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Insect Science 2024年第4期31卷 1026-1040页

作者： Wei Wang Linshengzhe Ji Xinyuan Jing Ping Zhao qingyou xia Integrative Science Center of Germplasm Creation in Western China(Chongqing)Science City Biological Science Research CenterSouthwest UniversityChina Key Laboratory for Germplasm Creation in Upper Reaches of the Yangtze River Ministry of Agriculture and Rural AffairsChongqingChina Engineering Laboratory of Sericultural and Functional Genome and Biotechnology Development and Reform CommissionChongqingChina

MicroRNAs play critical roles in multiple developmental processes in *** previous study showed that CRISPR/Cas9-mediated knock down of the microRNA let-7 in silkworms increased the size of larvae and silk glands,thereby improving the silk production *** this study,we elucidate the molecular mechanism underlying of let-7 regulates *** of differentially expressed genes in response to let-7 knock down revealed enrichment of pathways associated with cell proliferation and DNA ***-7 dysregulation affected the cell cycle and proliferation of the Bombyx mori cell line ***-luciferase and target site mutation assays showed that BmCDK1 is a direct target gene of let-7,with only 1 binding site on its 3′-untranslated *** interference of BmCDK1 inhibited cell proliferation,but this effect was counteracted by co-transfection with let-7 ***,let-7 knock down induced BmCDK1 expression and promoted cell proliferation in multiple tissues,and further induced endomitosis in the silk gland in *** down of BmCDK1 resulted in abnormal formation of a new epidermis,and larval development was arrested at the 2nd or 3rd molt *** together,our results demonstrated that BmCDK1 is a novel target of let-7 in cell fate determination,possessing potential for improving silk yield in silkworm.

关键词： BmCDKI CRISPR/Cas9 cell proliferation let-7 silkworm

SAA-Cas9: A tunable genome editing system with increased bio-safety and reduced off-target effects

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Journal of Genetics and Genomics 2019年第3期46卷 145-148页

作者： Sanyuan Ma Jianduo Zhang Wei Lu Yue Liu qingyou xia State Key Laboratory of Silkworm Genome Biology Southwest University Chongqing Engineering and Technology Research Center for Novel Silk Materials Southwest University Chongqing Key Laboratory of Sericulture Southwest University

The site-specific nature and ease of handling of clustered regularly interspaced palindromic repeat(CRISPR)/Cas9 has fulfilled the decades-long goal of genome engineering,and thus has pushed the biological sciences into a new era in which site-directed manipulation of DNA is no longer a short slab(Doudna and Charpentier,2014).The incomparable power of this system also enabled unprecedented new opportunities for practical applications in agriculture,ecosystem and human health(Liu,2017).However,with the extremely rapid development and application of CRISPR system,concerns about its bio-safety have been increasing(Caplan et al.,2015;Taning et al.,2017).While multiple studies have addressed this issue,alternative options for regulating Cas9 are of great *** has been proved by increasing evidences,another well?known concern is the off-target effects,which could not only confound research experiments or breeding,but also cause unwanted side effects in the forthcoming therapeutic uses(Fu et aL,2013;Taning et al.,2017;Zhang et al.,2018).

关键词： site-specific nature handling of clustered experiments or breeding

Analysis of cytochrome P450 genes in silkworm genome (Bombyx mori)

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Science China(Life Sciences) 2005年第4期48卷 414-418页

作者： LI Bin xia qingyou LU Cheng ZHOU Zeyang xiaNG Zhonghuai Key Sericultural Laboratory of Agricultural Ministry College of Sericulture&BiotechnologySouthwest Agricultural UniversityChongqing 400716China

We have searched the Bombyx mori genome for members of the major enzyme family,the Cytochrome P450s,which carry out multiple reactions to enable organisms to rid themselves of foreign *** a result,86 putative P450s were discovered in silkworm genome,which are thought to belong to 32 subfamilies.A comparative genomic analysis with Drosophila melanogaster reveals that the two insects have some similar P450 distribution pat-terns but still have some obvious ***,the diverse distribution exists in 7 p450 subfamilies,which are CYP4A,CYP4C,CYP4D,CYP6A,CYP6AE,CYP6B and ***-thermore,we collected expression sequence tag(EST)evidence for 49 putative P450s genes,which are expressed at the transcriptional level and more likely to be true P450s.

关键词： cytochrome P450 silkworm genome.

