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gene cloning and expression analyses of WBC genes in the developing grapevine seeds

Journal of Integrative Agriculture 2018年第6期17卷 1348-1359页

作者： TANG Yu-jin WANG Qian XUE Jing-yi LI Yan LI Rui-min Steve Van Nocker WANG Yue-jin ZHANG Chao-hong Key Laboratory of Biology and genetic Improvement of Horticultural Crops (Northwest Region) Ministry of Agriculture/State Key Laboratory of Crop Stress Biology for Arid Areas/College of HorticultureNorthwest A&F University College of Life Sciences Northwest A&F University Department of Horticulture Michigan State University

White-brown complex （WBC） transporters, also called half-size ATP binding cassette G （ABCG） transporters, are involved in many biological processes, including seed development; however, the WBC transporters in grapevines received less attention to date. To reveal the molecular characteristics of WBCs and the connection between WBCs and agronomic traits of stenospermocarpic （seedless） grapevine, we carried out a genomic census and analysis of ovule-associated expression for VvWBC genes in grapevine. We identified 30 VvWBC genes and cloned full-length complementary DNAs （cDNAs） for 20 of these. The tissue or organ-specific expression analysis showed that several VvWBCs exhibited distinct expression patterns with some showing tissue specificity. Twelve VvWBC genes were found to be expressed in the developing ovules. Moreover, the results of quantitative real-time PCR （qRT-PCR） suggested that four of twelve ovule-expressed VvWBCs have distinct expression profiles during the development of ovules between seeded and stenospermocarpic grapevines. These four genes might be involved in ovule abortion. Meanwhile, chromosome mapping, multiple sequence alignments, exon/intron structure analyses and synteny analyses were preformed on VvWBC genes. Our experiments provide a new perspective on the mechanism of stenospermocarpic seedlessness and put forward a framework for further study of WBC transporters.

关键词： Vitis vinifera stenospermocarpy VvWBCs half-size VvABCGs gene cloning expression analysis

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gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp. SM9913

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Acta Oceanologica Sinica 2007年第6期26卷 91-100页

作者： ZHAO Dianli CHEN Xiulan HE Hailun SHI Mei ZHANG Yuzhong State Key Laboratory of Microbial Technology Marine Biotechnology Research Center Shandong University Jinan 250100 China

Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.

关键词： cold-adapted chaperone DnaJ DnaK deep-sea Pseudoalteromonas sp. SM9913 gene cloning sequence analysis

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gene cloning of Murine α-Fetoprotein gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2003年第4期23卷 392-395页

作者：易继林田耕 Department of general Surgery

To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

关键词： gene cloning α fetoprotein gene eukaryotic expression vector CHO cells

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gene cloning and Expression Analysis of G Protein αq Subunit from Helicoverpa assulta (Guenée)

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Agricultural Sciences in China 2008年第2期7卷 187-192页

作者： QIAO Qi LI Hai-chao YUAN Guo-hui GUO Xian-ru LUO Mei-hao College of Plant Protection Henan Agricultural University Zhengzhou 450002 P.R.China

The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta （Guen6e） by reverse transcription polymerase chain reaction （RT-PCR） and named as HassGαq. Sequencing analysis showed that the fulllength of HassGαq open reading frame （ORF） is 1 062 bp, 353 amino acid residues are encoded. The predicted molecular weights （MW） and isoelectric point （PI） are 41.5 kD and 5.15, respectively. HassGαq gene was then constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside （IPTG）, the GST-HassGαq fusion protein is expressed in Escherichia coil BL21, and its MW was found to be about 66 kD nearly equal to the predicted. In addition, RT-PCR analysis showed that the expressions of HassGαq are not tissue specific.

