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Integrative analysis of transcriptomic profile reveals potential roles of miRNAs in regulating development of Marsupenaeus japonicas

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Journal of Oceanology and Limnology 2024年第1期42卷 201-215页

作者： Jing WANG Longjun PU Xiaojuan ZHANG Cuicui LIANG Dandan DONG Jiantao GUAN huarong guo Ministry of Education Key Laboratory of Marine Genetics and Breeding College of Marine Life SciencesOcean University of ChinaQingdao 266003China Institute of Evolution&Marine Biodiversity Ocean University of ChinaQingdao 266003China Yellow Sea Fisheries Research Institute Chinese Academy of Fishery SciencesQingdao 266071China

Regulation of microRNAs(miRNAs)on various biological processes has been a surprising and exciting *** of miRNAs is the first step to comprehensively understand their ***,attempts on global identification and functional verification of miRNAs are very limited in penaeid shrimp Marsupenaeus japonicus,an economically important aquatic *** performing an integrated analysis of transcriptomic profile from gastrula embryos of ***,21conserved miRNAs in ***(mja-miRNAs),belonging to 19 miRNA families,were identified and *** the 21 mja-miRNAs,15 miRNAs were successfully verified to be predominantly expressed in gastrula stage,where they displayed dynamic expression patterns compared with those in naupliuin *** on perfect or near-perfect match to target region,120 target genes were predicted at transcriptome-wide ***,gene ontology(GO)classification and metabolic pathway annotation revealed eight targets that were actively involved in developmental *** the predicted miRNA-mRNA pairs,six targets were then randomly selected and experimentally validated by dual luciferase reporter assay,where three pairs were proved with potential targeting ***,to search for conserved miRNAs potentially involved in early development of ***,we combined in silico and experimental methods,which can be applied in other organisms as *** data implied important roles of miRNAs in the early embryonic development and also suggested the presence of complex miRNA-mRNA functional networks in ***.

关键词： microRNAs(miRNAs) identification Marsupenaeus japonicus embryo transcriptome

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Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus

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海洋湖沼学报(英文) 2023年第5期41卷 1918-1939页

作者： Liwen guo Yaqi ZHAO huarong guo Key Laboratory of Marine Genetics and Breeding Ministry of EducationCollege of Marine Life SciencesOcean University of ChinaQingdao 266003China Institute of Evolution&Marine Biodiversity Ocean University of ChinaQingdao 266003China

Crab cell line,especially continuous crab cell line,can provide us a useful tool for studies on the virology,immunology,and molecular biology of ***,no continuous crab cell line has been available due to the lacking of suitable medium and the occurrence of *** this study,long-term in vitro culture conditions for both two-(2D)and three-dimensions(3D)were successfully developed for the circulating hemocytes of swimming crab Portunus trituberculatus,designated as PTH *** 2D culture,a novel crab basic medium in osmolarity of 990–1100 mOsm/kg was optimized for the first time,which is different from Leibovitz's L-15 medium in mainly the components of amino acids,containing double strengths of the contents of free amino acid mixture in the crab *** an optimal crab growth medium was developed by supplementing 5%fetal bovine serum,50-g/L yeast extract powder,20-μg/L basic fibroblast growth factor and epidermal growth factor into the optimal crab basic medium,and found that it could support a long-term survival of PTH cells in a healthy monolayer up to 347 days and partially break through the mitosis-arrest of crab cells evidenced by the obvious increase of proliferating potential detected in the 10-d primarily cultured PTH *** 2D cultured PTH cells could be successfully sub-cultured for 11 times by physical flushing method and well cryopreserved in liquid *** 3D culture,using the same crab growth medium,the PTH cell aggregates could be easily formed and healthily maintained on the surface of solidified Matrigel or in the ultra-low-attachment plate with a survival rate of 50%–60%on Day *** work largely improved the primary culture and subculture of crab cells and will facilitate the establishment of continuous crab cell line.

