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Construction and detection of expression vectors of microRNA-9a in BmN cells

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2011年第7期12卷 527-533页

作者： Yong HUANG Quan ZOU Sheng-peng WANG Shun-ming TANG Guo-zheng ZHANG Xing-jia SHEN key laboratory of silkworm and mulberry genetic improvement Ministry of Agriculture Jiangsu University of Science and Technology Zhenjiang 212018 China School of lnformation Science and Technology Xiamen University Xiamen 361005 China Sericultural Research Institute Chinese Academy of Agricultural Sciences Zhenjiang 212018 China Animal Science and Technology College Henan University of Science and Technology Luoyang 471003 China

MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21–23 nucleotides in length,which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target ***,the functions of only a few miRNAs in organisms are ***,the expression vector of artificial miRNA has become a promising tool for gene function ***,a method for easy and rapid construction of eukaryotic miRNA expression vector was *** cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant *** enhanced green fluorescent protein (EGFP) gene was used as reporter *** Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post *** RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern *** showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.

关键词： miRNA-9a (miR-9a) EGFP gene Bombyx mori N (BmN) Cells Expression vector

PLA_(2) mediates the innate immune response in Asian corn borer, Ostrinia furnacalis

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Insect Science 2022年第1期29卷 245-258页

作者： Jia-Yue Ji Zhao-Hua Yin Sha-Sha Zhang Dong-Xu Shen Chun-Ju An Department of Entomology College of Plant ProtectionChina Agricultural UniversityBeijing100193China Jiangsu key laboratory of Sericutural Biology and Biotechnology School of BiotechnologyJiangsu University of Science and TechnologyZhenjiangJiangsu212018China key laboratory of silkworm and mulberry genetic improvement Ministry of Agriculture and Rural AffairsSericultural Research InstituteChinese Academy of Agricultural SciencesZhenjiangJiangsu212018China

The eicosanoid signaling pathway mediates insect immune reactions to a wide range of stimuli. This pathway begins with the biosynthesis of arachidonic acid (AA) from the hydrolysis of phospholipids catalyzed by phospholipase A_(2) (PLA_(2)). We report here that the PLA_(2) inhibitor, dexamethasone (DEX), impaired the innate immune response including nodulation, encapsulation, and melanization in Ostrinia furnacalis larvae, while AA partially reversed these effects of DEX. We cloned a full-length complementary DNA encoding a PLA_(2), designated as OfsPLA_(2), from O. furnacalis. The open reading frame of OfsPLA_(2) encodes a 195-amino acid residue protein with a 22-residue signal peptide. Sequence alignment analyses indicated that O. furnacalis PLA_(2) might be a Group III secretory PLA_(2). The highest transcript levels of OfsPLA_(2) were detected in the fat body, and its transcript levels increased dramatically after infection with Escherichia coli, Micrococcus luteus, or Beauveria bassiana. Recombinant OfsPLA_(2) significantly induced prophenoloxidase (PPO) activation in larval hemolymph in the presence of Ca^(2+) and encapsulation of agarose beads. Injection of recombinant OfsPLA_(2) into larvae resulted in increased transcript levels of attacin, defencin, and moricin-3 genes. Our results demonstrate the involvement of the eicosanoid signaling pathway in the innate immune response of O. furnacalis larvae and provide new information about the roles of O. furnacalis secretory PLA_(2) in activating PPO and antimicrobial peptide production.

关键词： eicosanoid signal pathway innate immune response Ostrinia furnacalis phospholipase A_(2)(PLA_(2))

Identification of a PP2A gene in Bombyx mori with antiviral function against *** nucleopolyhedrovirus

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Insect Science 2020年第4期27卷 687-696页

作者： Zhi-Gang Hu Zhan-Qi Dong Fei-Fan Dong Yan Zhu Peng Chen Cheng Lu Min-Hui Pan State key laboratory of silkworm Genome Biology Southwest UniversityChongqingChina key laboratory of Sericultural Biology and genetic Breeding Ministry of AgricultureSouthwest UniversityChongqingChina

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 mon' PP2A

Evolutionary and functional analyses of the interaction between the Bombyx mori inhibitor of apoptosis(IAP)and nucleopolyhedrovirus IAPs

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Insect Science 2020年第3期27卷 463-474页

