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Construction of mini-Tn4001 transposon vector for Mycoplasma gallisepticum

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Science China(Life Sciences) 2010年第11期53卷 1340-1345页

作者： CHEN HongJun, ZHAO ChunMei, SHEN XieYue, CHEN DanQing, YU ShengQing & DING Chan Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Shanghai China

The detailed genetic analysis of mycoplasmas has long been hampered by the lack of appropriate tools for genetic manipulation. In this study, the transposon vector, mini-Tn4001tetM, was constructed containing the tnp gene, encoding a transposase gene in Staphylococcus aureus, two copies of the IS256 inverted repeat sequence (inner and outer) and the tetM gene, from the Enterococcus faecalis Tn916 transposon, conferring resistance to tetracycline. This vector was electro-transformed into Mycoplasma gallisepticum (MG). The recombinant cells were screened by tetracycline selection. The results indicated that the transposon vector could replicate in MG strain R by successive passages, indicating that MG is a potential vector for expressing protective antigens of other pathogens.

关键词： Mycoplasma gallisepticum mini-Tn4001 transposon vector mycoplasma transformation

Identification of Two transposon-like Elements in Rice Wx Gene

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Science China Chemistry 1994年第4期37卷 437-447页

作者：王宗阳郑靠琴高继平王小全吴敏张景六洪孟民 Shanghai Institute of Plant Physiology Academia Sinica Shanghai 200032 PRC

Rice Wx genes have been cloned and sequenced from Oryza saliva subsp. japonica (Wx-R-jp) and subsp. indica (Wx-R-id) and wild rice Oryza saliva L. f. spontanea (Wx-R-sp). Comparison of the nucleotide sequences of these three Wx genes indicates that Wx-R-jp and Wx-R-id genes contain two types of transposon-like elements, designated by RTL-1 and RTL-2. in the region of their two introns and 5’ upstream, RTL-1 shares sequence homology with valine tRNA molecules, and contains an internal promoter of RNA polymerase Ⅲ. RTL-2 forms a stem-loop structure. Both RTL-1 and RTL-2 are flanked by direct repetitive sequences. Compared with the elements that have been known transposable, RTL-1 resembles the short interspersed-repeated DNA elements (SINEs) and RTL-2 is similar to the fold-back (FB) element of trahsposon of Drosophila.

关键词： transposon SINEs FB(fold-back) rice Wx gene.

Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)

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Animal Diseases 2021年第4期1卷 249-261页

作者： Janine T.Bossé Yanwen Li Leon G.Leanse Liqing Zhou Roy R.Chaudhuri Sarah E.Peters Jinhong Wang Gareth A.Maglennon Matthew T.G.Holden Duncan J.Maskell Alexander W.Tucker Brendan W.Wren Andrew N.Rycroft Paul R.Langford on behalf of the BRaDPT consortium Section of Paediatric Infectious Disease Department of Infectious DiseaseImperial College LondonSt.Mary’s CampusLondonUK Wellman Center for Photomedicine Harvard Medical SchoolBostonUSA The Applied Diagnostic Research and Evaluation Unit St George’s University of LondonLondonUK Department of Veterinary Medicine University of CambridgeCambridgeUK Department of Molecular Biology and Biotechnology University of SheffieldSheffieldUK Department of Pathology and Pathogen Biology The Royal Veterinary CollegeHatfieldUK The Wellcome Trust Sanger Institute CambridgeUK School of Medicine University of St AndrewsSt AndrewsUK The University of Melbourne ParkvilleVictoriaAustralia s Faculty of Infectious&Tropical Diseases London School of Hygiene&Tropical MedicineLondonUK

Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.

关键词： Mariner transposon TraDIS Pasteurellaceae Actinobacillus pleuropneumoniae Pasteurella multocida

The RAG key to vertebrate adaptive immunity descended directly from a bacterial ancestor

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National Science Review 2022年第8期9卷 18-21页

作者： Xin Tao Ziwen Huang Fan Chen Xinli Wang Tingting Zheng Shaochun Yuan Anlong Xu State Key Laboratory of Biocontrol Guangdong Key Laboratory of Pharmaceutical Functional GenesSouthern Marine Science and Engineering Guangdong Laboratory (Zhuhai)School of Life SciencesSun Yat-sen University Center for Infection and Immunity School of MedicineSun Yat-sen University Shanghai Institute of Immunology College of Basic Medical SciencesShanghai Jiao Tong University School of Medicine Laboratory for Marine Biology and Biotechnology Qingdao National Laboratory for Marine Science and Technology School of Life Sciences Beijing University of Chinese Medicine

The emergence of RAG recombinase to mediate V(D)J recombination has been considered an important milestone in the evolution of adaptive immunity in jawed vertebrates. In past decades,hypotheses, including the invasion of viral or bacterial genes and the transposition of mobile elements, have been proposed to shed light on the origin and evolution of the RAG genes and V(D)J recombination.