关键词： crab Portunus trituberculatus hemocyte long-term cell culture subculture 3D culture

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Evaluation of Cytotoxicity and Genotoxicity of Insecticide Carbaryl to Flounder Gill Cells and Its Teratogenicity to Zebrafish Embryos

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Journal of Ocean University of China 2015年第2期14卷 362-374页

作者： EY Manish Raj guo huarong Laboratory of Evolution & Development Institute of Evolution & Marine Biodiversity Ocean University of China Key Laboratory of Marine Genetics and Breeding of Ministry of Education College of Marine Life SciencesOcean University of China

In this study, we determined the cytotoxicity and genotoxicity of carbamate insecticide carbaryl to flounder gill(FG) cells and its teratogenicity to zebrafish embryos. The cytotoxicity of carbaryl to FG cells was determined with methods including MTT and neutral red uptaking(NRU), lactate dehydrogenase(LDH) releasing and Hoechst 33342 and propidium idodide(PI) double staining. Moderate cytotoxicity in a concentration-dependent manner was observed. The 24 h-IC50 value of 53.48 ± 1.21, 59.13 ± 1.19 and 46.21 ± 1.24 mg L-1 carbaryl was obtained through MTT, NRU and LDH assays, respectively. Double fluorescence staining demonstrated that carbaryl induced the death of FG cells mainly through necrosis. There was no significant genotoxicity found in the FG cells exposed to the highest testing concentration of carbaryl(20 mg L-1, P > 0.05) as was demonstrated by Comet assay. Zebrafish embryos exposed to carbaryl at concentrations ≥10 mg L-1 displayed moderate toxic effects on the survival, spontaneous movement, hatching, heart rates of the embryos and their development, which were evidenced by yolk and pericardial sac edemas, body length reduction and tail flexure in time- and concentration-dependent manners at specific stages. The 24 h-, 48 h- and 96 h-LC50 values of carbaryl to zebrafish embryos were 41.80 ± 1.10, 17.80 ± 1.04 and 14.46 ± 1.05 mg L-1, respectively. These results suggested that carbaryl is moderately toxic to FG cells cultured in vitro and zebrafish embryos, and the FG cells were similar to zebrafish embryos in their sensitivity to carbaryl as 24 h-IC50 and LC50 indicated.

关键词：氨基甲酸酯类杀虫剂遗传毒性细胞毒性西维因鳃细胞斑马鱼致畸性胚胎

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Comparison of the In Vitro and In Vivo Toxic Effects of Three Sizes of Zinc Oxide(ZnO)Particles Using Flounder Gill(FG)Cells and Zebrafish Embryos

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中国海洋大学学报：英文版 2017年第1期16卷 93-106页

作者： HAN Li ZHAI Yanan LIU Yang HAO Linhua guo huarong Key Laboratory of Marine Genetics and Breeding(Ocean University of China) Ministry of EducationCollege of Marine Life SciencesQingdao 266003P.R.China Institute of Evolution and Marine Biodiversity Ocean University of ChinaQingdao 266003P.R.China First Institute of Oceanography State Oceanic AdministrationQingdao 266061P.R.China

Nano-sized zinc oxide(n ZnO) particles are one kind of the most commonly used metal oxide nanoparticles(NPs). This study compared the cytotoxic and embryotoxic effects of three increasing sized ZnO particles(φ30 nm, 80-150 nm and 2 μm) in the flounder gill(FG) cells and zebrafish embryos, and analyzed the contribution of size, agglomeration and released Zn^(2+) to the toxic effects. All the tested ZnO particles were found to be highly toxic to both FG cells and zebrafish embryos. They induced growth inhibition, LDH release, morphological changes and apoptosis in FG cells in a concentration-, size-and time-dependent manner. Moreover, the release of LDH from the exposed FG cells into the medium occurred before the observable morphological changes happened. The ultrasonication treatment and addition of serum favored the dispersion of ZnO particles and alleviated the agglomeration, thus significantly increased the corresponding cytotoxicity. The released Zn^(2+) ions from ZnO particles into the extracellular medium only partially contributed to the cytotoxicity. All the three sizes of ZnO particles tested induced developmental malformations, decrease of hatching rates and lethality in zebrafish embryos, but size-and concentration-dependent toxic effects were not so obvious as in FG cells possibly due to the easy aggregation of ZnO particles in freshwater. In conclusion, both FG cells and zebrafish embryos are sensitive bioassay systems for safety assessment of ZnO particles and the environmental release of ZnO particles should be closely monitored as far as the safety of aquatic organisms is concerned.

