| Silk fabrics are popular because of their lightness,comfort,skin-friendliness and health.China is the origin of sericulture,and silk has been exported to Europe in ancient times.China is the largest silk producer and trading country in the world.The current sericulture industry still maintains a good development trend,playing an important role in poverty alleviation and the revitalization of rural areas.In the breeding of silkworm,the important reason that affects the stable development of production is silkworm disease.Among various silkworm diseases,blood type pus caused by Bombyx mori nuclear polyheydrosis virus（BmNPV）accounts for more than 70%of the total loss of silkworm disease.The study of the pathogenesis of BmNPV-induced blood type pus in silkworm and the molecular mechanism of BmNPV resistance inheritance in different silkworm species is of great significance in breeding of resistant varieties and reducing the loss of blood type pus in the sericulture industry.At the same time,it can also be used as a reference for the prevention and control of silkworm virus diseases.In this study,the resistant strain AN and the susceptible strain C108,which have great differences in resistance to BmNPV,were used to construct BmNPV resistant isolates and resistant near-isogenic lines by means of genetic crossing.Based on genomic linkage analysis and mapping analysis,2b-RAD genome sequencing analysis,fine mapping of BmNPV resistance was performed.Transcriptome sequencing analyzes the difference in host（Bombyx mori）and pathogen（BmNPV）gene expression.Targeting the silkworm Bmvncp gene with significant expression differences is over-expression and CRISPR/Cas9knock-down expression in BmN cells to analyze its effect on BmNPV infection,replication and proliferation Impact.In addition,the possible mechanism of the high expression of Bmvncp gene on the inhibitory effect of virus replication and proliferation was also analyzed from the expression of related genes in the silkworm IMD signaling pathway.The relevant results help to deepen the understanding of the resistance mechanism of silkworm BmNPV.The main research content and experimental results of this subject are as follows:（1）Genome fine mapping of BmNPV resistance in silkworm AN strainBased on the genetic analysis of BmNPV resistance genome mapping of AN strain and2b-RAD genome sequencing conducted by our team in the early stage,this study used BmNPV susceptible strain Bombyx mori C108（P2）as the backcross variety and the resistant strain（P1）repeatedly crossed 1500 heads of BC4M positioning group were constructed for fine positioning.Among the molecular markers S2800-9 and S2801-35 on the nearest sides of the obtained AN’s resistance to BmNPV,8 new molecular markers of SNP and In Del that can have HRM polymorphism were newly obtained.The BmNPV resistance trait of the AN strain was finely mapped between the C8911 and C8866 markers on chromosome 27.The physical distance between the two molecular markers was 45 kb,and the genetic distance between the mapping gene and the C8911 marker and C8866marker was 0.1 c M and 0.4c M,there are 10 genes in the 45kb range of the marked interval.Cross the susceptible silkworm strain C108 with the resistant silkworm strain AN,and then repeat backcross C108.At the same time,each generation of BmNPV-resistant silkworm was screened,and a near-isogenic line C108＿AN with high resistance to BmNPV was constructed;C108 and C108＿AN were used to attack the 5th instar silkworm by feeding mulberry leaves impregnated with 1×108/m L suspension of BmNPV polyhedons orally,and transcriptome sequencing was performed on the midgut tissues at 12h,24h,36h,48h and60h after infection.The expression analysis of 10 genes in the two marker intervals of C8911 and C8866 showed that no stable differential expression was found in the 10 genes,and no sense mutation was found in the m RNA coding region of each gene.Based on this,it is believed that these 10 genes have no relationship with the characteristics of AN strain’s high resistance to BmNPV,and the main BmNPV resistance genes of AN strain still need to be further explored and determined by new research strategies.（2）Analysis of host and viral gene expression in the midgut transcriptome of the silkworm infected with BmNPVAnalysis of the number of BmNPV genes detected in the midgut tissue transcriptome at different times of BmNPV infection showed that 115 BmNPV genes were detected in the midgut tissue of PI12h in the C108 strain.The results show that it may have reached the budding virus particle replication stage.At PI24h,all 137 genes of BmNPV were detected,and the proliferation and replication of inclusion body virus had appeared in the midgut tissue.In addition,101 BmNPV genes were detected in the PI12h midgut tissue of the BmNPV resistance near-isogenic line C108＿AN,which lacked some genes required for budding virus particle formation;At PI24h,there were only 15 BmNPV genes detected in the samples,and the average number of reads detected in each sample was only 6.7,indicating that the gene expression and DNA replication of BmNPV in the midgut tissue of the AN strain was extremely inhibited;At PI36h～PI60h,the number of virus genes detected in the samples was only 15-91,which did not reach the number of genes required to complete BV virus replication.Therefore,it can be considered that although BmNPV can establish infection in the midgut tissue of the resistant strain C108＿AN,it cannot effectively complete genome replication,nor reach the budding virus proliferation stage,and cannot achieve secondary infection between cells.The transcriptome of the midgut tissues infected with BmNPV at different times found that there were many differentially expressed genes in C108 and C108＿AN.Among them,KWMTBOMO12431,KWMTBOMO04643,KWMTBOMO04149,KWMTBOMO04878,KWMTBOMO08182,etc.had relatively stable up-regulated expression at PI12h,PI24h,and PI36h,which may be in the AN variety has an important role in the resistance performance of BmNPV.（3）Identification and analysis of the correlation between Bmvncp gene expression in silkworm and BmNPV infection and proliferationThe KWMTBOMO12431 gene significantly up-regulated in C108＿AN samples was the nucleocapsid protein gene encoding 230Aa of Bombyx mori virus.qRT-PCR analysis showed that the expression of this gene in the midgut,fat body,body wall and other tissues of AN strain was higher than that of C108.Gene cloning and sequencing results showed that the coding region sequence of Bmvncp gene of C108,AN and p50T was consistent,and KWMTBOMO12431 was named Bmvncp.Furthermore,at the cellular level,through the construction of an overexpression vector system p IZT/V5-His-m Cherry-Bmvncp and a gene editing system CRISPR/Cas9,the expression analysis of the Bmvncp gene was performed and the function of the Bmvncp gene was verified by BmN silkworm cells.The experimental results show that overexpression of Bmvncp gene in BmN cells can significantly reduce the replication and proliferation of BmNPV that infects cells;BmN cells were transfected with Bmvncp-targeted CRISPR/Cas9plasmid.Some cells were able to achieve plasmid co-transfection,but the stable increase of BmNPV proliferation and gene expression in the cells was not found.It is speculated that the increase of Bmvncp gene expression has an inhibitory effect on the replication of virus genes and the proliferation of virus particles.It is speculated that the increase of Bmvncp gene expression is beneficial to the inhibition of viral gene replication and viral particle proliferation.In addition,the original BmN silkworm cells that are sensitive to BmNPV further reduce the expression of Bmvncp gene and cannot promote the replication and proliferation of the virus.（4）Correlation analysis between Bmvncp gene expression and IMD pathwayThe immunodeficiency（IMD）signaling pathway plays a vital role in the immune defense of the silkworm.After overexpression of Bmvncp gene in BmN cells,it can significantly increase the expression level of Dredd gene in the host IMD signaling pathway,but has no effect on the transcription level of FADD gene.The expression levels of PGRP-LB and PGRP-LF genes are so low that they cannot be detected by qRT-PCR.Comprehensive analysis of the above results,it is believed that high-level expression of Bmvncp gene in silkworm cells or tissues cannot prevent BmNPV infection,but can reduce viral gene expression and virus proliferation after BmNPV infection.Bmvncp gene product may trigger immune mechanism through IMD signaling pathway. |