关键词： transposon

Functional characterization of piggyBac-like elements from Nilaparvata lugens(St?l)(Hemiptera:Delphacidae)

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2022年第6期23卷 515-527页

作者： Jun LYU Qin SU Jinhui LIU Lin CHEN Jiawei SUN Wenqing ZHANG State Key Laboratory of Biocontrol School of Life SciencesSun Yat-sen UniversityGuangzhou 510275China

Piggy Bac is a transposable DNA element originally discovered in the cabbage looper moth(Trichoplusia ni).The T.ni piggyBac transposon can introduce exogenous fragments into a genome,constructing a transgenic organism.Nevertheless,the comprehensive analysis of endogenous piggyBac-like elements(PLEs)is important before using piggyBac,because they may influence the genetic stability of transgenic lines.Herein,we conducted a genome-wide analysis of PLEs in the brown planthopper(BPH)Nilaparvata lugens(St?l)(Hemiptera:Delphacidae),and identified a total of 28 PLE sequences.All N.lugens piggy Bac-like elements(NlPLEs)were present as multiple copies in the genome of BPH.Among the identified NlPLEs,NlPLE25 had the highest copy number and it was distributed on five chromosomes.The full length of NlPLE25 consisted of terminal inverted repeats and sub-terminal inverted repeats at both terminals,as well as a single open reading frame transposase encoding546 amino acids.Furthermore,NlPLE25 transposase caused precise excision and transposition in cultured insect cells and also restored the original TTAA target sequence after excision.A cross-recognition between the NlPLE25 transposon and the piggy Bac transposon was also revealed in this study.These findings provide useful information for the construction of transgenic insect lines.

关键词： Nilaparvata lugens piggyBac-like elements transposon NlPLE25

Waxy allele diversity in waxy maize landraces of Yunnan Province, China

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Journal of Integrative Agriculture 2022年第2期21卷 578-585页

作者： WU Xiao-yang LONG Wen-jie CHEN Dan ZHOU Guo-yan DU Juan WU Shao-yun CAI Qing Biotechnology and Germplasm Resources Institute Yunnan Academy of Agricultural SciencesKunming 650223P.R.China

Waxy maize is one of the main fresh-eating maize types,and a mutation of the waxy gene causes the waxy character of maize grains.China is rich in waxy maize landraces,and Yunnan and its surrounding areas,are the place of origin and genetic diversity center of Chinese waxy maize.The six known waxy alleles of Chinese waxy maize are wx-D7,wx-D10,wx-Cin4,wx-124,wx-Reina,and wx-Xuanwei.The mutation sites of these alleles all occur in the coding region of the waxy gene,however,the mechanism by which the waxy characteristic is caused by the mutation in the regulatory region has only been reported rarely in maize.In this study,405 waxy maize landraces from Yunnan were used as materials to identify the insertion and deletion of a large sequence fragment in the upstream~3.5 kb regulatory region of the waxy gene by molecular marker detection.Three different waxy alleles were identifed in this study:wx-PIF/Harbinger,wx-hAT and wxElote2.These three types of mutations all represented transposons inserted into the regulatory region of the waxy gene.Wx-PIF/Harbinger was a 304-bp MITE class transposon insertion belonging to the PIF/Harbinger family,while wx-hAT was a 560-bp MITE class transposon insertion belonging to the hAT family,and wx-Elote2 was a 6560-bp LTR-like transposon insertion.In this study,the alleles were identifed for more than 70%of the waxy maize landraces in Yunnan,which provids a basis for the utilization of these waxy maize landraces.

关键词： waxy maize landraces Waxy allele transposon gene regulatory region Yunnan

Establishment of Real-Time Quantitative PCR Method for the Determination of transposon Copy Number in Cronobacter sakazakii

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Agricultural Biotechnology 2012年第1期1卷 40-43页

作者： Fei WANG Xinjun DU Rong ZHANG Guixiang XU Shuo WANG Key Laboratory of Food Nutrition and Safety Ministry of EducationTianjin University of Science&Technology

[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, recombinant plasmid containing both single-copy housekeeping gene atpD and EZ-TN5 transposon was constructed; based on the established standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon, copy number of atpD gene and EZ-TN5 transpason in three C. sakazakii mutants was detected and the ratio was calculated. [ Result] Correlation coefficients of the standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon were 0. 999 and 0.998, respectively ; the ratios of copy number of atpD gene and EZ-TN5 transposon in three C. sakazakii mutants were 0.98, 1.17 and 0.91, respectively, which indicates that EZ-TN5 transpeson in C. sakakii mutants is a single-copy. [ Conclusion] Real-time quantitative PCR method established in this study had high availability and could replace the Southern blot method to detect the copy num- ber of EZ-TN5 transposon in different bacteria.

