Advanced biotechnology最新文献

Direct detection of meiotic recombination events in the highly heterozygous amphioxus genome. 高度杂合文昌鱼基因组减数分裂重组事件的直接检测。

Advanced biotechnology Pub Date : 2025-10-06 DOI: 10.1007/s44307-025-00083-7

Lei Tao, Jing Xue, Junwei Cao, Guang Li, Cai Li

{"title":"Direct detection of meiotic recombination events in the highly heterozygous amphioxus genome.","authors":"Lei Tao, Jing Xue, Junwei Cao, Guang Li, Cai Li","doi":"10.1007/s44307-025-00083-7","DOIUrl":"10.1007/s44307-025-00083-7","url":null,"abstract":"Amphioxus, a basal chordate with highly heterozygous genomes (3.2 ~ 4.2% in sequenced species), represents a key model for understanding vertebrate origins. However, the extreme heterozygosity poses challenges for many genomic analyses, including studying meiotic recombination. Here, we present a novel bioinformatic pipeline that enables direct detection of crossover (CO) and non-crossover (NCO) recombination events using short-read whole-genome sequencing of a two-generation pedigree (two parents and 104 F1 offspring) of the amphioxus Branchiostoma floridae. Using parental assemblies generated by Platanus-allee as a custom reference for read alignment, we tracked inheritance patterns in offspring and phased contig-level haplotypes in parents, allowing us to detect recombination events. We identified 2,329 paternal and 2,288 maternal COs, yielding recombination rates of 4.66 cM/Mb and 4.57 cM/Mb, respectively. We found CO coldspots spanning > 140 Mb in each parent and these are likely associated with large-scale heterozygous inversions. CO rates were positively correlated with transposable element and gene density in both sexes, but showed weak or no correlation with GC content. We further identified ~ 10,000 paternal and ~ 5,800 maternal NCO events, predominantly shorter than 200 bp in tract length, and found evidence of GC-biased gene conversion. This work provides the first direct and genome-wide measurement of recombination in amphioxus and demonstrates how high heterozygosity, often considered a barrier, can be leveraged for fine-scale recombination mapping. Our findings illuminate conserved and divergent features of recombination in chordates and establish a framework for studying recombination in other highly heterozygous organisms.","PeriodicalId":519913,"journal":{"name":"Advanced biotechnology","volume":"3 4","pages":"30"},"PeriodicalIF":0.0,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12500494/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145234841","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Integrated proteomics and transcriptomics analysis of dynamic changes in muscle fiber types in different regions of porcine skeletal muscle. 猪骨骼肌不同区域肌纤维类型动态变化的综合蛋白质组学和转录组学分析。

Advanced biotechnology Pub Date : 2025-09-24 DOI: 10.1007/s44307-025-00080-w

Zhiting Feng, Xiaoyu Wang, Qingshuang Zhou, Yihao Liu, Rong Xu, Ziyun Liang, Chong Zhang, Xiaohong Liu, Yunxiang Zhao, Yaosheng Chen, Delin Mo

{"title":"Integrated proteomics and transcriptomics analysis of dynamic changes in muscle fiber types in different regions of porcine skeletal muscle.","authors":"Zhiting Feng, Xiaoyu Wang, Qingshuang Zhou, Yihao Liu, Rong Xu, Ziyun Liang, Chong Zhang, Xiaohong Liu, Yunxiang Zhao, Yaosheng Chen, Delin Mo","doi":"10.1007/s44307-025-00080-w","DOIUrl":"10.1007/s44307-025-00080-w","url":null,"abstract":"Skeletal muscle serves as a valuable source of nutrition, with distinct muscle fiber types exhibiting different physicochemical properties that influence both meat quality and muscle function. Bama miniature pigs (BM) are recognized for their superior meat quality and their relevance as models for human medical research. Therefore, investigating the differences between slow and fast muscles at various developmental stages (from 57 days post-fertilization to 120 days postnatally) in BM is crucial for both the pork industry and biomedical studies. In this study, we employed a non-targeted data-independent acquisition (nDIA) -based proteomic approach for the first time to porcine embryonic skeletal muscle fibers. A total of 616 differentially expressed genes (DEGs) and 272 differentially abundant proteins (DAPs) were identified in the fast-twitch longissimus dorsi (LD) and slow-twitch semitendinosus (SD) muscles of BM. Domain enrichment analysis and in vitro experiments demonstrated that the NEK3 gene, containing the S_TKc domain, inhibits fast-twitch muscle fiber differentiation postnatally. Additionally, cross-species analysis showed upregulation of skeletal muscle development organ genes in pigs at postnatal day 28. In summary, our results provide both fundamental data and novel insights to further uncover the mechanisms underlying pig skeletal muscle development and muscle fiber transition.","PeriodicalId":519913,"journal":{"name":"Advanced biotechnology","volume":"3 4","pages":"29"},"PeriodicalIF":0.0,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12460891/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145133349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Advanced biotechnology Pub Date : 2025-09-23 DOI: 10.1007/s44307-025-00081-9

