| The pine wood nematode (Bursaphelenehus xylophilus)，the pathogen of pinewilt disease transmitted by Monochamus spp., is a kind of quarantine pest toconiferous trees, which has caused huge economic losses in some countries andregions in Asian. In the life cycle of B. xylophilus, two distinct phases are involved,a propagating phase and a dispersal phase, and the two stages can transform andinterchange. The pine wood nematode keep highly survival rate under favorableenvironment and propagate with Monochamus spp by this transformation. Howeverthe mechanism of this transformation is not clear to this day. Therefore，it is essentialand significant to understand the diapause in pine wood nematode.The research onthe genes controlling the diapause and the switch between propagation and dispersalphases of B.xylophilus under environment stress are necessary. In this study, wecloned and sequenced four important genes daf-3, daf-9, daf-12and daf-16in B.xylophilus based on the result of model nematode (Caenorhadits.elegans), andanalyzed their gene structure. We detected the mRNA expression patterns of the fourgenes in different stages and ecological environments. Meanwhile， and theexpression of daf-3, daf-9, daf-12and daf-16were suppressed by Agrobacteriummediated transformation of Botyfis cinerea expressing dsRNA of daf-3, daf-9, daf-12and daf-16. The development speed, reproduction number, life span and triglycerideaccumulation of B.xylophilus were counted. In this study, the protoplaststransformation system was established. Dicer-1of B. cinerea was successfullyknockdowned by protoplasts transformation methods. The main results were asfollows:1. Based on the genes involved in diapause in C. elegans, daf-3, daf-9, daf-12and daf-16full-length cDNA and DNA fragment were obtained from B. xylophilusby RACE. The GenBank acession number of daf-9, daf-12, daf-16-1and daf-16-2were GU256938、GU584879、JN628938and GU584878, respectively. According tothe analysis of gene structure, daf-3gene consists of9exons and8introns andbelongs to MH protein family, daf-9gene consists of6exons and5introns and belongs to P450protein family; daf-12gene contains6exons and5introns andbelongs to NHR protein family; daf-16gene belongs to FOXO transcription factorfamily including two genes and has two different shear mechanisms. A single copyof daf-3, daf-9, daf-12and daf-16in the genome of B. xylophilus was revealed bysouthern blot experiments. By multiple sequences alignment of daf-3, daf-9, daf-12and daf-16, the results indicated that they were highly conserved with its homdogy,respectively.2. The real-time qPCR was used to detect the mRNA expression levels of daf-9and daf-12in propagation stages of eggs, larvae and adults, as well as the twodispersal stages, LIIIand LIVjuveniles of B. xylophilus. The result showed that daf-9and daf-12expressed in all development stages. The expression levels of daf-9waslow in the third dispersal stage as0.1times as larvae in propagation stages, but theexpression of dispersal LIVjuveniles recovered high level. There were no obviousdifferences in the expression of daf-12in larvaes in propagation stage and in thethird dispersal stages. However, expression of daf-12in the fourth dispersal stagewas much significantly higher than that in any other stages, more than20times ofthose the third dispersal stages. It is possible that the low expression of daf-9gene isthe reason of B. xylophilu transformation betweem propagation and dispersal stagesand keeping dispersal stages LIIIjuveniles. B. xylophilu reserved fats, stop growingand taking food at dispersal stages. At dispersal stages of LIVjuveniles, theexpression levels of daf-9gene was recovered to the levels as larvaes in propagationstages, and the biosynthesis of DA was recovered and provided the ligand for daf-12finishing transformation from dispersal stages LIIIjuveniles to LIVjuveniles. Sterolprovided by food is the material for biosynthesis of DA. When intracorporal sterolwas consumed, the biosynthesis of DA is stop. The lack of DA was resulted in B.xylophilu keeping dispersal stages LIVjuveniles.3. The change of mRNA expression of daf-3, daf-9, daf-12and daf-16in differentenvironments, such as starvation, high temperature, low temperature and crowding,were analyzed. The expression of the genes increased under favorable environments(suitable temperature, enough food and crowding). Under condition of no food, daf-3, daf-9, daf-12and daf-16gene were all downregulated. Under condition of crowding,the expression of daf-3, daf-9, daf-12and daf-16gene were all upregulated. Undercondition of low temprture, daf-3, daf-9, daf-16-1gene were upregulated and daf-9,daf-16-2gene were downregulated. Under condition of high temprture, daf-16-1genewas downregulated and others were upregulated. The expression of daf-16-2proteinwas downregulated under condition of no food and low tempreture, but was littlechange under condition of high tempreture.4. The PHD-RH vector expressing dsRNA in filamentous fungi wasconstructed. The dsRNA of daf-3、daf-9、daf-12、daf-16-1、daf-16-2gene of B.xylophilus and non-target sex gene sGFP were expressed by Botryis cinerea withthe PHD-RH vector. The pine wood nematode were cultured on those transformersand transformer of PHD-RH vector for10genenration and analyse the geneexpression level, development speed, reproduction number, life span andtriglyceride accumulation. The expression level of target gene wasdeclined by20times and the sGFP has no effect on expression of target gene. Thedevelopment speed of B. xylophilus were declined with the RNAi treatment of daf-9,daf-12, daf-16-2gene, and other gene RNAi treatment have no effect on developmentspeed. The reproduction number of B. xylophilus were declined with the RNAitreatment of daf-9、 daf-12gene, and increased with the RNAi treatment ofdaf-16-1and daf-16-2gene, but daf-3and sGFP gene RNAi treatment haveno effect on the reproduction number. The life span of the nematode increased withthe RNAi treatment，and other gene RNAi treatment have no effect on life span.Thetriglyceride accumulation were increased with the RNAi treatment of daf-3、daf-9、daf-12、daf-16-1、daf-16-2gene, and sGFP gene RNAi treatment have no effect ontriglyceride accumulation.5. The protoplasts transformation system was established and successfully used toknockout the Dicer-1of B. cinerea. The transformation system can provide thetechnical support for studying on the gene function of B. xylophilus by B. cinereaexpressing dsRNA of target gene. |
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