Cloning and Expression Analysis of a Mitogen-Activated Protein Kinase Gene OsMPK14 in Rice

Mitogen activated-protein kinases （MAPKs） are important components in signal transduction pathways responding to various biotic and abiotic stresses. An MAPK gene, OsMPK14 （GenBank Accession No. GQ265780） from rice （Oryza sativa L.）, was cloned by RT-PCR. The full-length cDNA of OsMPK14 consists of 1660 bp in size, containing an open reading frame of 1629 bp, which encodes a 542-amino-acid polypeptide and has a typical protein kinase domain and a phosphorylation activation motif TDY. Sequence alignment and analysis revealed that OsMPK14 was located on rice chromosome 5, and composed of nine exons and eight introns in the coding region. Semi-quantitative RT-PCR was performed to detect the expression patterns of OsMPK14 in rice shoots and roots under darkness, drought, high salinity, low temperature and abscisic acid treatments. The OsMPK14 mRNA was induced by abscisic acid, low temperature and high salinity, but weakly inhibited by drought. In addition, the expression of OsMPK14 was up-regulated in roots, but down-regulated in shoots by light. The results indicate that OsMPK14 could be implicated in diverse rice stimuli-responsive signaling cascades, and its expression might be regulated by multiple factors.

Construction of recombinant plasmid and prokaryotic expression in E. Coli and biological activity analysis of human placenta arresten gene

BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerpera's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5α, BL21 and BL21 (DE3) by the CaCl_2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purlfled, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM). RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E. coil have laid a foundation for further study of its anti-angiogenic function and may pave the way for future antitumor application.

Molecular cloning and expression under abiotic stresses and hormones of the ethylene response factor Ⅶ gene FmRAP2.12 from Fraxinus mandshurica

RELATED TO AP2.12(RAP2.12)is one of the Ethylene Response Factors(ERF)transcription factor and plays a key role in controlling plant root bending and responding to multiple abiotic stresses including hypoxia stress.In this study,FmRAP2.12 gene was isolated and characterized from Fraxinus mandshurica Rupr.The open reading frame(ORF)of FmRAP2.12 was 1170 bp and encoded a protein of 389 amino acids.The conserved domains,three-dimensional phylogenetic relationship of FmRAP2.12 was also investigated.Quantitative real-time(qRT-PCR)analyzed the expression of FmRAP2.12 in different tissues.The expression level of FmRAP2.12 was highest in roots followed by leaves,and lowest in male flowers.Abiotic stress and hormone signal-induced expression was established using qRT-PCR.Salt stress induced FmRAP2.12 to a double peak pattern:the first peak value was at 6 h and the second at 72 h.Drought stress also induced FmRAP2.12 to a double peak pattern:the first at6 h and the second at 48 h.FmRAP2.12 was up-regulated after initiation of gibberellic acid(GA3)treatment,with a one peak pattern at 24 h.FmRAP2.12 may not respond to cold stress and Abscisic acid(ABA)treatment.The transient overexpression of FmRAP2.12 caused the up-expression of downstream key genes of abiotic stress response and gibberellin pathway.Our research reveals the molecular characteristic and expression patterns under abiotic stress and hormone condition of FmRAP2.12,providing support for the genetic improvement of F.mandshurica at a molecular level.

Cloning and Expression Analysis of OsNADPH1 Gene from Rice in Drought Stress Response

An experiment was conducted to compare the mRNA expression difference in rice leaves and roots under drought stress and normal conditions us, ng Fluorescent Differential Display （FDD） method. One positive fragment was isolated by combination of the H. A. Yellow-PAGE （cont,~ined 0.1% H. A. Yellow） separation and macroarray screening methods. Compared to Arabidopsis thaliana NADPH oxidoreductase gene, it has 96% identity. The cDNA was 1423 bp, and contained a complete open reading frame of 1048 bp encoding a protein with 345 amino acid residues. Moreover, the gene expression level was higher under drought stress than that under normal conditions. The possible role of NADPH oxidoreductase gene under drought response was also discussed.

Gene Cloning and Expression Analysis of G Protein αq Subunit from Helicoverpa assulta (Guenée)

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.