Jia-Yi Luo, Ju-Feng Wang, Jiong Yang, Peng Zhang, Geoff Hide, De-Hua Lai, Zhao-Rong Lun

{"title":"Thermo-related degeneration of stumpy forms of Trypanosoma brucei, the pathogen of African sleeping sickness.","authors":"Jia-Yi Luo, Ju-Feng Wang, Jiong Yang, Peng Zhang, Geoff Hide, De-Hua Lai, Zhao-Rong Lun","doi":"10.1007/s44307-025-00081-9","DOIUrl":"10.1007/s44307-025-00081-9","url":null,"abstract":"Trypanosoma brucei, the causative agent of African trypanosomiasis, develops from the long slender (LS) to the short stumpy (SS) form in the mammalian host. The SS trypanosomes are critical for transmission to the insect vector but face significant challenges within the vertebrate host. The role of the immune response in controlling the parasitaemia is well studied, however, the mechanism underpinning the rapid degeneration of SS trypanosomes during the first parasitaemic peak in mice remains somewhat elusive. We demonstrate that fever is a critical yet underexplored factor in facilitating the clearance of SS trypanosomes, suggesting that temperature may play a critical role in regulating the natural turnover of SS trypanosomes. The elevated body temperature correlates with the parasitaemic dynamics, accelerating SS trypanosome elimination in the mammalian host. The SS trypanosomes exhibited high thermo-sensitivity to elevated temperatures, accompanied with apoptosis-like events, mitochondrial damage and oxidative stress. Metabolomic profiling also revealed disruptions in glycolysis and the TCA cycle, shedding light on the processes in compromising the SS trypanosomes. Interestingly, antibodies during the acute phase did not directly cause SS trypanosomes death, but the combination of elevated temperature and antibodies enhanced the clearance of SS trypanosomes, highlighting the critical role of fever in eliminating the first parasitaemic peak. Our findings detail the mechanism of vulnerability of SS trypanosome to elevated temperatures and suggest that host fever serves as a neglected, but critical mechanism, for T. brucei SS trypanosome clearance.","PeriodicalId":519913,"journal":{"name":"Advanced biotechnology","volume":"3 4","pages":"28"},"PeriodicalIF":0.0,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457267/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145126828","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Uncovering essential lncRNAs through transcriptome-scale CRISPR-Cas13 screening. 通过转录组级CRISPR-Cas13筛选发现必要的lncrna。