关键词： Helicoverpa assulta G protein α subunit gene cloning prokaryotic expression expression pattern

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gene cloning,expression and purification of human mitochondrial tRNA^(Leu(UUR)) and its mutant

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Science China(Life Sciences) 2001年第2期44卷 113-120页

作者：韩伟国陈莉刘静查锡良金由辛王德宝 State Key Laboratory of Molecular Biology Shanghai Institute of Biochemistry Chinese Academy of Sciences Shanghai 200031 China Department of Biochemistry Shanghai Medical University Shanghai 200032 China State Key Laboratory of Molecular Biology Shanghai Institute of Biochemistry Chinese Academy of Sciences Shanghai 200031 China Department of Biochemistry Shanghai Medical University Shanghai 200032 China

genes of human mitochondrial tRNALeu(UUR) (mtRNALeu(UUR)) and itsmutant (mtRNALeu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively. *** JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNALeu(UUR) and mtRNALeu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters for *** LeuRS were measured. The results showed that the value of kcal / Km of mtRNALeu(M) was about one fifth of that of mtRNALeu(UUR) and indicated the leucine acceptability of mtRNALeu(M) was much lower than that of mtRNALeu(UUR)

关键词： human mitochondrial tRNALeu(UUR) mutation gene cloning expression purification.

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The Construction of the Probe for Amylase n gene cloning from Bacillus halodurans Strain 38C1-1

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Journal of Northeast Agricultural University(English Edition) 2005年第2期12卷 108-111页

作者： QU Juan-juan YANG Guo-hui Aoki Ken-ji College of Resource and Environment Northeast Agricultural University Harbin tteilongjiang 150030 PRC Agriculture Faculty Kobe University Kobe 657-8501Japan

Primers and probes were established according to the sequences of the alpha-amylase genes of Bacillus. halodurans C-125, Therrnus sp. IM6501, B. stearothermophilus ET-1, and B, acidopullulytics. Primers were designed and a 0.2 kb DNA fragment was amplified, the fragment was successfully used for the detection of the amylase Ⅱ gene in a 2 842 bp region from Bacillus halodurans strain 38C1-1.

关键词： probe amylase Ⅱ gene cloning

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Bacillus subtilis strain Czk1 lipopeptide related gene cloning and its active product analysis

Bacillus subtilis strain Czk1 lipopeptide related gene cloni...

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中国植物病理学会2015年学术年会

作者：贺春萍 FAN Lanyan ZHENG Fu-cong TAN Hong-zhi Environment and Plant Protection Institute CATAS Hainan Engineering Research Center for Biological Control of Tropical Crops Diseases and Insect Pests Key Laboratory of Integrated Pest Management on Tropical Grops Ministry of Agriculture Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests College of Environment and Plant Protection Hainan University

With four specific primers of lipopetide antibiotics synthesis related genes,the Bacillus subtilis Czkl genome genetic was tested,and crude extracts by Sephadex G25 column for purification,the results show that the strain Czkl genome of surface active element(Surfactin) synthesis related genes sfp,fengycin element synthesis related genes fenB(Fengycin),develop the hay cephalosporins(Iturin) synthesis related genes ituD and Ipa- 14.5 active component resulting from the separation and purification,MALDI-TOP- MS detection analysis,comprehensive analysis of the results speculated that Bacillus subtilis Czkl peptide may have produced by lipopeptide C Surfactin or C SurfactinBN CSurfactin or C Surfactin A,CIturinB、 C IturinB、 CturinB,C Iturin A、 C IturinA,and CFengycinB、 CFengycinB、 CFengycinB or C Fengycin A,CBacillomycinD、 CBacillomycinD.

关键词： Bacillus subtilis strain Czkl lipopeptide gene cloning MALDI-TOP-MS

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Mulberry（MORUSL.） pyttoline-5-carboxylate syntase（MP5CS） gene cloning,Sequence Analysis,and Expression in Plant Development and Stress Response

Mulberry（MORUSL.） pyttoline-5-carboxylate syntase（MP5CS）...

