| Pine wood nematode(Bursaphelenchus xylophilus),is a kind of extremely dangerous invasive harmful organism that seriously endangers pine forests in China and many countries and regions in the world.Pine wilt disease caused by pine wood nematode can cause pine tree decay and wilt,and eventually wither and die,which has a serious impact on forest health and ecological balance in China and many countries and regions in the world.Low temperature is one of the important environmental signals that affect the survival,development and diffusion of pine wood nematode under natural conditions.It is also the environmental condition that pine wood nematode needs to adapt to and overcome when it continuously invades into the cold climate area and survive through the winter.At the same time,low temperature is also one of the important environmental signal factors that induce the second instar larvae of pine wood nematode to change into the third instar larvae,and then promote the pine wood nematode to develop into the fourth instar larvae,which are carried and diffused by Alternaria alternata.However,there are few researches on the molecular mechanism of low temperature response of pine wood nematode.The study of the molecular mechanism of the response of pine wood nematode to the low-temperature environment signal will help to fill the blank of the molecular mechanism research in related fields,improve the theoretical system of the basic biological research of pine wood nematode,and provide the theoretical basis for the development of the control means based on the low-temperature molecular mechanism of pine wood nematode response and the development of new molecular control targets.In this paper,pine wood nematode was taken as the research object.Through the statistics of the survival rate of pine wood nematode at low temperature and normal temperature,it was found that the survival rate of pine wood nematode at 5℃ constant low temperature had significantly increased.In order to further study the molecular mechanism of low-temperature response of pine wood nematode,high-throughput transcriptome sequencing technology was used to obtain the transcriptome data of pine wood nematode in low-temperature treatment group and normal temperature control group.Through the analysis of differentially expressed genes,several metabolic pathways and gene families closely related to the response of pine wood nematode to low temperature were screened out.Six GPCR genes,three cGMP signal pathway genes and one Bx-SCD gene related to fatty acid metabolism were cloned and identified.The low temperature response patterns of 6 GPCR genes,3 cGMP signal pathway genes,4 IIS signal pathway genes and 9 stress resistance related genes were analyzed.The key functions of one GPCR gene,three cGMP signal pathway genes and three cytochrome P450 family genes in the response of pine wood nematode to low temperature were verified.The main results are as follows:1.Through the statistics of the survival rate of pine wood nematode under low temperature,it is found that the survival rate of pine wood nematode under 5℃C non-freezing constant low temperature is significantly higher than that under 25 ℃ normal temperature,which indicates that 5℃ constant low temperature can prolong the life of pine wood nematode,which provides a basic reference for the subsequent research on the molecular mechanism of low temperature response of pine wood nematode.2.Through the transcriptome sequencing and library construction of pine wood nematode treated with low temperature and normal temperature,312138316 high-quality reads were obtained,and 432 significantly different expression genes were identified.Through the analysis of differentially expressed genes,we found several metabolic pathways and several important genes that may be closely related to the response of pine wood nematode to low temperature,including genes and pathways related to energy metabolism,such as environmental signal perception,stress adaptation amino acids,sugars and fats,and some genes regulating the secondary metabolic process.These results provide basic data support and reference for the identification and functional verification of key genes in the subsequent cold response process.3.Six GPCR genes of pine wood nematode were screened and identified according to the characteristics of 7 times transmembrane receptors.The results of fluorescence quantitative PCR showed that the six GPCR genes were positively responsive to low temperature stress.DsRNA produced by specific primers can significantly reduce the transcription level of BxGPCR17454,and RNAi of BxGPCR17454 can significantly reduce the survival rate of pine wood nematode at low temperature.The GPCR gene of pine wood nematode is closely related to the low temperature response process.BxGPCR17454 is one of the key genes regulating the low temperature response process of pine wood nematode.4.Three important genes Bx-daf-11,Bx-tax-2 and Bx-tax-4 in the cGMP signaling pathway of pine wood nematode were cloned and identified by homologous sequence retrieval.The Bx-DAF-11,Bx-TAX-2 and Bx-TAX-4 proteins showed high identity with the homologous proteins of other nematodes,and the sequence characteristics are consistent with the typical characteristics of homologous genes.Bx-daf-11 may encode a membrane binding form of guanylate cyclase,which can bind to extracellular ligands and synthesize cGMP.Bx-tax-2 and Bx-tax-4 may encode membrane-bound ion-gated channels with cyclic nucleotide binding domains,which may be regulated by cGMP to control ion flow inside and outside cells.The results of fluorescent quantitative PCR showed that the three cGMP signaling genes were positively responsive to low temperature stress,and the transcription trend was the same.The transcription levels of four genes Bxdaf-16,Bxage-1,BxAkt and BxSgk-1 in IIS signaling pathway have significant changes,but the response time and trend are not the same,indicating that IIS signaling pathway is probably one of the important pathways involved in the response process of pine wood nematode to low temperature.The results of RNAi test of Bx-daf-11,Bx-tax-2 and Bx-tax-4,three genes of cGMP signaling pathway,showed that the dsRNA of Bx-daf-11,Bx-tax-2 and Bx-tax-4 produced by the design of specific primers could significantly reduce the transcription level of corresponding genes.The survival rate of pine wood nematode in Bx-daf-11,Bx-tax-2 and Bx-tax-4 gene silenced group decreased significantly at low temperature,which indicated that these three genes of cGMP signaling pathway were the key genes regulating the response process of pine wood nematode to low temperature.5.The gene Bx-SCD of stearoyl coenzyme A desaturase of pine wood nematode was cloned and identified by homologous sequence search.The Bx-SCD protein encoded by Bx-SCD has high homology with the homologous proteins of other nematodes,and its sequence has the same typical characteristics as other homologous genes.It contains a conserved fatty acid desaturase catalytic domain,which may have the function of catalyzing the decrease of fatty acid saturation.It belongs to the plasma membrane fatty acid desaturase domain superfamily.The results of fluorescence quantitative PCR showed that Bx-SCD,which are related to lipid metabolism,and BxPrx,Bxy-ctl-1,Bx-sod-1,Bx-sod-2 and Bx-sod-4,BxCYP33C9,BxCYP33C4 and BxCYP33D3 are related to the resistance to cold stress.The process of fatty acid metabolism regulated by Bx-SCD,antioxidant regulation regulated by BxPrx,Bxy-ctl-1,Bx-SOD-1,Bx-SOD-2 and Bx-SOD-4,and related metabolism regulated by BxCYP33C9,BxCYP33C4 and BxCYP33D3 may be one of the important cytoprotective mechanisms of pine wood nematode at low temperature.The results of gene silencing test showed that the gene silencing of BxPrx could not reduce the survival rate of pine wood nematode at low temperature.RNAi of BxCYP33C9,BxCYP33C4 and BxCYP33D3 can significantly reduce the survival rate of pine wood nematode at low temperature,which is one of the key genes regulating the response process of pine wood nematode to low temperature,and further proves the potential value of these three cytochrome P450 genes as control targets of pine wood nematode. |