BmSLC7A5 is essential for silk protein synthesis and larval development in Bombyx mori

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Insect Science 2024年第5期31卷 1425-1439页

作者： Xin Tang Huawei Liu Xin Wang Li Chang Qingsong Liu qingyou xia Ping Zhao Integrative Science Center of Germplasm Creation in Western China(CHONGQING)Science City Biological Science Research CenterSouthwest UniversityChongqingChina Chongqing Key Laboratory of Chinese Medicine&Health Science Chongqing Academy of Chinese Materia MedicaChongqing College of Traditional Chinese MedicineChongqingChina Key Laboratory for Germplasm Creation in Upper Reaches of the Yangtze River Ministry of Agriculture and Rural AffairsChongqingChina

Insects produce silk to form cocoons,nests,and webs,which are important for their survival and ***,little is known about the molecular mecha-nism of silk protein synthesis at the translation *** solute carrier family 7(SLC7)genes are involved in activating the target of rapamycin complex 1(TORC1)signaling pathway and protein translation process,but the physiological roles of SLC7 genes in silk-producing insects have not been ***,we found that amino acid signaling regulates silk protein synthesis and larval development via the L-type amino acid trans-porter 1(LAT1;also known as SLC7A5)in Bombyx mori.A total of 12 SLC7 homologs were identified in the silkworm genome,among which BmSLC7A5 was found to be a silk gland-enriched gene and may be involved in leucine *** analy-sis indicated that SLC7A5 displays high homology and a close phylogenetic relationship in silk-producing ***,we found that leucine treatment significantly in-creased silk protein synthesis by improving the transcription and protein levels of silk ***,systemic and silk gland-specific knockout of BmSLC7A5 led to de-creased silk protein synthesis by inhibiting TORC1 signaling,and somatic mutation also resulted in arrested development from the 5th instar to the early pupal ***,our study reveals that BmSLC7A5 is involved in regulating silk protein synthesis and larval development by affecting the TORC1 signaling pathway,which provides a new strategy and target for improving silk yield.

关键词： amino acid signaling Bombyx mori larval development SLC7A5 silk pro-tein synthesis TORC1 signaling pathway

Homeodomain proteins POU-M2, antennapedia and abdominal-B are involved in regulation of the segment-specific expression of the clip-domain serine protease gene CLIP13 in the silkworm, Bombyx mori

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Insect Science 2022年第1期29卷 111-127页

作者： Huawei Liu Jingya Heng Luoling Wang Youshan Li Xin Tang Xuan Huang qingyou xia Ping Zhao State Key Laboratory of Silkworm Genome Biology Southwest UniversityChongqing400716China Biological Science Research Center Southwest UniversityChongqing400716China Chongqing Key Laboratory of Sericultural Science Chongqing Engineering and Technology Research Center for Novel Silk MaterialsSouthwest UniversityChongqing400716China College of Biological Science and Engineering Shaanxi University of TechnologyHanzhongShaanxi Province723001China

Clip-domain serine proteases (CLIPs) play important roles in insect innate immunity and development. Our previous studies indicated that CLIP13, an epidermis-specific gene, was involved in cuticle remodeling during molting and metamorphosis in the silkworm, Bombyx mori. However, the transcriptional regulatory mechanism and regulatory pathways of CLIP13 remained unclear. In the present study, we investigated CLIP13 expression and the regulation pathway controlled by 20-hydroxyecdysone (20E) in the silkworm. At the transcriptional level, expression of CLIP13 exhibited pronounced spatial and temporal specificity in different regions of the epidermis;homeodomain transcription factors POU-M2, antennapedia (Antp), and abdominal-B (Abd-B) showed similar expression change trends as CLIP13 in the head capsule, thorax, and abdomen, respectively. Furthermore, results of cell transfection assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation demonstrated that POU-M2, Antp, and Abd-B were involved in the transcriptional regulation of CLIP13 by directly binding to their cis-response elements in CLIP13 promoter. RNA interference-mediated silencing of POU-M2, Antp, and Abd-B led to a decrease of CLIP13 expression in the head capsule, the epidermis of the 1st to 3rd thoracic segments and the 7th to 10th abdominal segments, respectively. Consistent with CLIP13, 20E treatment significantly upregulated expression of POU-M2, Antp, and Abd-B in the silkworm epidermis. Taken together, these data suggest that 20E positively regulates transcription of CLIP13 via homeodomain proteins POU-M2, Antp, and Abd-B in different regions of the silkworm epidermis during metamorphosis, thus affecting the molting process. Our findings provide new insight into the functions of homeodomain transcription factors in insect molting.

