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Identification and characterization of adult alpha-and beta-globin genes and their genomic arrangement in Pseudosciaena crocea

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Acta Oceanologica Sinica 2006年第4期25卷 65-76页

作者： chu wuying QIAN Ronghua WANG Lianshen YU Xiameng YOU Zhenqiang YU Lian Department of Biomedical Engineering Zhejiang University Hangzhou 310027 China Institute of Preventive Veterinary Medicine Zhejiang University Hangzhou 310029 China

The α- and β-globin genes from Pseudosciaena crocea were cloned by rapid amplification of cDNA 3 ＇-end （ 3 ＇-RACE）. The cDNA of the α-globin is 595 bp with the ATG start codon located at Position 37, the TAA stop codon at Position 469 and the AATAAA polyadenylation signal at Position 560, which codifies 145 amino acids. The entire open reading frame of the β-globin gene is 447 bp long, which encodes 148 amino acids. Amino acid identity of the α- globin or β-globin gene compared with those reported in other fish species, ranged from 31.9% to 76.4%. When comparing with human α- and β-globins, three important alterations in the structural regions can be noted： ct39 Thr→Gln, α113 His→Tyr and β117 His→Lys. The α-globin has a unique inserted amino acid residue in the 47th position. To understand the process of globin gene duplication and identify the regulatory elements present in the intergenic and intragenic regions of globin genes, the genomic arrangement of α- and β-globin genes was investigated. The results showed that the orientation of the two genes was head-to-head relative to each other. The intergenic region between the translation initiation codons of the linked α- and β-globin genes contains classical promoter elements and the length of it is much shorter than that reported in other fish.

关键词： Pseudosciaena crocea globin gene cloning structural region head-to-head regulatory elements

Intragenic and intergenic sequences regulating the expression of the 5'-to-5' linked adult α-and β-globin genes from large yellow croaker Pseudosciaena crocea

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Acta Oceanologica Sinica 2008年第5期27卷 126-133页

作者： chu wuying YU Lian QIAN Ronghua MENG Tao ZHOU Ruixue FU Guihong CHEN Jia ZHANG Jianshe Department of Bioengineering and Environmental Science Changsha University Changsha 410003 China Institute of Preventive Veterinary Medicine Zhejiang University Hangzhou 310029 China

One adult α-globin gene and one β-globin gene have been cloned from the large yellow croaker Pseudosciaena crocea. Linkage analysis indicated that the α- and β-globin genes were oriented head-to-head relative to each other. To identify the regulatory elements present in the intergenic and intragenic regions of the globin complex, the intergenic region alone or together with the β-globin gene first intron was cloned into the luciferase-reporter vector pGL3-Basic respectively, and the chimeric constructs were tran- siently transfected into Vero cells and primary fish erythrocytes. The intergenic region cannot support the high-level expression of luciferase. However, the promoter activity of the intergenic region was strongly stimulated by the positive regulatory elements （PRE） located in the β-globin gene intron 1. Thus, it is proposed that the intergenic promoters and intragenic PRE were necessary for the effective expression of the linked α- and β-globin genes.

关键词： globin intergenic intragenic intron large yellow croaker promoter regulation

EST-Based Identification of Genes Expressed in Skeletal Muscle of the Mandarin Fish (Siniperca chuatsi)

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Genomics, Proteomics & Bioinformatics 2011年第1期9卷 30-36页

作者： Feng Ding wuying chu Peng Cui Meng Tao Ruixue Zhou Falan Zhao Songnian Hu Jianshe Zhang CAS Key Laboratory of Genome Sciences and Information Beijing Institute of Genomics Chinese Academy of Sciences Beijing 100029 China Department of Bioengineering and Environmental Science Changsha University Changsha 410003 China

To enrich the genomic information of the commercially important fish species, we obtained 5,063 high-quality expressed sequence tags （ESTs） from the muscle cDNA database of the mandarin fish （Siniperca chuatsi）. Clustering analysis yielded 1,625 unique sequences including 443 contigs （from 3,881 EST sequences） and 1,182 sin- gletons. BLASTX searches showed that 959 unique sequences shared homology to proteins in the NCBI non-redundant database. A total of 740 unique sequences were functionally annotated using Gene Ontology. The 1,625 unique sequences were assigned to Kyoto Encyclopedia of Genes and Genomes reference pathways, and the results indicated that transcripts participating in nucleotide metabolism and amino acid metabolism are relatively abundant in S. chuatsi. Meanwhile, we identified 15 genes to be abundantly expressed in muscle of the mandarin fish. These genes are involved in muscle structural formation and regulation of muscle differentiation and development. The most remarkable gene in S. chuatsi is nuclease diphosphate kinase B, which is represented by 449 EST sequences accounting for 8.86% of the total EST sequences. Our work provides a transcript profile expressed in the white muscle of the mandarin fish, laying down a foundation in better understanding of fish genomics.

