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cloning,Identification and Differential Expression Analysis of Full-length cdna of Carp Interleukin-1β

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Agricultural Science & Technology 2011年第3期12卷 405-408,412页

作者：何江帅卢强李伟赵晓冯祥汝陈义龙吉林大学人兽共患病教育部重点实验室吉林长春130062 黑龙江生物科技职业学院黑龙江哈尔滨150025

[Objective] The research aimed to carry out the cloning,identification and differential expression analysis of carp interleukin-1β （IL-1β） cdna. [Method] By using DD-RTPCR method,the differential expression cdna fragments were gained. The cdna library of carp peripheral blood leucocytes which was stimulated by the mitogen was screened,and the full length cdna of carp IL-1β was cloned. Moreover,the sequence analysis and differential expression analysis were carried out. [Result] The positive clone which had a whole ORF that encoded 276 amino acids was obtained. The cluster analysis showed that the amino acid sequence of carp IL-1β and Japanese carp closely gathered as a branch,and the homoeology of amino acid sequence reached 95%. The clustering order was the carassius,zebra fish,pig,cattle,horse,human and mouse in turn. The differential expression analysis showed that the expression of IL-1β in the leucocytes significantly increased in the prior period （4 h） after the mitogen stimulated. But as the time went by （12 and 24 h）,it didn＇t increase in the same period. The total trend of expression amount presented the peak type. [Conclusion] The research laid the foundation for further studying the expression manner,function characteristic,regulation mechanism of IL-1β in vivo and its action mechanisms in the inflammatory reaction,emergency reaction and immune response.

关键词： Carp Interleukin-1β cdna cloning Identification Differential expression analysis

cloning and molecular characterization of a novel lectin gene from Pinellia ternata

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Cell Research 2003年第4期13卷 301-308页

作者： JIAN HONG YAO, XIU YUN ZHAO, ZHI HUA LIAO, JUAN LIN, ZHONG HAI CHEN, FEI CHEN, JUN SONG, XIAO FEN SUN, KE XUAN TANG State Key Laboratory of Genetic Engineering, School of Life Sciences, Morgan- Tan International Center for Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R and D Center, Fudan University, Shanghai 200433, ChinaFudan-SJTU-Nottingham Plant Biotechnology R and D Center, Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200030, China State Key Laboratory of Genetic Engineering School of Life Sciences Morgan-Tan International Center for Life Sciences Fudan-SJTU-Nottingham Plant Biotechnology R and D Center Fudan University Shanghai 200433 China

The full-length cdna of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cdna sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was succ

关键词： araceae cdna cloning lectin Pinellia ternata RACE-PCR.

Validation of Reference Genes for Quantitative Real-Time PCR in Laodelphax striatellus

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Journal of Integrative Agriculture 2014年第4期13卷 811-818页

作者： HE Xiu-ting LIU Cheng-cheng LI Zhao-qun ZHANG Zan LI Guo-qing LI Fei DONG Shuang-lin Key Laboratory of Integrated Management of Crop Diseases and Pests Ministry of Education/College of Plant ProtectionNanjing Agricultural University

The normalization of quantitative real-time PCR （qPCR） is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions, qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes （LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9） in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were fiarther confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.

关键词： cdna cloning housekeeping gene qPCR reference gene small brown planthopper

cloning and Characterization of Porcine TSARG7 Gene and Analysis of Its Tissue-Specific Expression

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Agricultural Sciences in China 2009年第11期8卷 1384-1391页

作者： LI Mei-li LI Gui-qiang FANG Wei WANG Wei SONG Xiao-guang LI Er-lin JIA Chao XU Yin-xue College of Animal Science and Technology Nanjing Agricultural University Nanjing 210095 P.R.China

TSARG7 is a novel member of the acyltransferase family since its sequence possesses the highly conserved phosphate acyltransferase （PlsC） domain existing in all acyltransferase-like proteins. The porcine TSARG7 had been identified by cloning in silico but had not been confirmed experimentally. The full-length mRNA of porcine TSARG7 gene was sequenced and two splice variants were discovered. The full-length cdna of TSARG7 variant 1 was 2 513 bp and variant 2 was 2 634 bp. The putative porcine TSARG7 proteins, which were located in the cytoplasm, encoded 458 and 456 amino acids, respectively. Real-time PCR analysis showed that TSARG7 gene was expressed in various tissues, but at different levels. The expression levels of this gene were higher in the skeletal muscle, heart, and testis than that in other tissues, suggesting that the TSARG7 gene played a role in procine skeletal muscle, heart, and testis functions.