Isolation of a Plasmalemma Aquaporin Encoding Gene StPIP1 from Solanum tuberosum L. and Its Expression in Transgenic Tobacco

Aquaporin （AQP） belongs to a highly conserved group of membrane proteins considered as major intrinsic proteins, which facilitate water transport across biological membranes. The discovery of AQPs in plants has resulted in a paradigm shift in the understanding of plant-water relations, however, the potential relationship between the role of aquaporins in regulating plant water balance and drought tolerance still remains elusive. In this study, the gene encoding potato AQP cDNA, StPIP1 （GenBank accession no. DQ999080）, was cloned from the leaf of potato cultivar Gannongshu 2 by reverse transcription-PCR （RT-PCR）. Sequence alignment was made by BLASTn in GenBank, the phylogenetic analysis was conducted using PHYLIPWY, the 3D structure was predicted in Swiss-Model server. Subcellular localization of StPIP1 was performed by constructing CaMV35S-StPIP1-GFP and rd29A-StPIP1-GFP fusion proteins and transient expression in onion epidermis. To understand StPIP1 physiological functions in potato under various stress conditions, the StPIP1 gene in a reverse orientation was transformed into tobacco driven by the Cauliflower mosaic virus （CMV） 35S promoter. The expression levels of transgenic and wild-type plants were assessed under various abiotic stress conditions using semi-quantitative RT-PCR, and the morphological and physiological responses of transgenic plants to different stress conditions were investigated. The expression of StPIP1 mRNA decreased in transgenic plants under non-stress and stress conditions, however, the reduction was more severer under drought stress. In both non-stress and stress conditions, StPIP1 was expressed predominantly in root. The morphological and physiological investigation showed no significant differences in growth rate, germination rate, and root fresh weight （FW） between transgenic and wild-type plants when grown under favorable conditions. In contrast, under drought stress, the reduction in StPIPI expression leads to a delay in seed germination and seedling growth, accelerated seedling wilt, and leaf morphological abnormity. Under ＂enough＂ water conditions （i.e., water culture）, the aerial parts of anti-sense plants showed no differences. However, for the aerial parts to accumulate the same amount of biomass, transgenic plants needed about 3 times more abundant root system to transport water for plant growth than wild-type plants. Morphological investigation showed that the reduction in StPIP1 expression increased the root system in transgenic plants under drought stress. As a result, the increase of root mass might compensate the reduced cellular water permeability in order to ensure a sufficient water supply for the plant. Results demonstrated that StPIP1 plays an important role for water transportation in potato, especially under drought stress conditions. The reduced expression of StPIP1 decreases the cellular water transport and influences the expression of endogenous AQPs genes and thereby, has impacts on seed germination, seedling growth, and stress responses of potato to drought conditions.

Cloning and Expression Profile of Deoxyhypusine Snyhtase Gene and Deoxyhypusine Hydroxylase Gene in Silkworm, Bombyx mori

Deoxyhypusine snyhtase （DHS） and deoxyhypusine hydroxylase （DOHH） are the two enzymes that catalyze the synthesis of hypusine within eukaryotic initiation factor 5A （eIF5A）. Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we described the cloning and expression of two full-length cDNAs, encoding respectively DHS-like protein and DOHH-like protein from Bombyx mori by using the methods of bioinformatics, RACE, and RT-PCR technology, named as BmDHS and BmDOHH. Sequencing results indicate that they are 1 311 and 1 874 bp in length including complete open reading frame （ORF） 1 116 and 915 bp, which encode 371 amino acids （molecular weight is about 41.11 kD and isoelectric point is 5.84） and 304 amino acids （molecular weight is about 34.30 kD and isoelectric point is 4.86）, respectively. BmDHS contains only 1 exon, and BmDOHH contains 4 exons and 3 introns. The deduced amino acid sequence of BmDHS contains a deoxyhypusine synthase domain from 47 to 361 amino acid residues, and the deduced amino acid sequence of BmDOHH contains 6 E-Z type HEAT repeat domains （23-52, 54-83, 87-116, 177-206, 208-237, and 241- 270）. Compared to DHS and DOHH amino acid sequences from other species, such as Homo sapiens and Drosophila melanogaster, both silkworm DHS protein and DOHH protein have more than 55% identity. The conservative regions are very similar with each other. The phylogenetic tree analysis indicated that not only DHS but also DOHH from different species has genus-specific features. The expressions of BmDHS and BmDOHH are no tissue and stage specific in our tested samples.