关键词： mandarin fish cDNA library EST muscle

Isolation and Identification of Bacterial Pathogens of Proteus vulgaris and Chryseobacterium meningosepticum from Peodiscus sinensis and Observation on the Histopathology

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Agricultural Biotechnology 2021年第2期10卷 61-65页

作者： Guomin JIANG Li LIU wuying chu Zhigang HE Li ZOU Dongwu WANG Shiming DENG Fisheries Research Institute of Hunan Province Changsha 410153China Hunan Provincial Key Laboratory of Nutrition and Quality Control of Aquatic Animals Changsha 410003China Department of Bioengneering and Environmental Science Changsha UniversityChangsha 410003China

Two dominant strains of bacteria Lb18-01 and Lb18-02 were isolated and purified from scabies,liver and intestine of diseased Peodiscus *** artificial infection test,the two strains were verified as pathogenic strains with similar characters to that of natural infectious *** strains Lb18-01 and Lb18-02 showed strong pathogenicity to healthy *** in the artificial infection experiment,so they were the pathogenic strain of the *** to the morphology,physicochemical characteristics,16 S rDNA sequence analysis and phylogenetic tree clustering,the pathogenic strains Lb18-01 and Lb18-02 were identified as Proteus vulgaris and Chryseobacterium meningosepticum which were tolerant to 20 drugs such as penicillin,tetracycline and *** observation on diseased *** showed the pathological symptoms of sepsis such as hemorrhage and congestion of liver,spleen and intestine,and glomerular disintegration.

关键词： Peodiscus sinensis Proteus vulgaris Chryseobacterium meningosepticum Drug susceptibility test Histopathology

Expression,Characterization and Immunogenicity of a Major Outer Membrane Protein from Vibrio alginolyticus

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Acta Biochimica et Biophysica Sinica 2007年第3期39卷 194-200页

作者： Ronghua QIAN~(1,2) wuying chu~3 Zhijuan MAO~(1,4) Chongwen ZHANG~(1,2) Yongwei WEI~2 Lian YU~2* 1 Department of Biomedical Engineering,Zhejiang University,Hangzhou 310027,China 2 Institute of Preventive Veterinary Medicine,Zhejiang University,Hangzhou 310029,China 3 Institute of Subtropical Agriculture,Chinese Academy of Sciences,Changsha 410125,China 4 Ningbo Oceanography and Fishery Institute,Ningbo 315012,China Department of Biomedical Engineering Zhejiang UniversityHangzhou 310027 China Institute of Preventive Veterinary Medicine Zhejiang UniversityHangzhou 310029 China Institute of Subtropical Agriculture Chinese Academy of SciencesChangsha 410125 China Ningbo Oceanography and Fishery InstituteNingbo 315012 China

Vibrio alginolyticus is one of the Vibrio pathogens common to humans and marine *** infection and induction of the host immune response,outer membrane proteins of bacteria play animportant *** this study,an outer membrane protein gene(ompW)was cloned from *** andexpressed in Escherichia *** 645 bp open reading frame(ORF)encodes a protein of 214 amino acidresidues with a predicted molecular weight of 23.3 *** amino acid sequence showed a high identitywith that of Photobacterium damselae(96.2%)and Vibrio parahaemolyticus(94.4%).The alignment analy-sis indicated that OmpW was highly *** dodecyl sulfate-polyacrylamide gel electrophoresisshowed that the gene was over-expressed in *** BL21(DE3).Western blot analysis revealed that theexpressed protein had *** recombinant protein was purified by affinity chromatographyon Ni-NTA Superflow *** yellow croaker vaccinated with the purified OmpW showed significantlyincreased antibody to OmpW,which could resist the infection by ***.A specific antibody wasdetected by enzyme-linked immunosorbent *** study suggested that the conserved OmpW could bean effective vaccine candidate against infection by ***.

