Bombyx mori cytoplasmic polyhedrosis virus(BmCPV)is a major pathogen of the economic insect silkworm,Bombyx mori.Virus‐encoded microRNAs(miRNAs)have been proven to play important roles in host–pathogen interactions....Bombyx mori cytoplasmic polyhedrosis virus(BmCPV)is a major pathogen of the economic insect silkworm,Bombyx mori.Virus‐encoded microRNAs(miRNAs)have been proven to play important roles in host–pathogen interactions.In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA,BmCPV‐miR‐1,from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR(qPCR)and investigated its functions with qPCR and lentiviral expression systems.Bombyx mori inhibitor of apoptosis protein(BmIAP)gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′untranslated region.It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae.At the same time,it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics.Furthermore,BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm.In the midgut of BmCPV‐infected larvae,BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene.With the viral genomic RNA segments S1 and S10 as indicators,BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm.These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression,providing the virus with a better cell circumstance for its replication.展开更多
dsRNA segment Ⅴ was separated from Bombyx mori cytoplasmic polyhedrosis virus(BmCPV)of Chinese Guangzhou strainAfter ligation with a ssDNA adaptor,the cDNA fragments overlapping full-length segment Ⅴ of BmCPV ...dsRNA segment Ⅴ was separated from Bombyx mori cytoplasmic polyhedrosis virus(BmCPV)of Chinese Guangzhou strainAfter ligation with a ssDNA adaptor,the cDNA fragments overlapping full-length segment Ⅴ of BmCPV were acquired by RT-PCR amplificationSegment Ⅴ is 2852 nucleotides long and possesses a single open reading frame encoding a putative protein of 881 amino acidsComparison of amino acid sequences shows 97% homology with BmCPV Japanese isolate segments Ⅴ,86% and 36% with LdCPV-1 and LdCPV-14 segments Ⅴ respectivelyPart of sequence of BmCPV segment Ⅴ exhibits high homology with 2Apro motif of picornavirus members,which may suggest that there might exist some affinities evolutionally between both of展开更多
EcoR I-P fragment has been cloned from Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) genomic DNA and used as a probe. 0.5-kb and 1.1-kb fragments including p10 gene from Bombyx mori nuclear polyh...EcoR I-P fragment has been cloned from Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) genomic DNA and used as a probe. 0.5-kb and 1.1-kb fragments including p10 gene from Bombyx mori nuclear polyhedrosis virus (BmNPV) have been hybridized. The p10 ORF was located in the EcoR I-R fragment. Initiation codon ATG of p 10 from BmNPV has been mutated by PCR, and the ATG region became a Bgl II site. A novel transfer vector pBmAcPV-1 has been constructed using both the p10 5’-flanking region whose initiation codon ATG has been mutated with BmNPV and the p 10 3’-flanking region of AcMNPV. The vector can recombine with not only AcMNPV DNA to express foreign gene in Sf cells, but also BmNPV DNA to express foreign gene in Bm cells. CAT gene was expressed at high level in Bm cells under the control of the mutated p10 promoter of BmNPV.展开更多
【目的】探究家蚕Bombyx mori肠道特异表达的金属羧肽酶基因BmMCP 18的功能及对外源病毒侵染的抵抗机制。【方法】构建敲除BmMCP18的家蚕BmMCP18KO(C18KO),对C18KO和对照组野生型家蚕(C18KOC)的5龄幼虫中肠以及感染家蚕核型多角体病毒(B...【目的】探究家蚕Bombyx mori肠道特异表达的金属羧肽酶基因BmMCP 18的功能及对外源病毒侵染的抵抗机制。【方法】构建敲除BmMCP18的家蚕BmMCP18KO(C18KO),对C18KO和对照组野生型家蚕(C18KOC)的5龄幼虫中肠以及感染家蚕核型多角体病毒(Bombyx mori nuclear polyhedrosis virus,BmNPV)的家蚕BmMCP18KO(C18KOV)和对照野生型(C18KOCV)的5龄幼虫中肠进行转录组测序;分析差异表达基因并进行GO功能注释和KEGG通路富集;利用qRT-PCR验证相关基因表达。【结果】与对照组C18KOC相比,C18KO有75个基因上调表达,303个基因下调表达。与对照组C18KOCV相比,C18KOV有96个基因上调表达,57个基因下调表达。C18KOC vs C18KO比较组差异表达基因显著富集的GO条目为细胞外空间、细胞表面、肽交联和突触靶标识别等。C18KOCV vs C18KOV比较组差异表达基因富集最显著的GO条目为跨膜转运蛋白活性。C18KO 5龄幼虫中肠转录组中免疫通路、碳水化合物和能量代谢通路相关基因的表达比C18KOC的显著下调,包括Toll和Imd通路及MAPK通路相关基因,而泛素介导的蛋白质水解通路相关基因表达比C18KOC的显著上调。C18KOV 5龄幼虫中肠转录组中的能量代谢基因的表达比C18KOCV的显著上调。qRT-PCR验证结果表明转录组数据可靠。【结论】BmMCP18的功能涉及家蚕中肠的细胞识别、免疫调节和能量代谢,可能是通过参与中肠细胞免疫反应、能量和物质供应,从而影响蚕体对外源病原侵染的抵抗能力。展开更多
随着家蚕抗血液型脓病新品种‘华康’系列在全国的推广和应用,市面上出现了一些鱼龙混杂现象。针对这一问题,本研究用SLAF-seq(Specific-locus amplified fragment sequencing)技术对15个不同的家蚕品种(7个抗脓病品种和8个非抗脓病品种...随着家蚕抗血液型脓病新品种‘华康’系列在全国的推广和应用,市面上出现了一些鱼龙混杂现象。针对这一问题,本研究用SLAF-seq(Specific-locus amplified fragment sequencing)技术对15个不同的家蚕品种(7个抗脓病品种和8个非抗脓病品种)进行简化基因组测序,结果共获得所有检测品种的447 359个SNP位点,分析这些SNP标记在家蚕的28条染色体上的分布以及与抗脓病性能的相关性,得到第27群上的50个与抗BmNPV相关的SNP分子标记;经过PCR验证的结果与SLAF-seq测序结果一致,认为这些SNP可以作为鉴定和判别‘华康’系列品种的分子标记。展开更多
基金This work was financially supported by the National Natural Science Foundation of China(Grant No.31572463).
