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中国蚕学会第十届青年学术研讨会

作者：胡志刚 dong Zhanqi dong feifan Zhu Yan Chen Peng Lu Cheng 潘敏慧 State Key Laboratory of Silkworm Genome Biology Southwest University Key Laboratory of Sericultural Biology and Genetic Breeding Ministry of AgricultureSouthwest University

Ser/Thr protein phosphatase 2A（PP2A） is one of the type 2 protein phosphatases,which is required for many intracellular physiological processes and pathogen ***,the function of PP2A is unclear in silkworm,Bombyx ***,we cloned and identified BmPP2A,a PP2A gene from ***,which has two HEAT domains and a high similarity to PP2A from other *** results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut,and that Bombyx mori nucleopolyhedrovirus（BmNPV） infection induces down-regulation of BmPP2A ***,up-regulation of BmPP2A via overexpression significantly inhibited BmNPV *** contrast,down-regulation of BmPP2A via RNA interference and okadaic acid（a PP2A inhibitor） treatment allowed robust BmNPV *** is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A,a potential anti-BmNPV mechanism,and a possible target for the breeding of silkworm-resistant strains.

关键词： antiviral BmNPV Bombyx mori PP2A

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CRISPR/Cas9-mediated ie-0 and ie-2 dual mutation confers resistance to BmNPV in Transgenic Silkworm

CRISPR/Cas9-mediated ie-0 and ie-2 dual mutation confers res...

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中国蚕学会第十四届暨国家蚕桑产业技术体系家（柞）蚕遗传育种学术研讨会

作者： Zhanqi dong Zhigang Hu Qi Qin feifan dong Liang Huang Jiangqiong Long Peng Chen Cheng Lu Minhui Pan State Key Laboratory of Silkworm Genome Biology Southwest University Key Laboratory of Sericultural Biology and Genetic Breeding Ministry of Agriculture Southwest University

The CRISPR/Cas9 system has been widely used against a variety of infectious diseases as a therapeutic strategy. Here, we used the baculovirus DNA-replicating early transcriptional activators, immediate early-0(ie-0) and ie-2 genes, as double target sites to disrupt the Bombyx mori nucleopolyhedrovirus(BmNPV) genome and inhibit viral replication. We constructed a transgenic line expressing sgIE0-sgIE2 and hybridized with Cas9 transgenic lines to obtain a double positive transgenic Cas9(+)/sgIE0-sgIE2(+) line that could activate the CRISPR gene editing system. We first demonstrated that the system is capable of efficiently editing target genes and result in fragment deletions in the BmNPV genome. Mortality statistics showed that the survival rate of the transgenic Cas9(+)/sgIE0-sgIE2(+) line could reach more than 65% after inoculation with 1×10 occlusion bodies(OBs)/larva. Molecular analysis showed that BmNPV DNA replication and viral gene expression level in the transgenic Cas9(+)/sgIE0-sgIE2(+) line were significantly inhibited compared with the control Cas9(-)/sgRNA(-) line. These results indicated that IE-0 and IE-2, as baculovirus early transcriptional activators, can be used as target sites for gene therapy and that multi-gene editing may more effectively disrupt the viral genome and help breed disease resistance strain.

关键词： Bombyx mori BmNPV Transgenic CRISPR/Cas9 Antiviral therapy sgIE0-sgIE2

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Excision of Nucleopolyhedrovirus form Transgenic Silkworm using the CRISPR/Cas9 System30

Excision of Nucleopolyhedrovirus form Transgenic Silkworm us...

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中国蚕学会第十四届暨国家蚕桑产业技术体系家（柞）蚕遗传育种学术研讨会

作者： Zhanqi dong feifan dong Xinbo Yu Liang Huang Yaming Jiang Zhigang Hu Peng Chen Cheng Lu Minhui Pan State Key Laboratory of Silkworm Genome Biology Southwest University College of Biotechnology Southwest University Key Laboratory of Sericultural Biology and Genetic Breeding Ministry of Agriculture Southwest University

CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus(BmNPV) immediate early-1(ie-1) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose(LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies(OB). The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

关键词： Bombyx mori BmNPV CRISPR/Cas9 Transgenic Antiviral therapy

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CRISPR/Cas9 Mediated Disruption of the ie-0 and ie-2 as a Therapeutic Approach to BmNPV in Transgenic Silkworm

CRISPR/Cas9 Mediated Disruption of the ie-0 and ie-2 as a Th...