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江苏省遗传学会2014年学术研讨会

作者： Li Feng Heng Wang Wei Tong Qian Jiao Li Long Rongjun Fang Weiguo Zhao School of Biology and Chemical Engineering Jiangsu University of Science and Technology Sericultural Research Institute Chinese Academy of Agricultural Sciences School of Life Sciences Nanjing University

Drought is one of the important factor limiting plant growth.?1-Pyrroline-5-Carboxylate Synthase is the key enzyme closely related in the synthesis of Proline under drought *** and function analysis of mulberry gene P5CS has a very important *** this report,we cloned the P5CS gene of mulberry by means of homology cloning,RT-PCR and RACE（rapid amplification of cDNA ends）,and we got the full sequence of c DNA named MP5CS（GenBank accession number:KC202259）.Homology and phylogenetic analysis of P5CS were carried out using Clustal X and MEGA4.1 *** addition,the expression level of P5CS in drought and salt stresses were performed by *** shows that the expression level of P5CS is up-regulated.

关键词： mulberry P5CS gene cloning expression analysis drought stress

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hEGFfr-PEII-scFv gene cloning into Pichia pastoris

hEGFfr-PEII-scFv gene cloning into Pichia pastoris

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2011 2nd International Conference on Biology,Environment and Chemistry（ICBEC 2011）

作者： Khalissa Deffar Institute of genetics and Cytology Northeast Normal UniversityKey Laboratory of Molecular Epigenetics of Ministry of Education Faculty of Nature and Life Sciences Department of BiochemistryFerhat Abbas University

Human acidic fibroblast growth factor（aFGF） is a member of the family of structurally related heparin-binding growth *** has important roles both in normal biological activities and in tumorgenesis,indicating that aFGF could be a target for the cancer *** explore the relationship between aFGF and tumors growth,a single-chain variable fragment（scFv） antibody specific for aFGF was *** scFv may block the activity of aFGF which leads to tumor cell growth ***,anti aFGF-scFv as a medicine might be harmful for the normal cells because the aFGF is not a tumor specific *** other hand,we have found that the scFv interacts with its antigen in the cytoplasm but delivering proteins with therapeutic potential activity into cells is ***,it has been difficult to utilize such proteins as therapeutic *** this work,we want to test a strategy for cancer targeting and intracellular delivery of anti aFGF-scFv by generating a fusion protein consisting of anti aFGF-scFv,a protein translocation domain（Pseudomonas exotoxin A domain II,PEII） and a ligand which can bind with cancer cells（human epidermal growth factor receptor binding fragment,hEGFfr）.In previous study the fusion gene（hEGFfr-PEII-scFv） encoding for this fusion protein was cloned into a prokaryotic expression *** fusion gene（6 His*Tag-hEGFfr-PEII-scFv） derived from the pET-28a（+） recombinant plasmid was successfully cloned into a Pichia pastoris expression system.

关键词： acidic fibroblast growth factor single-chain variable fragment gene cloning fusion gene Pichia pastoris

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genetic analysis,gene cloning and expression pattern of a capsule length mutant cl2 in sesame

Genetic analysis,gene cloning and expression pattern of a ca...

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中国作物学会油料作物专业委员会第八次会员代表大会暨学术年会

作者： WEI Li-bin MIAO Hong-mei LI Chun DUAN Ying-hui ZHANG Hai-yang Henan Sesame Research Center Henan Academy of Agricultural Sciences

The capsule length is an important characteristic closely related to the seed yield in sesame.A short capsule-length mutant cl2,induced from *** 11 via EMS mutagenesis,was studied in this *** background of the mutant cl1 was analyzed using the populations(i.e.,two parents,F,BC and F).The results showed that the capsule length trait is controlled by single recessive nuclear *** mapping showed that the mutant site was located in a 380 kb region on chromosome 5 of *** order to determine the mutated gene,the SNP sites in the 380 kb regions were screened using the genome re-sequencing data of 500 sesame germplasm *** a result,one specific SNP mutation site was *** candidate gene with the specific SNP is comprised of 7 exons and 6 introns and code for 163 amino acid *** alignment proved that the putative candidate gene with the target SNP site was a YABBY transcription *** gene family is involved in the regulation of the occurrence of plant pistil differentiation and ovule initiationand plays a role in the initiation of ovule *** addition,the expression pattern of the cl2 genein capsule,flower and other vegetative organs of the mutant and the wild type was analyzed and *** foundation of the cl2 gene related with capsule length supplies the important genetic information for further sesame capsule development and yield formation research in sesame.

关键词： sesame capsule length mutant gene cloning gene expression

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