Gene Expression & Regulation Of Dauer Related Genes,akt-1 And Daf-8 In Pinewood Nematode,Bursaphelenchus Xylophilus

Pine wilt disease is caused by nematode named Bursaphelenchus xylophilus.It undergoes dauer larvae in response to harsh environmental conditions.The dauer larvae are spread to another healthy pine tree mediated by Monochamus alternatus.The underlying mechanism of dauer formation is largely unknown in B.xylophilus.Two dauer related genes,akt-1 and daf-8,were chosen to elucidate the formation of dauer larvae in B.xylophilus.Real-time quantitative PCR(q PCR),m RNA in situ hybridization and microinjection were used to investigate spatio-temporal expression,and RNAi technique was utilized to examine the gene expression regulation at the key pre-dauer formation stage in B.xylophilus.1.q PCR showed that the similar expression pattern was found between akt-1 and daf-8 during embryogenesis and post-embryogenesis.The significant increase was seen from embryo to L2 stage in both akt-1 and daf-8.Their expression reached the peak at L2 stage and then gradually dropped from L2 to adult.It indicated that the akt-1 and daf-8 genes could activate the insulin/IGF-1 signal pathway and the TGF-? signal pathway prior to the dauer formation of B.xylophilus,and appears to inhibit the formation of dauer larvae.Interestingly,significant higher expression level in male were observed than that in female both akt-1 and daf-8,suggesting that there were gender differences between them.2.Based on above findings,L2 larvae were chosen to carry out m RNA in situ hybridization.The akt-1 gene was stably expressed in the digestive system of the middle part of L2 larvae in B.xylophilus,and the daf-8 gene was stably expressed in the middle esophagus bulb and its posterior part.It indicated that the akt-1 and daf-8 genes appears to be the key genes responsible for the formation of the dauer larvae in B.xylophilus.3.Promoters of the akt-1 and daf-8 genes of B.xylophilus were cloned into the upstream of the m Cherry of the nematode-specific expression vector p CFJ90 by In-fusion Cloning.The constructs were injected into the gonads of Caenorhabditis elegans,and F2 was screened by marker gene of myo-2.At the embryonic stage,the akt-1 gene was expressed in the middle and posterior part of the worm.The daf-8 gene was mainly localized in the neuronal ring near the middle esophagus bulb.Expression patterns of akt-1 and daf-8 were highly consistent with that of m RNA in situ hybridization.In postembryonic stages,the akt-1 and daf-8 genes were largely expressed in the head and tail ganglions.4.RNAi was used to interfere akt-1 and daf-8 expression during embryogenesis at 6 h and 18 h.The hatching rate were significantly decreased after akt-1 and daf-8 genes were interfered.The morphology was shown with obvious deformity.It suggested that akt-1 and daf-8 genes participated in B.xylophilus embryonic development.Interestingly,no significant phenotypic changes were observed when interfering expression of akt-1 and daf-8 at L2 stage,which might result from primary changes at the physiological metabolic level.5.The potential target genes including daf-12,daf-14,daf-16 and exp-2 were examined by q PCR at the different time points when treated with ds RNAs of akt-1 and daf-8 at L2 stages.The preliminary data showed that the significant changes of above potential genes were detected,suggesting that the akt-1 and daf-8 genes may be synergistic with these genes to regulate the formation of the dauer larvae in B.xylophilus.