作者： Peng Chen Tao-Tao Kang Xi-Yan Bao Zhan-Qi Dong Yan Zhu Wen-Fu Xiao Min-Hui Pan Cheng Lu State key laboratory of silkworm Genome Biology Southwest UniversityChongqingChina key laboratory of Sericultural Biology and genetic Breeding Ministry of AgricultureSouthwest UniversityChongqingChina

As an important insect immune response,apoptosis plays a critical role in the interaction between baculoviruses and insect *** reports have identified inhibitor of apoptosis(IAP)proteins in both insects and baculoviruses,but the relationship between these proteins is still not clearly ***,we found that insect IAP proteins were clustered with baculovirus IAP3,suggesting that the baculovirus iap3 gene might be derived from the Lepidoptera or *** demonstrated that Bombyx mori inhibitor of apoptosis(Bmiap)gene had an inhibitory effect on apoptosis in silkworm *** analysis of the effects of Bmiap genes on the proliferation of *** nucleopolyhedrovirus(BmNPV)showed that both the Bmiap and BmNPV iap genes increased BmNPV proliferation after BmNPV infected silkworm *** results also indicated that BmNPV IAP1 and IAP2 directly interacted with BmIAP in silkworm cells,implying that the Bmiap gene might be hijacked by BmNPV iap genes during BmNPV *** together,our results provide important insights into the functional relationships of iap genes,and improve our knowledge of apoptosis in baculoviruses and insect hosts.

关键词： baculovirus baculovirus-host interaction BmNPV Bombyx mori evolution inhibitor of apoptosis

Sob gene is critical to wing development in Bombyx mori and Tribolium castaneum

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Insect Science 2022年第1期29卷 65-77页

作者： Zhan-Feng Ye Pan Zhang Ting-Ting Gai Jing-Hou Lou Fang-Yin Dai Xiao-Ling Tong State key laboratory of silkworm Genome Biology Key Laboratory of Sericultural Biology and Genetic BreedingMinistry of Agriculture and Rural AffairsCollege of BiotechnologySouthwest UniversityChongqingChina

The development of insect appendages requires the expression of multiple genes in a strict spatial and temporal order. The odd-skipped family genes are vital transcriptional factors involved in embryonic development. The development and morphogenesis of the insect wing requires multiple transcription factors to regulate the expression of wing patterning genes at the transcriptional level. However, the function of odd-related genes in insect wing morphogenesis and development during postembryonic stages is unclear. We focused on the roles of the sister of odd and bowl (sob) gene, a member of odd-skipped family genes, during the wing morphopoiesis in Bombyx mori using the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 system and in Tribolium castaneum by RNA interference. The results showed that the wings were significantly smaller and degenerated, and wing veins were indistinct in the sob gene loss-of-function group in both B. mori and T. castaneum. Quantitative real-time polymerase chain reaction revealed that the Tcsob gene regulated the expression of wing development genes, such as the cht 7 and the vg gene. The findings suggest the importance of sob gene in insect wing morphology formation during postembryonic stages.

关键词： Bombyx mori sob gene transcription factor Tribolium castaneum wing

bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2016年第2期17卷 127-135页

作者： Chen CHEN Yang-yang FAN Xin WANG Fei SONG Tao JIANG Ping QIAN Shun-ming TANG Xing-jia SHEN Jiangsu key laboratory of Sericultural Biology and Biotechnology School of BiotechnologyJiangsu University of Science and Technology key laboratory of silkworm and mulberry genetic improvement Ministry of AgricultureSericultural Research Institute Chinese Academy of Agricultural Sciences

Based on bioinformatic analysis, we selected two novel micro RNAs（miRNAs）, bmo-miR-0001 and bmomiR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland（PSG）. Firstly, we examined the expression of bmo-miR-0001 and bmo-miR-0015 in 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of Bm Fib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pc DNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] and pri-bmo-miR-0015 expressing the plasmid pc DNA3.0 [ie1-egfp-pribmo-miR-0015-SV40]. Finally, the Bm N cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly（P〈0.05） in Bm N cells co-transfected by pc DNA3.0 [ie1-egfp-pri-bmomiR-0001-SV40] or pc DNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40] with p GL3.0 [A3-luc-Fib-L-3＇UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of Bm Fib-L in vitro.

