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Spatial and diel variations of the prokaryotic community in the Phaeocystis globosa blooms area of Beibu Gulf,China

Acta Oceanologica Sinica 2022年第12期41卷 87-97页

作者： Cheng He Sha Xu Shuqun Song Caiwen Li North China Sea Environmental Monitoring Center State Oceanic AdministrationQingdao 266033China Key Laboratory of Ecological Prewarning Protection and Restoration of Bohai SeaMinistry of Natural ResourcesQingdao 266033China Key Laboratory of Marine Ecology and Environmental Sciences Chinese Academy of SciencesQingdao 266071China Marine Ecology and Environmental Science Laboratory Pilot National Laboratory for Marine Science and Technology(Qingdao)Qingdao 266237China Center for Ocean Mega-Science Chinese Academy of SciencesQingdao 266071China Shaanxi Provincial Academy of Environmental Science Xi’an 710061China

While prokaryotes play key roles in nutrient cycling and energy flow during Phaeocystis globosa blooms,the information on the spatial and diel temporal distribution of the prokaryotic community during Phaeocystis blooms remains scarce.In January 2019,we used high-throughput sequencing of the 16S rRNA gene to explore the spatial and diel variations of particle-attached(PA)and free-living(FL)prokaryotic communities during the blooming phase of P.globosa in Beibu Gulf,Guangxi,China.The results suggested a significant spatial variation pattern in the horizontal distribution of prokaryotic communities,while there was no significant difference in the vertical direction.Both spatial distance and environmental variables shaped the horizontal distribution of the prokaryotic community structure,while environmental variables,particularly the abundance of P.globosa colony and Chl a,showed more significant influence and were closely related to the structure and variation of the prokaryotic community.Strong vertical mixing of the water column disrupted the vertical structure heterogeneity of the prokaryotic community in winter.There were significant differences in the diel samples of PA prokaryotic communities,but not in the FL prokaryotic communities.Nitrate,ammonium and the abundance of P.globosa colony were the key environmental variables impacting the diel variations of prokaryotic communities over the sampling period.The present study provided valuable information to depict the spatial-temporal variations of the microbial community and its association with environmental parameters during P.globosa bloom in the tropical gulf.

关键词： algal bloom 16S rRNA genes prokaryotic community diversity spatial patterns diel distribution

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Structural Basis of Gabija Bacterial Defense System

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Bulletin of the Chinese Academy of Sciences 2024年第1期38卷 53-54页

prokaryotic organisms have evolved sophisticated immune systems for self-protection to resist bacteriophage invasion.With further research on prokaryotic immune systems,several important molecular biology tools have been developed,including the widely used restriction endonuclease in molecular cloning experiments and the CRISPR-Cas system in gene editing.

关键词： prokaryotic invasion molecular

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Cloning, construction of prokaryotic expression vector and expression of Escherichia coli cytosine deaminase gene

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Chinese Science Bulletin 1998年第3期43卷 223-230页

作者： Shengjun Ren Huiqiu Jiang Jianren Gu Shanghai Canc Inst Natl Lab Oncogenes & Related Genes Shanghai 200032 Peoples R China

Cytosine deaminase gene of Escherichia coli strain H30 was cloned, and its initiation codon of ’GTG’ was mutated to ’ATG’ by PCR. prokaryotic recombinant expression vector pBV220CD was constructed. Clone with high enzyme activity were selected by detecting their specific activity of cytosine deaminase. 5FC（5FC, 5fluorocytosine） could induce the lethal toxicity to cells containing active CD gene. DNA sequence analysis indicated that there were 16 altered bases and 5 of them resulted in the alteration of amino acids in predicted peptide by comparing DNA sequence of the clone H30CD11 with high enzyme activity with CD gene reported in Gene Bank.

关键词： cytosine deaminase gene prokaryotic expression vector SDS_PAGE analysis cytosine deaminase activity assay high activity clone of CD.

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Cloning and prokaryotic expression of HGLP cDNA

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Chinese Science Bulletin 2001年第13期46卷 1089-1092页

作者： ZHOU Haijun HUANG Xiaowei ZHOU Yan LI Guangtao YIN Bin MA Yanling PENG Xiaozhong YUAN Jiangang QIANG Boqin Institute of Basic Medical Sciences Chinese Academy of Medical Sciences&Peking Union Medical CollegeNational Center of Human Genome ResearchBeijing 100005China

A novel cDNA, HGLP, which encodes a G-protein coupled receptor (GPCR) like protein, has been isolated and cloned. The coding region of the human HGLP predicts a 7-transmembrane region protein with motifs of rhodopsin-like GPCR superfamily. Northern blot analysis reveals a 3-kb transcript in various human tissues examined. The N- and C-terminal coding regions of HGLP, which are deduced as non-transmembrane regions, have been amplified by PCR and cloned into pET30a+ vector. Then the re-combinant proteins are highly expressed in E. coli.