Molecular Cloning and Expression Analysis of an Actin Gene from Chinese Licorice(Glycyrrhiza uralensis Fisch)

A PCR-based homologous cloning strategy was used to identify an actin gene from the roots of Chinese licorice（Glycyrrhiza uralensis Fisch）. Results of sequence analysis indicate that a 1137 bp cDNA with an open reading frame encoding 377 amino acids, actin ortholog, GuActin, was successfully cloned and characterized（GenBank accession No. EU190972）. Thus far, GuActin is the first actin of Chinese licorice that has been identified at a molecular level. Analysis by Northern blot shows that GuActin was expressed strongly in the roots, particularly in radicles than in stems and leaves. These results suggest that GuActin may be a member of the vegetative subfamily of the actin family.

Cloning and Expression of the vp39 Gene of Bombyx mori Nuclear Polyhedrosis Virus in E.coli

The nuclear capsid protein gene (vp39) ofBombyx mori nuclear polyhedrosis virus (BmNPV) was amplified successfully by PCR technique and inserted into pGEM 3zf(+). The 5′ and 3′ terminal area of the amplified vp39 gene were sequenced with silver-staining dideoxy method. Bmvp39 gene was sub-cloned into the expression vector pRSET-A, and transformed intoE. coli BL21. This gene was highly expressed by IPTG induction. SDS-PAGE analysis showed that the expressed protein is about 38 kd, and the expressed amount reached maxium in 4 h with IPTG induction.

Cloning and Expression Analysis of <i>RrGT2</i>Gene Related to Anthocyanin Biosynthesis in <i>Rosa rugosa</i>

At present, the research about flower color of Rosa rugosa is a very inno-vative and practical study. Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1422bp, encoding 473 amino acids, designated as RrGT2, were isolated from flowers of R. rugosa ‘Zizhi’ and then functionally characterized. According to online software prediction, the molecular formula of the protein encoded by the RrGT2 gene is C2334H3628N602O711S18, the relative molecular mass is 52,075.17 Da, and the theoretical isoelectric point is pI = 4.76. The result of the RrGT2 protein 3D model construction showed that it had the highest homology with the UDP-glycosyltransferase 74F2 protein model in the database (39.53%). Sequence alignments with the NCBI database showed that the RrGT2 protein is a member of the GTB superfamily. Homology analysis revealed that the coding regions of RrGT2 was highly specific among different species, but still had typical conserved amino acid residues called PSPG that are crucial for RrGT2 enzyme activity. RrGT2 transcripts were detected in five flowering stages and seven tissues of R. rugosa ‘Zizhi’, R. rugosa ‘Fenzizhi’ and R. rugosa ‘Baizizhi’, and their expression patterns corresponded with the accumulation of antho-cyanins. Therefore, we speculated that glycosylation of RrGT2 plays a crucial role in anthocyanin biosynthesis in R. rugosa.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control f gene expression in the gynogenetic fish embryogenesis.

Cloning and Expression Level Analysis of Melanocyte-stimulating Hormone Receptor 1 Gene(MC1R) in Alpacas with Different Coat Color

Specific primers for the MC1R gene of alpacas(GenBank EU1358800) were designed to amplify the cDNA sequence using RT-PCR to seek variation in the sequence and explore the relationship between the expression level of MC1R gene and alpaca coat color.The MC1R gene from white alpaca was cloned successfully and sequence analysis verified that the MC1R gene,encoding 317 amino acids,was 1081 bp in length.Compared with the existing sequence in GenBank,sequence identity was 99.9%and 7 mutations were found.Primers,designed from the sequence obtained,were used to assess the relative expression of MC1R in alpacas of different coat color using QRT-PCR and SPSS 13.0 software.Relative expression of MC1R in the skin of brown alpacas was 4.32 times higher than that in white alpacas after normalization with GAPDH(P【0.01),indicating that MC1R expression may be related to coat color of alpacas.

Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

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.