关键词： Bombyx mori MicroRNA bmo-miR-0001 bmo-miR-0015 BmFib-L Regulation of expression

Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in silkworms

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Virologica Sinica 2019年第4期34卷 444-453页

作者： Zhanqi Dong Qi Qin Zhigang Hu Peng Chen Liang Huang Xinling Zhang Ting Tian Cheng Lu Minhui Pan State key laboratory of silkworm Genome Biology Southwest UniversityChongqing 400716China key laboratory of Sericultural Biology and genetic Breeding Ministry of AgricultureSouthwest UniversityChongqing 400716China

Recently the developed single guide(sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease(CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector(pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus(BmNPV) in insect *** screened the immediate-early-1 gene(ie-1), the major envelope glycoprotein gene(gp64), and the late expression factor gene(lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector(PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.

关键词： Bombyx mori Bombyx mori nucleopolyhedrovirus(BmNPV) Antiviral therapeutic CRISPR/Cas9 Multigene editing

E2F4 regulates the cell cycle and DNA replication in the silkworm,Bombyx mori

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Insect Science 2022年第4期29卷 1006-1016页

作者： Peng Chen Ling Wang Yan-Bi Long Guang-Yan Liang Xiu Yang Zhan-Qi Dong Xia Jiang Yan Zhu Min-Hui Pan Cheng Lu State key laboratory of silkworm Genome Biology Southwest UniversityChongqingChina key laboratory of Sericultural Biology and genetic Breeding Ministry of Agriculture and Rural AffairsSouthwest UniversityChongqingChina

The E2F family of transcription factors is crucial for cell cycle progression and cell fate *** E2Fs have been widely studied in mammals,there have been few studies performed in ***,we determined the function of E2F4 in the silkworm,Bombyx *** demonstrate that E2F proteins are highly conserved among species from lower animals to higher *** of the BmE2F4 gene led to cell cycle arrest in the G1 phase,whereas interfering with the BmE2F4 mRNA led to accumulation of cells in the S *** results indicate that BmE2F4 is important in cell cycle *** also demonstrate that the BmE2F4 gene is involved in DNA replication of BmN-SWU1 cells and DNA synthesis in the silk ***,we identified a protein called Bm14-3-3ζthat can interact with BmE2F4 and allow it to localize in the *** of the Bm14-3-3ζgene led to cell cycle arrest in the G1 phase,while knocking down the gene increased the proportion of cells in S *** findings provide important insights into the function of E2F transcription factors and increase our understanding of their involvement in cell cycle regulation.

关键词： Bm14-3-35 Bombyx mori cell cycle E2F transcription factors E2F4 insect

Identification and characterization of genes involved in the jasmonate biosynthetic and signaling pathways in mulberry(Morus notabilis)

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Journal of Integrative Plant Biology 2014年第7期56卷 663-672页

作者： Qing Wang Bi Ma Xiwu Qi Qing Guo Xuwei Wang Qiwei Zeng Ningjia He State key laboratory of silkworm Genome Biology Southwest University

Jasmonate （JA） is an important phytohormone regulating growth, development, and environmental response in plants, particularly defense response against herbivorous insects. Recently, completion of the draft genome of the mulberry （Morus notabilis） in conjunction with genome sequencing of silkworm （Bombyx mori） provides an opportuni-ty to study this unique plant-herbivore interaction. Here, we identified genes involved in JA biosynthetic and signaling pathways in the genome of mulberry for the first time, with the majority of samples showing a tissue-biased expression pattern. The analysis of the representative genes 12-oxophy-todienoic acid reductase （OPRs） and jasmonate ZIM-domain （JAZs） was performed and the results indicated that the mulberry genome contains a relatively smal number of JA biosynthetic and signaling pathway genes. A gene encoding an＆amp;nbsp;important repressor, MnNINJA, was identified as an alternative splicing variant lacking an ethylene-responsive element binding factor-associated amphiphilic repression motif. Having this fundamental information wil facilitate future functional study of JA-related genes pertaining to mulberry-silkworm interactions.

关键词： Jasmonate mulberry (Morus notabilis) pathway silkwormCitation: Wang Q, Ma B, Qi x, Guo Q, wang x, zeng Q, He N (2014)Identification and characterization of genes involved in the jasmonatebiosynthetic and signaling pathways in mulberry (Morus notabilis). J