关键词： Bombyx mori clip-domain serine protease epidermis homeotic gene POU-M2 20-hydroxyecdysone Bombyx mori

A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana

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Science Bulletin 2015年第15期60卷 1332-1347页

作者： Wenshan Liu xiaohong Zhu Mingguang Lei qingyou xia Jose Ramon Botella Jian-Kang Zhu Yanfei Mao School of Life Sciences Chongqing University Shanghai Center for Plant Stress Biology Shanghai Institutes of Biological Sciences Chinese Academy of Sciences State Key Laboratory of Silkworm Genome Biology Southwest University Department of Horticulture and Landscape Architecture Purdue University School of Agriculture and Food Sciences University of Queensland

The newly developed CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas(CRISPR-associated) system has emerged as an efficient tool for genome-editing, providing an alternative to classical mutagenesis and transgenic methods to study gene function and improve crop traits. CRISPR/Cas facilitates targeted gene editing through RNA-guided DNA cleavage followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. Here we describe a detailed procedure for our previously developed and highly efficient CRISPR/Cas9 method that allows the generation of heritable-targeted gene mutations andcorrections in Arabidopsis. This protocol describes the strategies and steps for the selection of targets, design of single-guide RNA(sg RNA), vector construction and analysis of transgenic lines. We also offer a method to target two loci simultaneously using vectors containing two different sg RNAs. The principles described in this protocol can be applied to other plant species to generate stably inherited DNA modifications.

关键词： CRISPR/Cas9 Targeted gene editing Genome engineering Arabidopsis thaliana

In silico identification of silkworm selenoproteomes

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Chinese Science Bulletin 2006年第23期51卷 2860-2867页

作者： CHEN Ping DUAN Jun JIANG Liang LIU Qiong ZHAO Ping xia qingyou XU Huibi Key Sericultural Laboratory of Agricultural Ministry Southwest University Chongqing 400716 China College of Life Science Shenzhen University Shenzhen 518060 China Department of Chemistry Huazhong University of Science and Technology Wuhan 430074 China

Selenium (Se) is an essential trace element in vivo. Its biological function is mainly exerted through selenoproteins. Selenocysteine (Sec), the active site of selenoproteins, is incorporated into the protein at an in-frame TGA codon under the guidance of Sec insertion sequence (SECIS) element in the 3′-untranslated region (UTR) of the gene. In this work, a method was developed and a series of programs were edited by PERL language to in silico identify selenoproteomes from the genome of domesticated silkworm (Bombyx mori). Out of 18510 annotated genes, 6348 was terminated with TGA codons, 249 containing both in-frame TGAs and SECIS elements in the 3′-UTRs. Alignments of those selenoprotein candidates with their cysteine (Cys)-containing homologs revealed that 52 genes had TGA/Cys pairs and similar flanking regions around the in-frame TGAs. Restricted by the patterns of SECIS elements only 5 genes were screened out to fully meet the requirements for selenoproteins. Among them glutathione S-transferase (GST) has been reported as a microbial selenoprotein, the other four are novel selenoproteins annotated as CG6024, CG5195, ATP-binding cassette transporter subfamily A (ABCA), and nuclear VCP-like protein. Derived from the general properties of GST, ABCA and VCP, silkworm selenoproteins may play important roles in redox regulation, Se storage and transportation, as well as cell apoptosis.

关键词：桑蚕硒 ABCA 细胞凋亡

Genome-wide CRISPR screening reveals key genes and pathways associated with 20-hydroxyecdysone signal transduction in the silkworm(Bombyx mori)

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Insect Science 2024年第1期31卷 47-58页

作者： Hao Sun Jingya Chen Ruolin Wang Dan Liu Na Zhang Tong Zhang Ling Jia Sanyuan Ma qingyou xia State Key Laboratory of Silkworm Genome Biology Southwest UniversityChongqingChina Biological Science Research Center Southwest UniversityChongqingChina

Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone(JH)and 20-hydroxyecdysone(20E).Despite important progress in understanding various aspects of silkworm biology,the hormone signaling pathway in the silkworm remains poorly ***-wide screening using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-based libraries has recently emerged as a novel method for analyzing genome function,enabling further research into essential genes,drug targets,and virus-host ***,we constructed a genome-wide CRISPR/Cas9-based library of the silkworm(Bombyx mori)and successfully revealed the genes involved in biotic or abiotic stress factor *** this study,we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of *** annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and *** enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity,interfere with intracellular nutrition and energy metabolism,and eventually cause cell *** screening results were experimentally validated by generating cells with knockout alleles of the relevant genes,which had increased tolerance to *** findings provide a panoramic overview of signaling in response to 20E in the silkworm,underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.

