| Pine wilt disease is one of the destructive diseases of pine that infected by Bursaphelenchus xylophilus.There has caused great economic losses to forestry since the disease afterintruducting into our country,and the situation of preventing and controlling pine wilt disease is still existed.In this study,Transcriptome sequencing was used in this study,analysis genes and its route of participation that related with development in pine wood nematode.We hope to provide relevant theoretical and technical support for further study of pine wood nematode development characteristics,and strategies and techniques for prevention and control through developmental intervention,et al.Based on the transcriptome sequencing about B.xylophilus eggs,two instar larvae and adults which obtained in the condition of continuous cultivation in 25 ?,and the gene difference measured according to the function and regulation of the pathway enrichment summary,results show that B.xylophilus begins to enter the vigorous period of cell differentiation from egg development to two instar larvae,with the development of nematode to adult stage,its cell differentiation was decreased;with the development of eggs about B.xylophilus to two instar larvae,the genes let-60 and Ras-2 that related with sexual development up-regulated,and when developing into adults,the expression levels of the two were lower than those in the egg;there are important differences in the expression levels of genes related with nervous system in three insect states,With the development of eggs about B.xylophilus to two instar larvae,it is up-regulated in many genes associated with neural development.Transcriptome sequencing results showed that in the development process of pine wood nematode involved in different neurotransmitter and receptor related gene expression,so we analysis two genes related with neurotransmitter: 5-serotonin receptor gene cloning ser-1sequence measured online comparison results show that with the most similar is the CDs sequence of Caenorhabditis elegans 5-HT protein CBR-SER-1;On acetylcholinesterase gene sequencing of ACE-1 obtained clones were compared that ACE1 nematode acetylcholinesterase gene amino acid sequence similarity with the sequence of GeneBank ID:GU166345.1 to 99%.We predict the structure and characteristics of the product would give a hand for later function verification and interpretation.In this study,we design siRNA for acetylcholinesterase gene ACE-1And then we use the method of RNAi to investigate the effect of ace-1 on the motility of B.xylophilus.After siRNA exposure to two instar larvae of acetylcholinesterase gene ACE-1,the body bendingtimes of nematodes was 32.24 times /min,significantly lower than those of the matched group after 24 hours,and the frequency of head oscillation was significantly increased,which was2.22 times /min.After 120 hours,the adults showed the same trend,the body bending times reduced to 33.88 times/min and the head swing frequency upgraded to 3.34 times/min.The body bending times of larvae3 and adult was decreased compared with the untreated group of nematodes,respectively be 58.04times/min and 39.38times/min;The head swing frequency increased,respectively be 2.75times/min and 2.13times/min.Results show that the interference of ace-1,causing the behavior of Bursaphelenchus xylophilus peristalsis is abnormal.Therefore,there's the relevant between acetylcholine and pine wood nematode motility,interfering the normal metabolism of acetylcholine can lead to abnormal behavior of pine wood nematode motility and present regularly characteristics.This study makes the nervous system regulation of movement as a starting point,to lay a good foundation for the study of pine wood nematode migration diffusion regularity and pathogenic mechanism. |
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