关键词： Vibrio alginolyticus ompW expression characterization immunogenicity vaccine candidate

虾壳源蛋白水解物降糖降脂活性评价及肽序分析

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食品与机械 2024年第4期40卷 148-157页

作者：韩鹏薇易彤李虹辉吴昊李彦褚武英长沙理工大学食品与生物工程学院湖南长沙410114 长沙学院生物与化学工程学院湖南长沙410022

目的:采用酶解法制备克氏原螯虾壳蛋白水解物(Procambarus clarkii shell protein hydrolysates,PCSPHs),并分析其体外降糖降脂活性及肽序。方法:分别采用胃蛋白酶、碱性蛋白酶、胰蛋白酶、风味蛋白酶和木瓜蛋白酶水解制备不同虾壳蛋白... 详细信息

目的:采用酶解法制备克氏原螯虾壳蛋白水解物(Procambarus clarkii shell protein hydrolysates,PCSPHs),并分析其体外降糖降脂活性及肽序。方法:分别采用胃蛋白酶、碱性蛋白酶、胰蛋白酶、风味蛋白酶和木瓜蛋白酶水解制备不同虾壳蛋白水解物,分析其体外降糖降脂活性和肽序列;运用Peptide Ranker及BIOPEP-UWM网站在线分析,再以核受体PPARγ配体结合区域的晶体结构作为靶点,使用Autodock vina进行分子对接模拟,获得具有潜在降糖降脂活性的虾壳肽。结果:胃蛋白酶水解物(PEP-PCSPHs)对α-淀粉酶和α-葡萄糖苷酶活性具有较强的抑制作用,IC 50值分别为(5.42±0.05),(7.11±1.01)mg/mL;胰蛋白酶水解物(TRY-PCSPHs)对胰脂肪酶活性具有最强的抑制能力,IC 50值为(4.71±1.12)mg/mL,且对甘氨胆酸钠表现出最好的体外结合效果。此外,经质谱鉴定PEP-PCSPHs和TRY-PCSPHs中分别得到3391,2086条肽序;通过在线网站预测和分子对接筛选出多条均能与PPARγ结合的降糖降脂虾壳活性肽(PCSAPs)。结论:酶解克氏原螯虾壳制备的虾壳蛋白水解物具有潜在的降糖降脂活性,可能改善糖脂代谢紊乱。

关键词：克氏原螯虾壳蛋白水解物 PPARγ 分子对接降糖降脂活性

鳜twist2序列特征、时空表达模式及靶向miRNAs预测分析

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中国水产科学 2024年第2期31卷 144-154页

作者：程聪益朱鑫曾维宾琴王玮褚武英张建社湖南师范大学生命科学学院省部共建淡水鱼类发育生物学国家重点实验室长沙学院生物与化学工程学院水生动物营养与品质调控湖南省重点实验室

Twist2在调控动物发育过程发挥了重要作用。为解析鳜(Siniperca chuatsi) twist2基因的表达模式以及参与肌肉发育调节的功能和机制,本研究从鳜基因组数据库获得twist2基因序列,采用生物信息学方法对Twist2蛋白特征和同源进化进行了分析... 详细信息

Twist2在调控动物发育过程发挥了重要作用。为解析鳜(Siniperca chuatsi) twist2基因的表达模式以及参与肌肉发育调节的功能和机制,本研究从鳜基因组数据库获得twist2基因序列,采用生物信息学方法对Twist2蛋白特征和同源进化进行了分析。鳜twist2基因所编码的蛋白由162个氨基酸组成,具有一个保守功能域bHLH结构域。基于氨基酸序列的多序列比对结果显示,Twist2蛋白在脊椎动物中具有较高的相似性;采用RT-qPCR技术分析了鳜twist2基因的时空表达规律,发现twist2基因在不同发育阶段存在表达差异,原肠期之前表达水平较低,从神经胚期开始显著上升,尾芽期表达最高;在鳜组织中的表达情况显示,该基因在脾脏中表达量较高,其次是脑和红肌。采用整胚原位杂交技术检测其早期胚胎不同发育阶段的twist2基因定位,发现其主要在神经胚层和体节中特异性表达。采用Targetscan和RNAhybrid工具对可能靶向鳜twist2基因的miRNAs进行预测,结果发现miR-30a、miR-30b、miR-30e-5p和miR-204可能作用于twist2 3'UTR,提示twist2是miR-30a、miR-30b、miR-30e-5p和miR-204的潜在靶基因。鳜短期饥饿胁迫实验显示,在饥饿3 d时twist2的表达与上述miRNAs的表达呈明显的负相关,表明twist2与上述4个miRNAs存在潜在的调控关系。因此,本研究结果将有助于从分子水平了解twist2基因的序列特征、时空表达规律以及其调控肌肉生长发育的潜在功能,为鱼类发育生物学以及健康养殖提供理论依据。