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中国蚕学会第十届青年学术研讨会

作者： Zhanqi dong Zhigang Hu Qi Qin feifan dong Liang Huang Jiangqiong Long Peng Chen Cheng Lu 潘敏慧 State Key Laboratory of Silkworm Genome Biology Southwest University Key Laboratory of Sericultural Biology and Genetic Breeding Ministry of AgricultureSouthwest University

CRISPR/Cas9 system is a powerful tool for the treatment of infectious *** our previous study,we knocked out the Bombyx mori nucleopolyhedrovirus(BmNPV) key genes and BmNPV-dependent host factor to generate transgenic antiviral *** further expand the range of target genes for BmNPV and more effectively prevent and control pathogenic infections,we performed gene editing and antiviral analysis by constructing a target-directed baculovirus early transcriptional activator immediate early-0(ie-0) and ie-2 transgenic silkworm *** hybridized it with Cas9 transgenic line to produce a double positive transgenic Cas9(+)/sgIE0-sgIE2(+) line that could activate the CRISPR gene editing *** first demonstrated that the system is capable of efficiently editing target genes and resulting in fragment deletions in the BmNPV *** rate of the transgenic Cas9(+)/sgIE0-sgIE2(+) line reached 65% after inoculation with 1×10 occlusion bodies(OBs)/*** analysis showed that BmNPV DNA replication and viral gene expression level in the transgenic Cas9(+)/sgIE0-sgIE2(+) line were significantly inhibited compared with the control Cas9(-)/sgIE0-sgIE2(-) *** results indicated that IE-0 and IE-2,as baculovirus early transcriptional activators,can be used as target sites for gene therapy and that multi-gene editing could expand the range of target sites for research to create silkworm resistance breeds.

关键词： Transgenic silkworm BmNPV CRISPR/Cas9 Multi-gene editing sgIE0-sgIE2

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泉州盆地地震效应的二维非线性分析

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地震工程与工程振动 2013年第3期33卷 95-102页

作者：董菲蕃陈国兴金丹丹南京工业大学岩土工程研究所江苏南京210009 江苏省土木工程防震技术研究中心江苏南京210009

基于ABAQUS显式有限元并行计算平台,采用大尺度二维精细化有限元非线性分析方法,研究泉州盆地地震效应特征。结果表明:(1)与基岩输入地震动相比,地表峰值加速度总体呈放大效应,且盆地不同位置的放大效应存在明显差异;(2)地表或基岩剧烈... 详细信息

基于ABAQUS显式有限元并行计算平台,采用大尺度二维精细化有限元非线性分析方法,研究泉州盆地地震效应特征。结果表明:(1)与基岩输入地震动相比,地表峰值加速度总体呈放大效应,且盆地不同位置的放大效应存在明显差异;(2)地表或基岩剧烈起伏及土层横向分布极不均匀处,地震动易产生局部聚集效应,相应地表地震动呈显著放大或缩小效应;地表加速度反应谱产生双峰甚至多峰现象。(3)在土层竖向、横向分布不均匀处,峰值加速度沿深度方向呈非单调递减,加速度反应谱沿深度存在较大差异。场地地震效应的二维非线性分析法能在一定程度上反映特殊场地条件对地震动效应的影响,该结果可为泉州盆地及类似盆地的场地设计地震动参数的确定提供参考依据。

关键词：盆地地震效应大尺度场地二维精细化有限元非线性分析峰值加速度加速度反应谱聚集效应

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Establishment of baculovirus-inducible CRISPR/Cas9 system for antiviral research in transgenic silkworms

Establishment of baculovirus-inducible CRISPR/Cas9 system fo...

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中国蚕学会第十四届暨国家蚕桑产业技术体系家（柞）蚕遗传...

作者： Zhanqi dong Liang Huang feifan dong Zhigang Hu Qi Qin Jiangqiong Long Mingya Cao Peng Chen Cheng Lu Minhui Pan State Key Laboratory of Silkworm Genome Biology Southwest University Key Laboratory of Sericultural Biology and Genetic Breeding Ministry of Agriculture Southwest University

The CRISPR/Cas9 system is a powerful genetic engineering technology that has been widely used in gene therapy, as well as in the development of novel antimicrobials and transgenic insects. However, several challenges such as lack of effective host target genes and off-target efficiency limit its application to insects. To overcome these challenges, we established a highly efficient virus-inducible CRISPR/Cas9 system in transgenic silkworms. This system includes a baculovirus-inducible promoter 39 K, which initiates transcription Cas9 protein, and U6 promoter, a target sgATAD3 A site of the ATPase family AAA domain-containing protein 3（ATAD3 A） gene. The double-positive transgenic line sgATAD3 A×39 K-Cas9（ATAD3 A-KO） was obtained by hybridization, which is inducible upon Bombyx mori nucleopolyhedrovirus（BmNPV） infection in silkworm. Further analysis showed that the BmNPV-inducible transgenic lines could significantly reduce off-target efficiency and exert no effect on economically relevant. Most importantly, this novel system can efficiently and consistently edit target genes and inhibit BmNPV replication as well as the stably expressed CRISPR/Cas9 system after inoculation with occlusion bodies（OB）. These results indicate that we have established a highly efficient BmNPV-inducible ATAD3 A-KO transgenic line with improved specificity for gene targeting and antiviral efficiency in transgenic silkworm, thereby providing insights into the treatment of infectious diseases and insect pest control.