关键词： zinc oxide (ZnO) nanoparticles cytotoxicity embryotoxicity zebrafish fish cells

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The Toxic Mechanism of High Lethality of Herbicide Butachlor in Marine Flatfish Flounder,Paralichthys olivaceus

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Journal of Ocean University of China 2010年第3期9卷 257-264页

作者： guo huarong YIN Licheng ZHANG Shicui FENG Wenrong Department of Marine Biology and Key Laboratory of Marine Genetics and Breeding Ministry of Education Ocean University of China Qingdao 266003 P. R. China

The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder,Paralichthys Olivaceus,was analyzed by histopathological examination,antioxidant enzymes activities and ATP content *** examination of gill,liver and kidney of exposed fishes showed that gill was a target organ of *** butachlor seriously impaired the respiration of gills by a series of lesions such as edema,lifting and detachment of lamellar epithelium,breakdown of pillar cells,and blood *** dysfunction of gill respiration caused suffocation to the exposed flounder with extremely high acute *** enzyme activity assay of the in vitro cultured flounder gill(FG) cells exposed to butachlor indicated that butachlor markedly inhibited the antioxidant enzyme activities of Superoxide dismutase(SOD),catalase(CAT) and glutathione peroxidase(GPX).Furthermore,along with the decline of antioxidant enzyme activities,ATP content in the exposed FG cells decreased,*** infers that the oxidative stress induced by butachlor can inhibit the production of cellular *** decrease of ATP content was also observed in the exposed flounder gill *** together,as in FG cells,butachlor possibly induced a short supply of ATP in pillar cells by inhibiting the antioxidant enzyme activities and then affecting the contractibility of the pillar cells,which in turn resulted in the blood congestion and suffocation of exposed flounder.

关键词：毒性机制丁草胺比目鱼除草剂牙鲆海洋抗氧化酶活性谷胱甘肽过氧化物酶

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Cloning, Expression and Characterization of Translationally Controlled Tumor Protein (TCTP) Gene from Flatfish Turbot (Scophthalmus maximus)

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Journal of Ocean University of China 2008年第2期7卷 184-192页

作者： WANG Jian guo huarong ZHANG Shicui YIN Licheng guo Bin WANG Shaojie College of Marine Life Sciences Ocean University of China Qingdao 266003 P. R. China

A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot (Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5’ untranslated region (UTR) of 84 bp, a 3’ UTR of 451 bp and an open reading frame (ORF) of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5’UTR of SmTCTP started with a 5’-terminal oligopyrimidine tract (5’-TOP), a typical feature for translationally controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known verte- brate TCTPs in a range of 58.8% to 64.1%. The length of fish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch (Lateolabrax japonicus) is 170 aa in length, while that of zebrafish (Danio rerio) and rohu (Labeo rohita) is 171 aa in length. North- ern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET30a-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further inves- tigation of the biological function of TCTP in fish.

关键词：大比目鱼大菱鲆基因克隆肿瘤基因表达

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In vitro Acute Cytotoxicity of Abamectin to the Gill Cell Line of Flounder Paralichthy olivaceus

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Journal of Ocean University of China 2007年第4期6卷 369-372页

作者： XU Yuyan guo huarong XIAO Qin SU Feng YIN Licheng College of Life Science and Technology Ocean University of China Qingdao 266003 P. R. China

The cytotoxicity of abamectin to the Gill Cell Line of Flounder (FG cell line) was examined in this study. It was found that the exposure of FG cells to abamectin caused the decreases of both cell growth rate and antioxidant enzyme activities,and the increase of intracellular O2- content. It was proposed that the reduction of antioxidant enzyme activities in FG cells caused the accu- mulation of O2- content in FG cells, leading to the change of cell morphology and even the death of cells. The results showed that FG cell line is suitable for the evaluation of the acute toxicity of abamectin.

