Expression And Regulatory Functions Of W03G11.3 Gene In Two Nematodes

Bursaphelenchus xylophilus is the causative agent of pine wilt disease.At present,pine wilt disease is seriously occurring in China,causing huge economic losses.Previous studies have found that blue light can significantly inhibit the embryonic development of pine wood nematode.Further studies have found that the expression of many genes in pine wood nematode embryos is down-regulated after blue light treatment,and the expression of W03G11.3 gene is significantly reduced.On the basis of previous studies,this paper analyzes the evolutionary status of W03G11.3 gene with Bursaphelenchus xylophilus and model nematode Caenorhabditis elegans,and studies the expression and regulation of this gene in two nematodes;The expression of W03G11.3 gene in the whole life cycle of two nematodes was analyzed by Semi Quantitative PCR.The plasmid expression vector of W03G11.3 gene was constructed,and the temporal and spatial dynamic expression characteristics were analyzed by transgenic microinjection and in situ hybridization.RNAi technology was used to study the regulatory function of this gene in the development of two nematodes.The specific research results are as follows:1.The results of Semi Quantitative PCR showed that the W03G11.3 gene was expressed in various stages of the development of B.xylophilus and C.elegans.In the B.xylophilus,the expression level was stable from the embryonic stage to the 2nd instar,the expression level was significantly increased in the 3rd instar,and the expression level was stable in the 3rd instar to adult stage,while there was no differential expression between male and female adults.B.xylophilus,the expression level of C.elegans from embryonic stage to adult stage tends to be stable,but it shows differential expression between hermaphroditic individuals and male individuals,and the expression level is higher in hermaphroditic individuals.2.In-situ hybridization results indicated that the W03G11.3 gene was expressed in the early mulberry body wall muscle precursor cells of the B.xylophilus embryo.W03G11.3 gene was expressed in body wall muscle and intestinal.3.The results of transgenic microinjection study showed that the W03G11.3 gene was expressed in the early comma stage of C.elegans embryos,and it was stable from embryonic to adult stage,mainly concentrated in body wall muscle,tail cloaca control muscle and intestinal muscle.The expression is also expressed in the smooth muscle of the adult uterus.4.The results of RNAi showed that the expression of W03G11.3 gene was disturbed by double-stranded RNA,and the hatching rate of pine wood nematode within 30 h decreased from 85.36% to 50.07%,and resulted in 2 instar and adult nematode within 20 s.The frequency of head swing decreased from normal 16.5,20.1 times to 2.3,7.3 times,and the number of eggs laid by adults within 12 h decreased from the normal 13.7 to 2.8;in contrast,the hatching rate of C.elegans embryos was There was a decrease,but it was not very obvious,but the number of eggs laid by adults within 12 h decreased from 9.3 to 3.9,and the total number of eggs laid was significantly reduced.The above results not only clarified the temporal and spatial dynamic expression characteristics of W03G11.3 gene in pine wood nematode and C.elegans and its regulation of individual development,but also provided a theory for further revealing the intrinsic mechanism of blue light inhibiting the growth of two nematodes populations.