关键词：鳜 twist2 时空表达 miRNA 短期饥饿

翘嘴鳜MyomiRs时空表达特征及靶向Pax7的预测分析

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中国水产科学 2023年第8期30卷 933-941页

作者：曾维朱鑫程聪益孟杨杨张宇宾琴李灿褚武英陈韬湖南农业大学动物医学学院长沙学院生物与化学工程学院水产动物营养与品质调控湖南省重点实验室

MyomiRs为一类肌肉特异性microRNAs (miRNAs),对于肌细胞的增殖和分化具有重要作用。本研究旨在探究翘嘴鳜(Sinipercachuatsi)4种MyomiRs基因(miR-1a、miR-133a-3p、miR-206和miR-499)的时空表达及其在短期饥饿胁迫下的表达特征,并预测分析4种MyomiRs对Pax7的调控作用。应用实时荧光定量PCR检测4种MyomiRs在翘嘴鳜不同组织、胚后不同发育阶段的白肌以及饥饿5d白肌中的表达情况，并利用RNAhybrid对4种MyomiRs与Pax7 mRNA 3′UTR的靶向位点进行预测。结果显示, miR-1a、miR-133a-3p和miR-206在D60 (出膜后60 d)高表达, miR-499在D100高表达。miR-1a和miR-133a-3p在红肌、白肌和心肌中高表达,在其他组织中表达量低。miR-206在红肌和白肌中高表达,在其他组织中表达较量低。miR-499在心肌中表达量较高,在红肌和白肌中的表达量次之,其他组织中表达量低。饥饿5d后4种MyomiRs的表达均显著上升,表明鳜骨骼肌可能对饥饿胁迫做出应激反应,通过上调4种MyomiRs的表达来调节代谢等生理过程。靶基因预测发现miR-1a、miR-133a-3p和miR-206与Pax7的3′UTR存在结合位点,进一步通过荧光定量PCR验证, miR-206和Pax7在翘嘴鳜胚后不同发育阶段以及昼夜节律性的表达趋势相反。本研究结果表明,miR-1a和miR-133a-3p具有肌肉特异性,miR-206和miR-499为肌源性组织中富集型表达。此外, miR-206可能直接调控Pax7的表达,进而调控翘嘴鳜幼鱼时期肌细胞的增殖和分化从而影响肌肉的生长发育。

关键词：翘嘴鳜 MyomiRs 骨骼肌表达特征昼夜节律

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中华鳖疥疮病原菌的分离鉴定及病理组织观察

福建农林大学学报（自然科学版） 2020年第5期49卷 660-665页

作者：邓时铭刘丽褚武英李跃辉王江伟陈圆华湖南省水产科学研究所湖南长沙410153 长沙学院生物工程与环境科学学院湖南长沙410003 湖南农业大学动物科学技术学院湖南长沙410128

取疥疮体表病灶、肝脏、肠道组织进行病原菌分离纯化,获得优势菌株Lb18-01和Lb18-02,经人工感染试验验证,出现与自然发病相同的症状,确定其为致病菌株.通过形态学观察、生理生化指标测定、分子生物学鉴定得出,分离菌株Lb18-01和Lb18-02... 详细信息

取疥疮体表病灶、肝脏、肠道组织进行病原菌分离纯化,获得优势菌株Lb18-01和Lb18-02,经人工感染试验验证,出现与自然发病相同的症状,确定其为致病菌株.通过形态学观察、生理生化指标测定、分子生物学鉴定得出,分离菌株Lb18-01和Lb18-02为普通变形杆菌和脑膜脓毒性黄杆菌;药物敏感性试验显示,分离菌株对青霉素、四环素、氨苄西林等20种抗生素药物均产生了耐药性.经病理组织观察可见,肝、脾和肠出血、淤血,肾小球解体,呈现脓毒症病理症状.

关键词：中华鳖疥疮普通变形杆菌脑膜脓毒性黄杆菌药敏试验病理组织学