关键词： CRISPR/Cas9 CRISPR library screening ecdysone 20E signal transduction silkworm whole genome editing

Facile and Low-Cost Fabrication of a Thread/Paper-Based Wearable System for Simultaneous Detection of Lactate and pH in Human Sweat

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Advanced Fiber Materials 2020年第5期2卷 265-278页

作者： Gang xiao Jing He Yan Qiao Feng Wang qingyou xia Xin Wang Ling Yu Zhisong Lu Chang-Ming Li Institute for Clean Energy and Advanced Materials School of Materials and EnergySouthwest University1 Tiansheng RoadChongqing 400715People’s Republic of China State Key Laboratory of Silkworm Genome Biology Southwest University1 Tiansheng RoadChongqing 400715People’s Republic of China Chongqing Engineering and Technology Research Center for Novel Silk Materials Southwest University1 Tiansheng RoadChongqing 400715People’s Republic of China College of Food Science Southwest UniversityNo.2 Tiansheng RoadBeibei DistrictChongqing 400715People’s Republic of China Institute of Advanced Cross-Field Science Qingdao UniversityQingdao 266071People’s Republic of China

Wearable devices have received tremendous interests in human sweat analysis in the past few ***,the widely used polymeric substrates and the layer-by-layer stacking structures greatly influence the cost-efficiency,conformability and breathability of the devices,further hindering their practical ***,we report a facile and low-cost strategy for the fabrication of a skin-friendly thread/paper-based wearable system consisting of a sweat reservoir and a multi-sensing component for simultaneous in situ analysis of sweat pH and *** the system,hydrophilic silk thread serves as the micro-channel to guide the liquid *** papers were functionalized to prepare colorimetric sensors for lactate and *** smartphone-based quantitative analysis shows that the sensors are sensitive and *** pH may interfere the lactate detection,the pH detected simultaneously could be employed to correct the measured data for the achievement of a precise lactate *** being integrated with a hydrophobic arm guard,the system was successfully used for the on-body measurement of pH and lactate in the sweats secreted from the *** low-cost,easy-to-fabricate,light-weight and flexible thread/paper-based microfluidic sensing device may hold great potentials as a wearable system in human sweat analysis and point-of-care diagnostics.

关键词： Wearable sensors Thread/paper-based microfluidics Sweat analysis Point-of-care diagnostics Multi-sensing system

Transgenic PDGF-BB sericin hydrogel potentiates bone regeneration of BMP9-stimulated mesenchymal stem cells through a crosstalk of the Smad-STAT pathways

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Regenerative Biomaterials 2023年第1期10卷 181-193页

作者： Hui-Jie Zhang Fu-Shu Li Feng Wang Han Wang Tong-Chuan He Russell R.Reid Bai-Cheng He qingyou xia Key Laboratory of Biochemistry and Molecular Pharmacology of Chongqing Chongqing Medical UniversityChongqing 400016China Department of Pharmacology School of PharmacyChongqing Medical UniversityChongqing 400016China Department of Pharmacy Chongqing Emergency Medical CenterChongqing University Central HospitalChongqing 400014China Biological Science Research Center Chongqing Key Laboratory of Sericultural ScienceChongqing Engineering and Technology Research Center for Novel Silk MaterialsSouthwest UniversityChongqing 400715China Department of Pharmacy Panzhou People’s HospitalGuizhou 553599China Molecular Oncology Laboratory Department of Orthopaedic Surgery and Rehabilitation MedicineThe University of Chicago Medical CenterChicagoIL 60637USA Laboratory of Craniofacial Biology and Development Department of Surgery Section of Plastic SurgeryThe University of Chicago Medical CenterChicagoIL 60637USA

Silk as a natural biomaterial is considered as a promising bone substitute in tissue *** and fibroin are the main components of silk and display unique features for their programmable mechanical properties,biocompatibility,biodegradability and morphological *** has been reported that sericin recombinant growth factors(GFs)can support cell proliferation and induce stem cell differentiation through cross-talk of signaling pathways during tissue *** transgenic technology allows the productions of bioactive heterologous GFs as fusion proteins with sericin,which are then fabricated into solid matrix or hydrogel ***,using an injectable hydrogel derived from transgenic platelet-derived GF(PDGF)-BB silk sericin,we demonstrated that the PDGF-BB sericin hydrogel effectively augmented osteogenesis induced by bone morphogenetic protein(BMP9)-stimulated mesenchymal stem cells(MSCs)in vivo and in vitro,while inhibiting adipogenic *** gene expression and protein-protein interactions studies demonstrated that BMP9 and PDGF-BB synergistically induced osteogenic differentiation through the cross-talk between Smad and Stat3 pathways in ***,our results provide a novel strategy to encapsulate osteogenic factors and osteoblastic progenitors in transgenic sericin-based hydrogel for robust bone tissue engineering.

关键词： biomaterials growth factor BMP9 osteogenesis