Advanced biotechnology Pub Date : 2025-09-15 DOI: 10.1007/s44307-025-00082-8

Jiahua Si, Xinming Su, Zhuoyan Jin, Shiwei Duan

{"title":"Uncovering essential lncRNAs through transcriptome-scale CRISPR-Cas13 screening.","authors":"Jiahua Si, Xinming Su, Zhuoyan Jin, Shiwei Duan","doi":"10.1007/s44307-025-00082-8","DOIUrl":"10.1007/s44307-025-00082-8","url":null,"abstract":"Approximately 75% of the human genome is transcribed into RNA, yet less than 5% encodes proteins, with the majority producing non-coding RNAs (ncRNAs). Among them, long non-coding RNAs (lncRNAs) represent a major class that exerts broad regulatory influence across cellular processes, disease contexts, and developmental stages. Despite their potential as biomarkers and therapeutic targets, their low sequence conservation, limited abundance, and structural complexity present significant challenges for functional characterization. Traditional RNA interference and CRISPR-Cas9-based methods have offered partial insights but remain limited in efficiency, specificity, and scalability. To address these barriers, Neville E. Sanjana's team developed CaRPool-seq, a transcriptome-scale CRISPR-Cas13 screening platform that directly targets RNA. Applying this approach across diverse human cell lines, they identified 778 essential lncRNAs, including 46 universally required for survival, with distinctive structural features and functional independence from neighboring protein-coding genes. Integration with single-cell transcriptomics revealed their critical roles in cell-cycle regulation, apoptosis, and developmental gene expression, as well as aberrant expression patterns in cancer linked to patient outcomes. This study establishes CRISPR-Cas13 as a precise and scalable strategy for lncRNA functional discovery, expanding opportunities for biomarker identification, therapeutic development, and precision medicine.","PeriodicalId":519913,"journal":{"name":"Advanced biotechnology","volume":"3 3","pages":"27"},"PeriodicalIF":0.0,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12436244/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145067183","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Functional characterization of AarMIXTAs as essential regulators in T-shaped non-glandular trichome development of Artemisia argyi. armixtas在艾叶t型非腺毛发育中的功能特征

Advanced biotechnology Pub Date : 2025-09-12 DOI: 10.1007/s44307-025-00077-5

Xinlian Chen, Duan Wu, Chunyu Li, Baosheng Liao, Qi Shen

引用次数: 0

Arabidopsis MACP2 contributes to autophagy induction by modulating starvation-induced reactive oxygen species homeostasis. 拟南芥MACP2通过调节饥饿诱导的活性氧稳态参与自噬诱导。

Advanced biotechnology Pub Date : 2025-08-20 DOI: 10.1007/s44307-025-00078-4

Ying Zhou, Xue Zhang, Tiancong Qi, Zi-Han Wang, Yao Wang, Lin-Na Wang, Yong-Lun Zeng, Hanjie He, Liwen Jiang, Daoxin Xie, Shi Xiao, Lu-Jun Yu, Qin-Fang Chen

{"title":"Arabidopsis MACP2 contributes to autophagy induction by modulating starvation-induced reactive oxygen species homeostasis.","authors":"Ying Zhou, Xue Zhang, Tiancong Qi, Zi-Han Wang, Yao Wang, Lin-Na Wang, Yong-Lun Zeng, Hanjie He, Liwen Jiang, Daoxin Xie, Shi Xiao, Lu-Jun Yu, Qin-Fang Chen","doi":"10.1007/s44307-025-00078-4","DOIUrl":"10.1007/s44307-025-00078-4","url":null,"abstract":"In plants, autophagy is a conserved recycling system essential for development and stress responses by targeting cellular components for massive degradation in the vacuole. Our previous work suggested that autophagy contributes to Arabidopsis (Arabidopsis thaliana) stress responses by modulating NADPH-oxidase-mediated reactive oxygen species (ROS) homeostasis; however, the molecular link between extracellular ROS and autophagy remains unknown. We performed a yeast two-hybrid screen to identify components involved in autophagy, using the central autophagy component ATG8e as a bait. We identified MEMBRANE ATTACK COMPLEX/PERFORIN-LIKE 2 (MACP2) as an interactor of ATG8e via its the ATG8-interacting motif and confirmed this interaction by co-immunoprecipitation and bimolecular fluorescence complementation assays. MACP2-overexpressing lines showed enhanced sensitivity to nutritional starvation, accelerated leaf senescence, and increased hydrogen peroxide (H2O2) levels, resembling the phenotypes of atg mutants defective in autophagy. Conversely, macp2 knockouts exhibited diminished starvation-induced H2O2 accumulation and attenuated autophagosome formation and fully suppressed the starvation-hypersensitive phenotypes of the atg5-1 mutant. In particular, MACP2 was degraded through the autophagy machinery during prolonged starvation, suggesting a feedback regulatory mechanism for maintaining MACP2 homeostasis. Our findings suggest that MACP2 acts as a key regulator in autophagy induction by controlling influx of extracellular H2O2 in Arabidopsis.","PeriodicalId":519913,"journal":{"name":"Advanced biotechnology","volume":"3 3","pages":"25"},"PeriodicalIF":0.0,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12367599/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144985237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Engineering of autoactive NLRs: a big step toward breeding crops with durable and broad-spectrum resistance. 自动nlr的工程：向培育具有持久和广谱抗性的作物迈出了一大步。