关键词：抗氧化剂细胞毒性生命科学生物学

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Computational identification and characterization of microRNAs and their targets in Penaeus monodon

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Journal of Oceanology and Limnology 2018年第3期36卷 853-869页

作者：濮龙军王晶王玉左建伟郭华荣 Ministry of Education Key Laboratory of Marine Genetics and Breeding College of Marine Life Sciences Ocean University of China Institute of Evolution and Marine Biodiversity Ocean University of China

Study on shrimp miRNAs was limited and just 7 mature miRNA sequences of Marsupenaeus japonicus are deposited in mir Base database. In this study, miRNAs and their target gene candidates were computationally identified from shrimp Penaeu s monodon and then experimentally validated. Using 39 908 expressed sequence tags(ESTs) and 21 124 genome survey sequences(GSSs) of P. monodon(pmo) as reference dataset, a comprehensive approach based on inter-species homolog search was employed to investigate the candidate miRNAs(i.e. pmo-miRNA). A total of eight miRNAs belonging to 7 families were computationally identified and five out of them were subsequently validated by PCR and sequencing. Of these, pmo-miR-4961a, pmo-miR-4961b, pmo-miR-4979 and pmo-miR-3819 were first identified from shrimps. Both the mature pmo-miRNAs and the corresponding precursors were conserved among different species. Based on perfect or near-perfect match to the target region, the target gene candidates of pmomiRNAs were predicted from 10 331 mRNA sequences of P. monodon. A total of 20 genes were predicted as the targets of pmo-miR-4961a, pmo-miR-4961b, pmo-miR-4979 and pmo-miR-6492. Experimental validation by dual luciferase reporter assay confi rmed the targeting between 3 pmo-miRNAs and one or two of their target genes, especially the pmo-miR-4979 which could significantly down-regulate the expression of target gene(JR226772). This study updates the miRNAs and their targets in P. monodon and lays a solid foundation for future RNAi study.

关键词：碱基数据对虾生态系统生物学

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法国近代保持或重获君主权力的隐匿言行--梵蒂冈有关秘密档案的探讨

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世界历史 2009年第2期 37-45页

作者：郭华榕北京大学历史系教授北京100871

法国近代,波旁王族、奥尔良王族、波拿巴王族的更替浮沉的主要事实并不复杂,它们的政治原则也可以在其声明、宣言与宪法当中找到。但是,这些王族追逐与护卫政治权力的内情却极少见诸坊间的书刊。梵蒂冈秘密档案馆所藏的档案为后人揭开... 详细信息

法国近代,波旁王族、奥尔良王族、波拿巴王族的更替浮沉的主要事实并不复杂,它们的政治原则也可以在其声明、宣言与宪法当中找到。但是,这些王族追逐与护卫政治权力的内情却极少见诸坊间的书刊。梵蒂冈秘密档案馆所藏的档案为后人揭开了内幕:这些王族利用秘密手段,掌权时力求保持手中的权力,丧失权力后渴望重获国家权力。由于法国大多数人信仰天主教,上述王族曾不同程度地寻求教廷的支持。

关键词：梵蒂冈秘密档案尚博尔伯爵路易-菲利普拿破仑三世教皇国

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法兰西历史的误读

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四川师范大学学报（社会科学版） 2010年第2期37卷 117-125页

作者：郭华榕北京大学历史系北京100871

法国历史的重要意义毋庸置疑。尽管近年来法国史学研究呈现出"萧条"和"平淡"的状况,但在档案的开放和研究视野的开阔方面仍取得了不少进步和成就。我国学术界对法国历史的研究取得了一定的成绩,然而若干误读至今还在束缚着我们,比如左... 详细信息

法国历史的重要意义毋庸置疑。尽管近年来法国史学研究呈现出"萧条"和"平淡"的状况,但在档案的开放和研究视野的开阔方面仍取得了不少进步和成就。我国学术界对法国历史的研究取得了一定的成绩,然而若干误读至今还在束缚着我们,比如左翼史学遭到了"个人化"而忽视其对大革命的批评、对于档案与社会调查等的认识不足、未能汲取国际学术界已经澄清的史实的成果等等。我国学术界应不断加强对法国史的研究,多利用法国原始档案和调查资料,加强原文名著的翻译,努力避免对法国历史的误读。

关键词：法国史学研究趋势中国学术界误读

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