关键词： TSARG7 cdna cloning pig bioinformatic analysis gene expression

Characterization and Expression Analysis of Starch Branching Enzymes in Sweet Potato

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Journal of Integrative Agriculture 2013年第9期12卷 1530-1539页

作者： QIN Hua ZHOU Shuang ZHANG Yi-zheng College of Life Sciences Sichuan University

Spatial and temporal expression patterns of Sbel and Sbe2 that encode starch branching enzyme （SBE） Ⅰ and Ⅱ, respectively, in sweet potato （Ipomoea batatas L.） were analyzed. Expression of both genes in Escherichia coli indicate that both genes encoded active SBE. Analysis with real-time quantitative polymerase chain reaction technique indicates that IbSbel mRNA was expressed at very low levels in leaves but was the predominant isoform in tuberous root while the reverse case was found for lbSbe2. The expression pattern of IbSbel, closely resembles that of AGPase S, a gene coding for one of the subunits ofADP-glucose pyrophosphorylase, which is the key regulatory enzyme in the starch biosynthetic pathway. Western analysis detected at least two isoforms of SBE I in tuberous roots, those two isoforms showed adverse expression patterns with the development of the tuberous roots. Expression of the two IbSbe genes exhibited a diurnal rhythm during a 12-h cycle when fed a continuous solution of sucrose. Abscisic acid （ABA） was aother potent inducer of IbSbe expression, but bypassed the semidian oscillator.

关键词： sweet potato starch branching enzyme cdna cloning cdna expression expression patterns sucroseinduction ABA induction

Expression of the Tribolium castaneum (Coleoptera: Tenebrionidae) hsp83 gene and its relation to oogenesis during ovarian maturation

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Journal of Genetics and Genomics 2010年第8期37卷 513-522页

作者： Jingjing Xu 1, Juan Shu 1, Qin Zhang Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China [a] Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture College of Animal Science and Technology China Agricultural University Beijing 100193 China

Heat shock proteins （HSP） can protect organisms and cells from thermal damage. In this study, we cloned the full length cdna encoding the HSP83 protein （the homologue of HSP90） of Tribolium castaneum（red flour beetle）. The isolated cdna contains the full coding sequence, a partial 5＇ untranslated region of 55 bp and the complete 3＇ untranslated region. We found the hsp83 gene is located on chromosome 5 of the T. castaneum genome. The predicted HSP83 protein sequence has a high similarity （on average 86.77%） with that of other insect species. The expression of the hsp83 gene in the whole body and in the ovary could be induced with heat stress （40℃ for 1 h） in newly hatched （within 3 h post emergence） and mature （10 days post emergence） beetles. Under normal conditions, the hsp83 expression in the ovary is about 3-fold higher than in the whole body at both stages. No significant difference in hsp83 expression was observed between the two ovarian developmental stages regardless if the beetles were treated with heat shock or not. The expression of the HSP83 protein in the whole body could also be induced with heat stress in newly hatched and mature beetles. However, in the ovary, HSP83 was only expressed in the follicle cells of mature beetles and not in newly hatched beetles, regardless if the beetles were treated with heat shock or not. Furthermore, the females were not able to produce mature oocytes after knock-down of the hsp83 expression by injecting dsRNA. These results suggest that the HSP83 protein is involved in protection against heat stress and could be involved in oogenesis during ovarian maturation of T. castaneum.