Advanced biotechnology Pub Date : 2025-08-20 DOI: 10.1007/s44307-025-00079-3

Yan Wang, Shi Xiao

引用次数: 0

Mannose receptor RpMR1 of Manila clam (Ruditapes philippinarum) defense against Vibrio anguillarum infection. 马尼拉蛤抗鳗弧菌感染的甘露糖受体RpMR1。

Advanced biotechnology Pub Date : 2025-08-04 DOI: 10.1007/s44307-025-00075-7

Zhihui Yin, Hongtao Nie

{"title":"Mannose receptor RpMR1 of Manila clam (Ruditapes philippinarum) defense against Vibrio anguillarum infection.","authors":"Zhihui Yin, Hongtao Nie","doi":"10.1007/s44307-025-00075-7","DOIUrl":"10.1007/s44307-025-00075-7","url":null,"abstract":"The mannose receptor (MR) is a member of the C-type lectin superfamily and a type I transmembrane protein that functions as a pattern recognition receptor (PRR) in immune responses. In this study, we identified 13 MR genes (RpMR1-13) in the genome of Ruditapes philippinarum and investigated their expression profiles following Vibrio anguillarum challenge. Notably, RpMR1, RpMR2, RpMR3, and RpMR4 exhibited peak expression at 72 h post-infection. We successfully purified the recombinant RpMR1 protein and demonstrated its antibacterial activity against three Gram-negative bacteria (V. splendidus, V. alginolyticus, and V. anguillarum), though it had no effect on Gram-positive bacteria. Furthermore, in vivo injection of RpMR1 significantly reduced mortality in R. philippinarum following V. anguillarum infection. To explore role of RpMR1 in immune signaling, we performed RNA interference (dsRNA-RpMR1) and observed successful gene silencing. Subsequent qRT-PCR analysis revealed that RpMR1 knockdown significantly suppressed TLR4 expression (P< 0.05) under V. anguillarum stress, confirming an interaction between RpMR1 and TLR4 in the immune response. This study provides the first functional evidence of mannose receptor-mediated immunity in mollusks, offering new insights into the molecular defense mechanisms of R. philippinarum against bacterial infection.","PeriodicalId":519913,"journal":{"name":"Advanced biotechnology","volume":"3 3","pages":"23"},"PeriodicalIF":0.0,"publicationDate":"2025-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12321717/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144786270","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Effect of Claroideoglomous etunicatums on rhizosphere bacterial community of tobacco under low nutrient conditions. 低营养条件下烟根际细菌群落的影响。

Advanced biotechnology Pub Date : 2025-07-04 DOI: 10.1007/s44307-025-00071-x

Jin Chen, Xiaowan Geng, Qing Zhang, Keqing Lin, Zishan Li, Boyan Wang, Qingchen Xiao, Xiaoyu Li

