Expression And Regulation Of Lin-39 Genes In Pinewood Nematodes,Bursaphelenchus Xylophilus

Pine wilt disease is a devastating disease of forestry caused by Bursaphelenchus xylophylus,occurred in a number of provinces and cities in China,resulting in a huge economic losses.Numerous studies on B.xylophilus have been done in many aspects,however,research of Bursaphelenchus xylophilus itself,especially from the molecular level research is still in the initial stage.The HOX family is a group of conserved and developmental genes responsible for body plan along anterior-posterior axis and cell fate decision during embryogenesis and organogenesis in all animals.The lin-39 gene is one of the members of the Bursaphelenchus.xylophilus HOX family,study the expression and regulation of lin-39 gene contributes to reveal the reproductive mechanism of pine wood nematode.In this study,the lin-39 gene expression and regulation was investigated.Using bioinformatics tools,we analyzed the conserved promoter domain motifs,predicted the function structure of the protein coding regions,and constructed the phyologentic trees of lin-39.Using m RNA in situ hybridization and transgenic GFP labeling technique to analyze the characters of the time-space of gene expression,and real time quantitative PCR to show the dynamic expression of individual development.Through RNAi and behavioral measurement to further verify its function in the individual development of Bursaphelenchus xylophilus.The results are as followings:1.Promoter analysis shows that Bursaphelenchus xylophilus share three conserved motifs with C.elegans which are in the same order,suggesting that they have conservative regulation mechanism.Protein structure prediction shows that lin-39 is a transcription factor containing conserved homeodomain.Protein sequence alignment indicates that the conservative region is restricted to the homeodomain even within the nematodes branch.The results showed that the HOX genes have great difference in nematode even they are conserved,suggesting that there's a distant relative or a faster rate of evolution between different nematodes.Phylogenetic tree analysis,however,showed that lin-39 could well reflect the genetic relationships within the nematode.2.The lin-39 genes expression in different developmental periods and sexual individuals was cleared by m RNA in situ hybridization.For the egg stage,lin-39 gene was expressed in middle anterior part of body.For J3 nematode,lin-39 gene was expressed in middle and posterior part of the body.For the adult nematode,lin-39 gene was expressed in the female vulva and tip of the male spicula.For the J2 and J4 nematode,lin-39 gene was not detected by m RNA in situ hybriidzaiton.3.It is the first time to use microinjection technique to analyze Bursaphelenchus xylophilus lin-39 gene expression in C.elegans.The promoter region only or full length region including promoter,intron and exon of pine wood nematode lin-39 gene was cloned the upstream of reporter gene by in-fusion cloning method.The expression vector was constructed successfully.Due to the technical bottleneck of pine wood nematode,the expression vector was microinjected into C.elegans gonad to investigate its dynamic expression at protein levels.Comparing with the data of m RNA in situ hybridization,pine wood nematode lin-39 dynamic expression has been clarified.4.The expression level of lin-39 gene in different developmental periods and sexes was investigated by quantitative real-time PCR.Lin-39 gene expression was high in egg stage,then maintained its stable expression level,which suggested that it was playing a role in body position and axial in the early embryonic development and the late organ development(such as vulva).5.The effects of lin-39 gene RNA interference in different developmental periods and sexual individuals were analyzed.In the embryonic stage,the treatment resulted in deformed embryo and a significant decrease in hatching rate to ± 2.5%.For the J2 nematode,treatment resulted in abnormal vulva.No significant abnormal phenotype was found in the J3 and J4 nematode.After being treated by lin-39 gene RNAi in J2 and J4 stage,mating quality of B.xylophilus was significantly compromised,indicated by the shortened copulation time.However,there was no significant difference in the fecundity and hatching rate of the eggs after mating.