Cloning of Human Uroplakin Ⅱ Gene from Chinese Transitional Cell Carcinoma of Bladder and Construction of Its Eukaryotic Expression Vector

Summary: To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T 3N 0M 0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between XbaⅠ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UPⅡ for Uroplakin Ⅱ was successfully constructed. After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

Cloning and Expression Analysis of <i>RrGT1</i>Gene Related to Anthocyanin Biosynthesis in <i>Rosa rugosa</i>

Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1161 bp, encoding 386 amino acids, designated as RrGT1, was isolated from flowers of R. rugosa ‘Zizhi’ and then functionally characterized. According to online software prediction, the molecular formula of the protein encoded by the RrGT1 gene is C1879H2964N494O556S14, the relative molecular mass is 41,820.02 Da, and the theoretical isoelectric point is pI = 5.03. The result of the RrGT1 protein 3D model construction showed that it had the highest homology with the UDP-glucose: anthocyanidin 3-O-glucosyltransferase protein model in the database (47.01%). Sequence alignments with the NCBI database showed that the RrGT1 protein is a member of the GTB superfamily. Homology analysis revealed that the coding regions of RrGT1 was highly specific among different species, but still had typical conserved amino acid residues called PSPG that are crucial for RrGT1 enzyme activity. RrGT1 transcripts were detected in five flowering stages and seven tissues of R. rugosa ‘Zizhi’, R. rugosa ‘Fenzizhi’ and R. rugosa ‘Baizizhi’, and their expression patterns corresponded with the accumulation of anthocyanins. Therefore, we speculated that glycosylation of RrGT1 plays a crucial role in anthocyanin biosynthesis in R. rugosa.

A Plasmid vector encoding functional human keratinocyte growth factor gene in vitro—Functional human KGF gene expression in vitro

In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lung fibro-blast (HLF) was used for isolation of total RNA. Then the total RNA was purified and reverse- transcribed into cDNA using an oligo (dT) primer. A full PCR fragment for hKGF was generated and cloned. Restriction digestion and nucleo-tide sequence analysis validated the complete hKGF transcription. The hKGF cDNA fragment was inserted into pEGFP-C2 vector by means of recombinant DNA technology and verified by restriction analysis and sequencing. We have constructed pEGFP-C2-hKGF encoding the green fluorescent protein (GFP). Furthermore, hKGF had the effect on AEC II proliferation. These results suggest that the potential appli-cation of a hKGF plasmid of gene expression should be useful for sustained AEC proliferation, and its in vivo efficacy needs to be validated. Keywords:

CCR5 gene expression in fulminant hepatitis and DTH in mice

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Identification and Expression Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Peanut (Arachis hypogaea L.)

Phosphoenolpyruvate carboxylase （PEPC） is widely distributed in plants and bacteria, and catalyzes the carboxylation of phosphoenolpyruvate to form oxaloacetate and inorganic phosphate. To investigate the molecular mechanisms of the regulation and control of peanut oil, with the degenerated primers and RACE-PCR approach, five PEPC genes were cloned from peanut, and designated as AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5, respectively. The structure and phylogenetic analysis of PEPC protein indicated that AhPEPC1-4 genes encoded a typical plant-type PEPC-enzyme, and AhPEPC5 a bacterial-type. By real-time quantitative RT-PCR approach the expression pattern of each gene was detected in various tissues of normal and high oil-content peanut varieties. It was found that there was a lower expression level of AhPEPCs genes except for the AhPEPC2 in high-oil peanut than normal-oil peanut line. The results provide some fundamental information for the further investigation of plant PEPC proteins and their role in regulation of oil-content in peanut seeds.

Vitis amuerensis CBF3 Gene Isolation,Sequence Analysis and Expression

The full length cDNA sequence of CBF3 （CRT/DRE-binding factor） was cloned from Vitis amurensis by reverse transcription polymerase chain reaction （RT-PCR） using the primers designed based on CBF genes available in GenBank. Sequence analysis showed that the gene had 854 bp long and its coding sequence contained 720 bp, encoding a protein with 239 amino acids and an AP2 structural domain. The molecular mass of CBF3 was predicted to be 25.9 kDa and its theoretical isoelectric point was 7.02 and aliphatic index was 59.29. The average hydropathicity of the protein was -0.551. The tertiary structures of CBF3 were also analyzed. The prokaryotic expression vector pGEX-4T-CBF3 containing CBF3 gene was constructed and CBF3 fusion protein （52 kDa） was produced in Escherichia coli after induction with 1 mmol L-1 IPTG. Further studies are needed to evaluate its potential application for improving plant resistance to cold and other stress condition such as drought and salinity.

Cloning of Bile Salt Hydrolase Gene and Its Expression in Lactic Acid Bacteria

According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.