关键词： G-protein coupled receptor HGLP cDNA prokaryotic expression.

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prokaryotic expression, purification of a novel candidate tumor suppressor gene FUS1 and characterization of its polyclonal antibodies

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Journal of Biomedical Science and Engineering 2010年第4期3卷 397-404页

作者： Dong-Mei Zhang Han-Shuo Yang Xin-Yu Zhao Wen Zhu Zhi-Hua Feng Yang Wan Zhi-Wei Zhao Ming-Hai Tang Nong-Yu Huang Yu-Quan Wei 不详

FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 ×His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.

关键词： FUS1 Polyclonal Antibody prokaryotic Expression Recombinant Protein Tumor Suppressor Gene

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prokaryotic Expression of IBV N Protein and Development of Indirect IBV N Protein-mediated ELISA

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Journal of Northeast Agricultural University(English Edition) 2020年第1期27卷 69-79页

作者： Li Wei-qun Wang Xin Zhong Ming Sun Xiao-qi Zhao Lei Huang Xiao-dan Zhang Rui-li Li Guang-xing 无 Key Laboratory for Laboratory Animals and Comparative Medicine of Heilongjiang Province College of Veterinary MedicineNortheast Agricultural UniversityHarbin 150030China College of Pharmaceutical Engineering Jilin Agriculture Science and Technology CollegeJilin 132101China

Avian infectious bronchitis(IB)is an acute and highly contagious disease caused by infectious bronchitis virus(IBV).In the study,according to IBV gene sequences published in Gen Bank,specific primers were designed to clone N gene by RT-PCR,and this gene was inserted into p ET-30a(+)vector resulting in a prokaryotic expression plasma p ET-30a-N.The results of SDS-PAGE and Western Blot analysis showed that the recombinant protein was expressed successfully and had good reactivity with IBV positive serum.Using purified recombinant N protein as a coating antigen,the indirect ELISA protocol was established and optimized,in which N protein was 2.5μg·m L^-1 of concentration,sample serum of 1:40 dilution.For clinical specimen,the IBV antibodies could be detected by this method efficiently and got nearly the same results as those of IBV-mediated ELISA.It would provide a good tool for rapid diagnosis and epidemiological study of avian infectious bronchitis.

关键词： infectious bronchitis virus N protein prokaryotic expression indirect ELISA

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prokaryotic Horizontal Gene Transfer in Freshwater Lakes: Implications of Dynamic Biogeochemical Zonation

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Journal of Environmental Protection 2012年第12期3卷 1634-1654页

作者： Christopher N. Drudge Lesley A. Warren School of Geography and Earth Sciences McMaster University Hamilton Canada

The highly adaptive nature of prokaryotic communities in the face of changing environmental conditions reflects in part their ability to share advantageous genetic information through horizontal gene transfer (HGT). Natural freshwater lacustrine (lake) systems are a vital and finite resource, and the influence of HGT on their quality (e.g. enabling the spread of antibiotic resistance and xenobiotic catabolism genes) is likely significant. Laboratory and in situ studies indicate that the dynamic physical, chemical, and biological conditions that structure freshwater systems can influence HGT within freshwater prokaryotic communities. Thus, understanding how biogeochemical parameters impact HGT in freshwater lakes is an emerging knowledge gap with potential implications for ecosystem and human health on a global scale. In this review, we provide a general synopsis of what is known about HGT in freshwater prokaryotic communities, followed by an integrated summary of current knowledge identifying how biogeochemical factors may influence prokaryotic HGT in freshwater lacustrine systems.

