycetes, Saprolegnia hasstrong adaptability to the host and region. It is endemic to all freshwater habitats andeggs around the world and is a serious problem in the aquaculture industry. Thedisease symptoms include white or grey patches of flamentous mycelium on the bodyor the fns of freshwater fsh. Saprolegnia parasitica host-targeting protein SpHtp1has been confirmed that it plays an important role in infection of Saprolegniasis. Thisgene encodes asecreted protein which containing an RxLR motif. This proteinweakened the host’s immune function and increasing the infection of pathogenic. Butlittle is known about the molecular and cellular mechanisims under lying the infectionprocess of fish pathogenic Oomycete.In this study, we identified15saprolegniastrains to know their species. The starins were separatedand purified by our lab.Thenwe adopted the motheds of Polymerase Chain Reactionto detect distribution of thegene in different sources and different host species. And we usefluorescencequantitativeto know the expression of SpHtp1of hyphae and spores under thetreatment of different temperature and different salinity. The main research of thispaper and the results are as follows:1) the research of identification of saprolegniastrainsThrough morphology observation and comparison of ITS genetic sequence,weidentifiedthese strain‘s species. The result showed that most strains belonged toSaprolegnia parasitica(Y7, O9, O10, O15, Y4, Y6, Y3) and Saprolegnia ferax (11, Y8,O12, O17), one strain belonged to Saprolegnia australis (O4), and two starinsbelonged to Saprolegnia.spp (Y1, Y5).2) distribution of SpHtp1gene in different Saprolegnia strainsAccording to the published genome CDS seguences of S.parasitica host-targeting protein SpHtp1(GenBank accession number: GU345745) a pair ofspecial-primers was designed and a PCR method was developed for the detection ofthis gene from ATCC. And then optimize parameters of this PCR. The PCR productwas extracted and linked to pMD18T vector and then was cloned into DH5α. Therecombination plasmid was identified by PCR and sequencing analysis to prove theamplified target gene. With this method, a224bp DNA fragment can be amplicfiedfrom SpHtp1genome. The homology of nucleotide sequence between the PCRproduct and other SpHtp1strains was99%. Seventeen samples from all parts of Chinause this method to identify SpHtp1gene. SpHtp1gene was present in8(47%) of the17isolates. These results showed that the SpHtp1gene was expressed highly specificof S. parasitica.3) detection of SpHtp1of S.parasitica under environmental factorswithfluorescence quantitative RT-PCR methodMycelium and spores of stain ATCC(200013) which were cultivate under20℃were usedas templates. The cDNA were synthesized by reverse transcription reactionfrom the mycelium and spores. The plasmid which contained SpHtp1and SpTub-bgene fragment were constructed as standard for real-time quantitative PCR toinvestigate the expression of SpHtp1in strain (ATCC200013) treated under differenttemperature (0℃,5℃,10℃,15℃,20℃,25℃,30℃) and different salinity(0.12%,0.25%,0.5%,1%,2%). The results showed that the expression of SpHtp1ofdifferent temperature was different. At20℃, the expression reached the maximumand the expression was higher at15-30℃than at0-10℃. The expression of sporesfrom S.parasitica was higher than in mucelium. And the expression of SpHtp1frommycelia and spores was dropped with the dropping of salinity.In summary, SpHtp1gene was existing extensively in S. parasitica. It has nostrict selectivily for the host source and region. Specially exists in S.parastica.SpHtp1was expressed in mycelium and spores, different environmental factors,different expression. The expression of SpHtp1was highly expressed under suitablegrowth temperature and lowsalinitySpHtp1is highly expressed in zoospores from S.parasitica when compared with in mycelium. |
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