{"title":"Effect of Claroideoglomous etunicatums on rhizosphere bacterial community of tobacco under low nutrient conditions.","authors":"Jin Chen, Xiaowan Geng, Qing Zhang, Keqing Lin, Zishan Li, Boyan Wang, Qingchen Xiao, Xiaoyu Li","doi":"10.1007/s44307-025-00071-x","DOIUrl":"10.1007/s44307-025-00071-x","url":null,"abstract":"Arbuscular mycorrhizal fungi (AMF) have the potential to enhance plant tolerance to abiotic stresses. However, the impact of AMF on the rhizosphere bacterial community of tobacco under conditions of low nutrient availability remains unclear. This study investigated the influence of inoculating Claroideoglomus etunicatum on the tobacco rhizosphere bacterial community and the microbial mechanisms by which AMF enhanced plants antioxidant capacity, employing Illumina MiSeq high-throughput sequencing. The findings indicated that AMF significantly increased both the aboveground and belowground fresh weight, as well as the plant height of tobacco. AMF inoculation led to elevated activities of catalase (CAT) and superoxide dismutase (SOD), a reduction in malondialdehyde (MDA) content, and an overall enhancement of the plants antioxidant capacity. Phylogenetic analysis demonstrated that AMF modified the bacterial community structure and significantly enriched beneficial rhizosphere bacteria, predominantly from the phyla Proteobacteria, Chloroflexi, Actinobacteriota, and Myxococcota, thereby facilitating tobacco growth. The network analysis revealed that the incorporation of arbuscular mycorrhizal fungi (AMF) contributed to increased stability within the bacterial community, enriched species diversity, and more intricate ecological networks. AMF enhanced interactions and positive correlations among bacterial species, indicating that heightened microbial synergy is associated with improved symbiotic relationships. Furthermore, the structural equation model demonstrated that AMF bolstered the plants antioxidant capacity by modulating the rhizosphere bacterial community. This study elucidates the impact of AMF on the tobacco rhizosphere bacterial community, providing a theoretical basis for promoting tobacco growth.","PeriodicalId":519913,"journal":{"name":"Advanced biotechnology","volume":"3 3","pages":"22"},"PeriodicalIF":0.0,"publicationDate":"2025-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12227407/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144562525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Rare and abundant taxa in Artemisia desertorum rhizosphere soils demonstrate disparate responses to drought stress. 荒漠蒿根际土壤中稀有和丰富的类群对干旱胁迫表现出不同的响应。

Advanced biotechnology Pub Date : 2025-07-03 DOI: 10.1007/s44307-025-00070-y

Mei-Xiang Li, Wen-Hui Lian, Zheng-Han Lian, Xiao-Qing Luo, Ling-Xiang Yue, Jia-Rui Han, Chao-Jian Hu, Shuai Li, Wen-Jun Li, Lei Dong

{"title":"Rare and abundant taxa in Artemisia desertorum rhizosphere soils demonstrate disparate responses to drought stress.","authors":"Mei-Xiang Li, Wen-Hui Lian, Zheng-Han Lian, Xiao-Qing Luo, Ling-Xiang Yue, Jia-Rui Han, Chao-Jian Hu, Shuai Li, Wen-Jun Li, Lei Dong","doi":"10.1007/s44307-025-00070-y","DOIUrl":"10.1007/s44307-025-00070-y","url":null,"abstract":"The growth and adaptability of desert plants depend on their rhizosphere microbes, which consist of a few abundant taxa and numerically dominant rare taxa. However, the differences in diversity, community structure, and functions of abundant and rare taxa in the rhizosphere microbiome of the same plant in different environments remain unclear. This study focuses on the rhizosphere microbial communities of Artemisia desertorum, a quintessential desert sand-stabilizing plant, investigating the diversity patterns and assembly processes of rare and abundant taxa across four Chinese deserts: Mu Us, Kubuqi, Tengger, and Ulan Buh. The results show that climatic factors, especially aridity and mean annual precipitation (MAP), significantly influence bacterial community composition and microbial network complexity. The interactions between rare and non-rare taxa are non-random, forming a modular network in which rare taxa serve as central nodes, and their loss could destabilize the network. Rare taxa are primarily shaped by heterogeneous selection, whereas abundant taxa are mainly influenced by dispersal limitation. Functionally, abundant taxa exhibit higher metabolic potential, whereas rare taxa are more involved in processes such as cell motility, indicating distinct ecological roles. These results provide new insights into the ecological functions of rare and abundant taxa in desert rhizosphere communities and highlight the importance of microbial management for desert plant health.","PeriodicalId":519913,"journal":{"name":"Advanced biotechnology","volume":"3 3","pages":"21"},"PeriodicalIF":0.0,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12229302/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144556418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0