| Pine wilt disease,is a worldwide quarantine disease.The disease is a complex disease system dominated by Bursaphelenchus xylophilus and integrated with host pine,associated bacteria,vector insects,and associated fungi,as well as environmental factors.Rapid development and efficient mating are the basis of the population expansion and disease epidemic of B.xylophilus.Our team analyzed the sequencing results of the B.xylophilus transcriptome and found that the expression levels of Bxy-egl-30varied among different ages and sexes,its homolog gene encoding Gαqsubunit plays a key role in the development and signal transduction of various species including plant and animal.However,no one has reported on the role of the gene in B.xylophilus.In this thesis,the Bxy-egl-30 gene was cloned for the first time,after a deep study on the spatiotemporal dynamic expression characteristics and biological functions of the Bxy-egl-30 gene,the results are as follows:1.The Bxy-egl-30 gene and its amino acid sequence and protein structure were analyzed by the bioinformatics method.The result of gene sequence analysis showed that the length of the coding region of Bxy-egl-30 is 1 128 bp,encoding 375 amino acids;Amino acid sequence analysis showed that Bxy-egl-30 belongs to G＿alpha and P-loop＿NTPase superfamily,and there were 20 kinds of amino acids encoded by the gene,among which the number of glutamic acids was the largest,and the secondary structure of the protein encoded by Bxy-egl-30 was mainly composed of alpha-helix,Bxy-egl-30 has three typical conserved domains of EGL-30 family proteins,and Bxy-EGL-30 has three conserved domains of EGL-30 family proteins,GTP/Mg2+binding domain,adenylate cyclase binding site and transmembrane receptor binding site;The phylogenetic tree showed that the Bxy-EGL-30 gene was highly homologous with the EGL-30 amino acids of nematodes.The Bxy-EGL-30 protein structure simulated with the Swiss database contains a GTP binding site.2.The results of in situ hybridization and RT-q PCR showed that the Bxy-egl-30 gene was expressed at different ages of B.xylophilus.The expression of Bxy-egl-30 in the embryonic stage,J2,and J3 were mainly in the midgut and body surface of the worm,and the expression level in J2 was the highest,while that in the embryonic stage and J3 decreased,in turn,it was concentrated expressed in the back end of the body of J4 and the tail,and the front end of the body was light,but the expression was low in the adult stage,male adult only expressed in the latter part of the intestine,and female only expressed in the vulva.3.Bxy-egl-30 gene is involved in the regulation of B.xylophilus growth,motility,mating,and pathogenicity by RNA interference.Compared with the normal developing embryos,the hatching rate of the embryos with Bxy-egl-30 gene interference was decreased by 10.88%（95.18%±1.59%in the control group,and 84.30%±0.01%in the interference group）,breaking through the eggshell is difficult;After the larvae of J2 and J3 were disturbed,their locomotion ability was decreased（J2 control group:12.70±0.93 times/min,interference group:8.35±0.88 times/min;J3 control group:20.62±0.41times/min,interference group:16.12±0.57 times/min）;The results of the study on mating behavior showed that compared with the two control groups,the average mating frequency,the spawning rate,and the average mating time of the female group were increased,and the mismatch time of the male group was increased,but the rate of mating error and oviposition decreased;The pathogenicity experiment showed that after the RNAi of Bxy-egl-30,the virulence of B.xylophilus decreased,and the onset of black pine in the RNAi group was later than that in the control group,and the death time was delayed by 9 days.These results suggest that the Bxy-egl-30 gene plays an important role in B.xylophilus development and mating,and indirectly affects the pathogenicity of B.xylophilus.The results not only reveal the spatiotemporal dynamic expression characteristics and biological functions of the Bxy-egl-30 gene but also provide the theoretical basis for further control of pine wilt disease at the molecular level. |