关键词： Inducible CRISPR/Cas9 Transgenic Antiviral therapy BmNPV ATAD3A-KO

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Generation of resistance nucleopolyhedrovirus material in silkworm by CRISPR/Cas9

Generation of resistance nucleopolyhedrovirus material in si...

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中国蚕学会第九届青年学术研讨会

作者：董战旗 feifan dong Liang Huang Yaming Jiang Zhigang Hu Peng Chen 鲁成潘敏慧 State Key Laboratory of Silkworm Genome Biology Southwest University Key Laboratory of Sericultural Biology and Genetic Breeding Ministry of AgricultureSouthwest University

CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the *** recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus（BmNPV） immediate early-1（ie-1） gene in the silkworm,respectively,*** and obtained four transgenic hybrid lines by G1 generation hybridization:Cas9（-）/sgRNA（-）,Cas9（+）/sgRNA（+）,Cas9（-）/sgRNA（-）,and Cas9（+）/sgRNA（+）.We demonstrated that the Cas9（+）/sgRNA（+） transgenic lines effectively edited the target site of the BmNPV genome,and fragment deletion was observed after BmNPV *** antiviral analysis of Cas9（+）/sgRNA（+） transgenic hybrid lines showed a significant increase in survival rate and inhibition of viral gene expression after BmNPV *** analysis of economic characters and off-target efficiency of Cas9（+）/sgRNA（+） transgenic hybrid line showed no significant difference compared with the normal *** findings indicated that CRISPR/Cas9-mediated genome engineering effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

关键词： Bombyx mori BmNPV CRISPR/Cas9 Transgenic Antiviral therapy

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Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication

Baculovirus LEF-11 nuclear localization signal is important ...

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中国蚕学会第九届青年学术研讨会

作者： Tingting Chen Zhanqi dong Nan Hu Zhigang Hu feifan dong Yaming Jiang Jun Li Peng Chen Cheng Lu Minhui Pan State Key Laboratory of Silkworm Genome Biology Southwest University Sichuan Provincial Suji Silkworm Farm Key Laboratory of Sericultural Biology and Genetic Breeding Ministry of AgricultureSouthwest University

Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA ***,the characteristics of its nuclear localization signal and its impact on viral DNA replication are *** the present study,systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences（cNLSs）,which were also detected in a few alphabaculovirus *** of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted ***,Bombyx mori nucleopolyhedrovirus（BmNPV） LEF-11 could be transported into the nucleus during viral infection in the absence of a *** investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA *** findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms.

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Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication

Baculovirus LEF-11 nuclear localization signal is important ...

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中国蚕学会第十四届暨国家蚕桑产业技术体系家（柞）蚕遗传育种学术研讨会

Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences(cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus(BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a c NLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms.

关键词： Bombyx mori Baculovirus BmNPV LEF-11 Nuclear localization signal

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河漫滩-阶地复合地形场地的非线性地震反应特征

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应用基础与工程科学学报 2015年第1期23卷 136-144页

作者：刘庆忠金丹丹陈国兴董菲蕃安徽省地震工程研究院安徽合肥230031 南京工业大学岩土工程研究所江苏南京210009 江苏省土木工程防震技术研究中心江苏南京210009

以滁州市地震小区划场地为原型,针对由河漫滩、阶地、波状起伏的低丘等组成的典型复合场地,基于ABAQUS软件显式有限元并行计算平台,建立了大尺度场地二维模型,考虑土体的非线性特征,以SH波作为输入地震动,分析了河漫滩-阶地复合场地地... 详细信息

以滁州市地震小区划场地为原型,针对由河漫滩、阶地、波状起伏的低丘等组成的典型复合场地,基于ABAQUS软件显式有限元并行计算平台,建立了大尺度场地二维模型,考虑土体的非线性特征,以SH波作为输入地震动,分析了河漫滩-阶地复合场地地震动力响应特征.结果表明,(1)与基岩地震动峰值加速度相比,土层近地表范围内(15m以浅)峰值加速度均表现出明显的放大现象,15m以深土层地震动放大效应随深度增加而逐渐减弱,且随深度增大,PGA放大系数值逐渐小于1.0;(2)土层峰值加速度随着深度增加非单一递减,局部土层深度地震动出现聚集效应,这一现象在河漫滩区表现比较明显;(3)场地不同区域地震动放大效应差异较大,河漫滩区地震动放大效应要明显比阶地低丘区更大;且输入地震动峰值相同时,河漫滩区反应谱特征周期要大于阶地区域,两者差距达0.05—0.15s;(4)随着输入地震动增加,反应谱谱峰向长周期方向移动明显.

关键词：非线性场地地震反应复合地形河漫滩阶地 PGA随深度变化特征周期

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