关键词： transposon Copy number Real-time quantitative PCR Cronobacter sakazakii

Introduction of Foreign Genes into Silkworm Eggs by Electroporation and Its Application in Transgenic Vector Test

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Acta Biochimica et Biophysica Sinica 2004年第5期36卷 323-330页

作者： Xiu-Yang GUO#, Liang DONG#, Sheng-Peng WANG, Ting-Qing GUO, Jian-Yang WANG, and Chang-De LU* Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China

Electroporation as a methodology to introduce foreign genes into silkworm eggs was system- atically analyzed. The foreign gene in both the newly hatched and 3rd instar larva DNA can be detected by PCR. The amount of foreign gene in 3rd instar larva DNA was about 1/1000 of that in newly hatched larva DNA. The ratio of foreign gene entering into silkworm eggs was voltage dependent and showed significant difference between the tested silkworm strains. When the piggyBac transposon system was applied, the effect of nuclear localization signal (NLS) peptide and the in vitro transcribed transposase mRNA on the transposi- tion rate has been measured. Results showed that the in vitro transcribed transposase mRNA facilitated trans- position to take place earlier and NLS could result in higher transposition probability and earlier transposition as well. When linearized vectors containing varied length of flanking homologous sequences around a re- porter gene were introduced into silkworm eggs by electroporation, the one with 2.6 kb total arm length gave higher G1 positive ratio than that with total arm length of 1.5 kb and 800 bp.

关键词： silkworm eggs gene introduction electroporation transposon homologous sequences

Natural Variation in CCD4 Promoter Underpins Species-Specific Evolution of Red Coloration in Citrus Peel

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Molecular Plant 2019年第9期12卷 1294-1307页

作者： Xiongjie Zheng Kaijie Zhu Quan Sun Weiyi Zhang Xia Wang Hongbo Cao Meilian Tan Zongzhou Xie Yunliu Zeng Junli Ye Lijun Chai Qiang Xu Zhiyong Pan Shunyuan Xiao Paul D.Fraser Xiuxin Deng Key Laboratory of Horticultural Plant Biology(Ministry of Education) Huazhong Agricultural University.WuhanChina School of Biological Sciences.Royal Holloway University of LondonEghamSurrey TW20 OEXUK Department of Plant Science and Landscape Architecture Institute for Bioscience and Biotechnology ResearchUniversity of MarylandRockvilleMDUSA Present address:College of Horticulture Agricultural University of Hebei.Baoding Hebei 071000China Present address:Key Laboratory of Biology and Genetic Improvement of Oil Crops Ministry of Agriculture.Oil Crops Research Institute of the Chinese Academy of Agricultural SciencesWuhan 430070China

Carotenoids and apocarotenoids act as phytohormones and volatile precursors that influence plant development and confer aesthetic and nutritional value critical to consumer preference.Citrus fruits display considerable natural variation in carotenoid and apocarotenoid pigments.In this study,using an integrated genetic approach we revealed that a 5;c/s-regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C3 0 apocarotenoids responsible for red coloration of citrus peel.Functional analyses demonstrated that in addition the known role in synthesizing 3-citraurin,CCD4b is also responsible for the production of another important C3 0 apocarotenoid pigment,p-citraurinene.Furthermore,analyses of the CCD4b promoter and transcripts from various citrus germplasm accessions established a tight correlation between the presence of a putative 5'c/s-regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C3 0 apocarotenoid-rich red-peeled accessions.Phylogenetic analysis provided further evidence that functional diversification of CCD4b and naturally occurring variation of the CCD4b promoter resulted in the stepwise evolution of red peels in mandarins and their hybrids.Taken together,our findings provide new insights into the genetic and evolutionary basis of apocarotenoid diversity in plants,and would facilitate breeding efforts that aim to improve the nutritional and aesthetic value of citrus and perhaps other fruit crops.

关键词： citrus apocarotenoid natural variation carotenoid cleavage dioxygenase promoter transposon