关键词： hsp83 cdna cloning Tribolium castaneum expression heat shock

cloning and Expression Analysis of Rice Sucrose Transporter Genes OsSUT2M and OsSUT5Z

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Journal of Integrative Plant Biology 2008年第1期50卷 62-75页

作者： Ai-Jun Sun Hong-Lin Xu Wan-Kui Gong Hong-Li Zhai Kun Meng Yong-Qin Wang Xiao-Li Wei Gui-Fang Xiao Zhen Zhu State Key Laboratory of Plant Genomics Institute of Genetics and Developmental Biology the Chinese Academy of Sciences Beijing 100101 China Graduate School of the Chinese Academy of Sciences Beijing 100049 China

Two sucrose transporter （SUT） cdnas, OsSUT2M and OsSUT5Z, were isolated from rice （Oryza sativa L.） by reverse transcription polymerase chain reaction （RT-PCR）. Sequencing results indicate they are 1 531 bp and 1 635 bp in length including complete open reading frame 1 506bp and 1 608 bp, which encode 502 amino acids and 536 amino acids, respectively. The TopPred program suggested that both sucrose transporter proteins, OsSUT2M and OsSUT5Z, consist of potentially 12 transmembrane domains. Semi-quantitative RT-PCR was carried out to investigate the gene expression patterns of OsSUT2M and OsSUT5Z. In vegetative organs, transcripts of OsSUT2M were higher in source leaf blades than in other organs at the same development stage, whereas transcripts of OsSUT5Z were less traceable in all organs investigated. In reproductive organs, both transcripts of these two genes were high in panicles from the booting stage to 7 days after flowering （DAF） and then sharply declined. The potential physiology functions of these two sucrose transporters are discussed.

关键词： cdna cloning expression pattern OsSUT2M OsSUT5Z rice sucrose transporter.

cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

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Tsinghua Science and Technology 2008年第4期13卷 466-473页

作者：范为民胡轶霖李长忠谢莉萍张荣庆 Institute of Marine Biotechnology Department of Biological Sciences and Biotechnology Tsinghua University Beijing 100084 China Institute of Marine Biotechnology Department of Biological Sciences and Biotechnology Tsinghua University Beijing 100084 China Protein Science Laboratory of the Ministry of Education Tsinghua University Beijing 100084 China

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cdna of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously expressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridization analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium, All these results suggest PCRT might be involved in Ca^2＋ transport and storage during oyster biomineralization.

关键词： calreticulin cdna cloning expression analysis biomineralization Pinctada fucata mollusc

Molecular cloning and Tissue-specific Expression of Cu/Zn and Mn-superoxide Dismutase in the Three-keeled Pond Turtle, Chinemys reevesii

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Asian Herpetological Research 2013年第2期4卷 79-89页

作者： Xiaojing JIA Cuijuan NIU Ministry of Education Key Laboratory of Biodiversity Science and Ecological Engineering College of Life SciencesBeijing Normal University

Both copper/zinc superoxide dismutase （SOD; Cu/Zn-SOD, SOD1） cdna and manganese SOD （Mn-SOD, SOD2） cdna were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE methods in this work. The SOD1 cdna was 749 bp long and consisted of a 32-bp 5＇-untranslated region （UTR）, a 249-bp 3＇-UTR, and a 468-bp open reading frame （ORF） encoding a 155-amino-acid protein with 16.0 kDa predicted molecular mass and 5.95 theoretical isoelectric point （p/）. The SOD2 cdna was 1687 bp long and comprised 94-bp of 5＇-UTR, 912-bp 3＇-UTR and 681-bp ORF encoding a 226-amino-acid protein with 25.0 kDa predicted molecular mass and 8.83 pI. The deduced amino acid sequence of SOD1 showed relatively high similarity （77.4%-87.1%） and identity （65.4%-74.4%） with the published sequences of SOD1 from other vertebrate species, whereas SOD2 protein shared slightly higher similarity （83.6%-95.6%） and identity （76.1%-88.9%） with other reported vertebrates SOD2s. Phylogenetic analysis revealed that the C. reevesii SOD1 and SOD2 were separately clustered together, and were highly conserved during evolution. Both SOD mRNA expression was detected widely in the brain, liver, muscle, kidney, gut, spleen, lung and heart at variable levels. The highest expression of the two SODs was observed in muscle, and followed in brain, liver, kidney, gut and heart, whereas low transcriptional levels were found in spleen and lung. Meanwhile, high activity of SOD 1 was kept in brain, liver, muscle, kidney and heart, and followed in gut, spleen and lung. The activities of SOD2 in brain, liver, muscle, kidney, gut and heart were significantly higher than those in spleen and lung.

关键词： Chinemys reevesii SOD 1 SOD2 cdna cloning mRNA expression