Expression Characteristics Of Madd-4 In Pinewood Nematode,Bursaphelenchus Xylophilus And Its Functional Validation In Embryonic Stage

Pine wilt disease is a quarantine disease caused by pinewood nematode,Bursaphelenchus xylophilus.In the research of reproductive biology of B.xylophilus,it was found that the embryogenesis of pinewood nematode,B.xylophilus,could be significantly prohibited by blue light.Results of RNA-sequence revealed that the expression of many metabolism-related genes,with particular respect to madd-4,was dramatically depressed when the eggs were treated under blue light.In the present study,madd-4 was cloned,its evolutionary status was analyzed,the corresponding plasmid expression vectors were constructed,and its spatio-temporal expression characteristics during the whole life from egg to adult of B.xylophilus were investigated by in situ hybridization test and transgene microinjection experiments.In addition,the crucial roles of madd-4 in regulating the embryogenesis of B.xylophilus were validated by RNA interference with soaking.Results of this paper will not only be able to illuminate the crucial roles of madd-4 during embryogenesis of B.xylophilus,but also to make a foundation for revealing the underlying mechanism of light inhibition on population growth of B.xylophilus,as well as to provide ideas for developing novel agents and designing specific measures against pine wilt disease at molecular level,and to be used as reference for related research on other organisms.The main results are as follows:1.The gene structure of madd-4 in B.xylophilus was highly similar with that of Ce-madd-4 in C.elegans since they both have two different transcripts of madd-4-long and madd-4-short,and madd-4-long shared the 2-10 exons of madd-4-short in both of these two species,suggesting that madd-4 may play the same regulatory roles in B.xylophilus and C.elegans.2.madd-4 is stably expressed in the ring of nervus centralis in head and pharynx and the motor neuron cells of the dorsal-ventral nerve cord during the whole life of B.xylophilus from embryo to adult.3.madd-4 plays important roles in 3 aspects during the course of embryogenesis,including regulating the extension of muscle arm toward the ring of nervus centralis and dorsal-ventral nerve cord,guiding the development of particular tissue in the head and pharynx,as well as controlling the axonal extension and the signal transduction toward postsynaptic muscle cells.In conclusion,madd-4 is an important gene in regulating the muscle development during the embryogenesis,and can be expressed stably in the whole life of B.xylophilus.