文摘Bombyx mori cytoplasmic polyhedrosis virus(BmCPV)is a major pathogen of the economic insect silkworm,Bombyx mori.Virus‐encoded microRNAs(miRNAs)have been proven to play important roles in host–pathogen interactions.In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA,BmCPV‐miR‐1,from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR(qPCR)and investigated its functions with qPCR and lentiviral expression systems.Bombyx mori inhibitor of apoptosis protein(BmIAP)gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′untranslated region.It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae.At the same time,it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics.Furthermore,BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm.In the midgut of BmCPV‐infected larvae,BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene.With the viral genomic RNA segments S1 and S10 as indicators,BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm.These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression,providing the virus with a better cell circumstance for its replication.
文摘dsRNA segment Ⅴ was separated from Bombyx mori cytoplasmic polyhedrosis virus(BmCPV)of Chinese Guangzhou strainAfter ligation with a ssDNA adaptor,the cDNA fragments overlapping full-length segment Ⅴ of BmCPV were acquired by RT-PCR amplificationSegment Ⅴ is 2852 nucleotides long and possesses a single open reading frame encoding a putative protein of 881 amino acidsComparison of amino acid sequences shows 97% homology with BmCPV Japanese isolate segments Ⅴ,86% and 36% with LdCPV-1 and LdCPV-14 segments Ⅴ respectivelyPart of sequence of BmCPV segment Ⅴ exhibits high homology with 2Apro motif of picornavirus members,which may suggest that there might exist some affinities evolutionally between both of
基金Project supported by the 8th Five-Year Plan Research Program of China.
文摘EcoR I-P fragment has been cloned from Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) genomic DNA and used as a probe. 0.5-kb and 1.1-kb fragments including p10 gene from Bombyx mori nuclear polyhedrosis virus (BmNPV) have been hybridized. The p10 ORF was located in the EcoR I-R fragment. Initiation codon ATG of p 10 from BmNPV has been mutated by PCR, and the ATG region became a Bgl II site. A novel transfer vector pBmAcPV-1 has been constructed using both the p10 5’-flanking region whose initiation codon ATG has been mutated with BmNPV and the p 10 3’-flanking region of AcMNPV. The vector can recombine with not only AcMNPV DNA to express foreign gene in Sf cells, but also BmNPV DNA to express foreign gene in Bm cells. CAT gene was expressed at high level in Bm cells under the control of the mutated p10 promoter of BmNPV.
文摘【目的】探究家蚕Bombyx mori肠道特异表达的金属羧肽酶基因BmMCP 18的功能及对外源病毒侵染的抵抗机制。【方法】构建敲除BmMCP18的家蚕BmMCP18KO(C18KO),对C18KO和对照组野生型家蚕(C18KOC)的5龄幼虫中肠以及感染家蚕核型多角体病毒(Bombyx mori nuclear polyhedrosis virus,BmNPV)的家蚕BmMCP18KO(C18KOV)和对照野生型(C18KOCV)的5龄幼虫中肠进行转录组测序;分析差异表达基因并进行GO功能注释和KEGG通路富集;利用qRT-PCR验证相关基因表达。【结果】与对照组C18KOC相比,C18KO有75个基因上调表达,303个基因下调表达。与对照组C18KOCV相比,C18KOV有96个基因上调表达,57个基因下调表达。C18KOC vs C18KO比较组差异表达基因显著富集的GO条目为细胞外空间、细胞表面、肽交联和突触靶标识别等。C18KOCV vs C18KOV比较组差异表达基因富集最显著的GO条目为跨膜转运蛋白活性。C18KO 5龄幼虫中肠转录组中免疫通路、碳水化合物和能量代谢通路相关基因的表达比C18KOC的显著下调,包括Toll和Imd通路及MAPK通路相关基因,而泛素介导的蛋白质水解通路相关基因表达比C18KOC的显著上调。C18KOV 5龄幼虫中肠转录组中的能量代谢基因的表达比C18KOCV的显著上调。qRT-PCR验证结果表明转录组数据可靠。【结论】BmMCP18的功能涉及家蚕中肠的细胞识别、免疫调节和能量代谢,可能是通过参与中肠细胞免疫反应、能量和物质供应,从而影响蚕体对外源病原侵染的抵抗能力。
文摘随着家蚕抗血液型脓病新品种‘华康’系列在全国的推广和应用,市面上出现了一些鱼龙混杂现象。针对这一问题,本研究用SLAF-seq(Specific-locus amplified fragment sequencing)技术对15个不同的家蚕品种(7个抗脓病品种和8个非抗脓病品种)进行简化基因组测序,结果共获得所有检测品种的447 359个SNP位点,分析这些SNP标记在家蚕的28条染色体上的分布以及与抗脓病性能的相关性,得到第27群上的50个与抗BmNPV相关的SNP分子标记;经过PCR验证的结果与SLAF-seq测序结果一致,认为这些SNP可以作为鉴定和判别‘华康’系列品种的分子标记。