关键词： Gene Transfer Freshwater Water Quality prokaryotic Biogeochemistry

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cDNA Cloning, prokaryotic Expression of Two Splicing Products of mLRG, a Mouse Gene of Lipopolysaccharide Response

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International Journal of Clinical Medicine 2015年第11期6卷 867-875页

作者： Zhongming Dai Zanguo Nie Liang He Lina Guan Yunsheng Yang Department of Digestive Disease Chinese PLA General Hospital Beijing China Department of Digestive Disease Urumuqi Military General Hospital Urumuqi China Department of General Surgery Urumuqi Military General Hospital Urumuqi China Department of Stomatology Urumuqi Military General Hospital Urumuqi China

Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Methods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely identical. We spliced some fragments to a whole mouse lrg-cDNA sequence and designed a pair of primers at completely homologous fragments in 5’-UTR and 3’-UTR, we amplified mouse lrg-cDNA by RT-PCR. Then the sequence encoding the mLRG protein was amplified by RT-PCR from the total RNA of NIH3T3 cell stimulated by lps (lipopolysaccharide), and we got two splicing products of mLRG (mLRGW, mLRGS) and two sequences encoding protein were cloned into the prokaryotic expression vector pTAT so as to construct the recombinant expression vector pTAT-MLRGW and pTAT-MLRGS. The proteins were expressed in E. coli BL21 (DE3). Results: We got a cDNA fragment with the length of 1905 bp. Its location is at chromosome X qF4 site and we amplified two encoding regions covered 1554 bp and 1404 bp respectively (mlrgW mlrgS). His-TAT-mLRGW and His-TAT-mLRGS fusion protein were expressed successfully. mlrgW is consist of 10 exons and 9 introns;mlrgS is consist of 11exons and 10 introns. Conclusion: Cloning of two splicing products of mouse novel gene MLRG and prokaryotic protein expressions are of help in the further study of this gene.

关键词： mLRG Lipopolysaccharide Response Gene cDNA Cloning prokaryotic Protein Expression

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SOME DNA SEQUENCES FROM ANIMAL VIRUSES AND E. coli POSSESS prokaryotic ENHANCER-LIKE FUNCTION

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Chinese Science Bulletin 1991年第3期36卷 230-234页

作者：吴淑华阮力潘卫张励刘向东金奇宇文镐张丽兰侯云德 National Laboratory of Molecular Virology and Genetic Engineering Institute of Virology Chinese Academy of Preventive Medicine Beijing 100052 PRC Shanghai Second Military Medical College

Since Banerji et al. first described an enhancer dement in SV40 genome in 1981, similar enhancer sequences have been discovered in many other viral genomes and eukaryotic cells. It is not dear, however, whether there is also a similar transcription-controlling element in prokaryotic cells. In 1985 Hou et al. and Wu et al. surprisingly found

关键词： vaccinia virus respiratory syncytical virus prokaryotic enhancer-like element.

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cDNA cloning and recombinant expression of the general odorant binding protein Ⅱ from Spodoptera litura

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Science China(Life Sciences) 2009年第1期52卷 80-87页

作者： JIN FengLiang,DONG XiaoLin,XU XiaoXia & REN ShunXiang Engineering Research Center of Biological Control,College of Natural Resources and Environment,South China Agricultural University,Ministry of Education,Guangzhou 510642,China Engineering Research Center of Biological Control College of Natural Resources and Environment South China Agricultural University Ministry of Education Guangzhou China

A cDNA encoding the general odorant binding protein Ⅱ(GOBP Ⅱ) was isolated from the antennae of Spodoptera litura(SlGOBP Ⅱ,GenBank Accession No.EU086371) by homologous cloning and rapid amplification of cDNA ends(RACE).Sequencing and structural analyses revealed that the open reading frame(ORF) of SlGOBP Ⅱ was 489 bp,encoding 162 amino acids with a predicted MW of 18.2 kD and pI of 5.72.SlGOPB Ⅱ shared typical structural features of odorant binding proteins with other insects,including the six conservative cysteine residues.The deduced amino acid sequence of SlGOPB Ⅱ shared significant identity with the GOBP Ⅱ from S.frugiperda and S.exigua.RT-PCR and Northern blot analyses showed that SlGOBP Ⅱ was specifically expressed in the antennae.cDNA encoding SlGOBP Ⅱ was constructed into the pET-32a vector and the recombinant protein was highly expressed in Escherichia coli BL21(DE3) after induction with IPTG.SDS electrophoresis and Western blot analysis confirmed the molecular weight of the recombinant SIGOBPⅡ i.e,32 kD,which has a 6×His tag at the N-terminus.The recombinant SlGOBP Ⅱ was purified by single-step Ni-NTA affinity chromatography and used to raise antiserum in rabbits.ELISA showed that the titer of antiserum was 1:12800,while Western blot analysis showed that the recombinant SlGOBP Ⅱ was recognized as anti-SlGOBP Ⅱ antiserum.

关键词： Spodoptera litura antennae general odorant binding protein gene cloning prokaryotic expression

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