Sweet potato is an important food crop that can also be used as an industrial raw material. Sucrose is the main form of long-distance carbohydrate transport in plants, and sucrose transporter (SUT) regulates the transmembrane transport and distribution of sucrose during plant growth and metabolism. Moreover, SUT plays a key role in phloem mediated source-to-sink sucrose transport and physiological activities, supplying sucrose for the sink tissues. In this study, the full-length cDNA sequences of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were obtained by rapid amplification of cDNA ends (RACE) cloning according to the transcripts of the two SUT coding genes which were differentially expressed in sweet potato storage roots with different starch properties. Phylogenetic analysis was performed to clarify the classification of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆. The subcellular localization of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ was determined by transient expression in Nicotiana benthamiana. The function of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in sucrose and hexose absorption and transport was identified using yeast functional complementarity system. The expression pattern of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in sweet potato organs were analyzed by real-time fluorescence quantitative PCR (RT-qPCR). Arabidopsis plants heterologous expressing IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ genes were obtained using floral dip method. The differences in starch and sugar contents between transgenic and wild-type Arabidopsis were compared. The results showed IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ encoded SUT proteins with a length of 505 and 521 amino acids, respectively, and both proteins belonged to the SUT1 subfamily. IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were located in the cell membrane and were able to transport sucrose, glucose and fructose in the yeast system. In addition, IbSUT₆₂₇₈₈ was also able to transport mannose. The expression of IbSUT₆₂₇₈₈ was higher in leaves, lateral branches and main stems, and the expression of IbSUT₈₁₆₁₆ was higher in lateral branches, stems and storage roots. After IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were heterologously expressed in Arabidopsis, the plants grew normally, but the biomass increased. The heterologous expression of IbSUT₆₂₇₈₈ increased the soluble sugar content, leaf size and 1 000-seed weight of Arabidopsis plants. Heterologous expression of IbSUT₈₁₆₁₆ increased starch accumulation in leaves and root tips and 1 000-seed weight of seeds, but decreased soluble sugar content. The results obtained in this study showed that IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ might be important genes regulating sucrose and sugar content traits in sweet potato. They might carry out physiological functions on cell membrane, such as transmembrane transport of sucrose, sucrose into and out of sink tissue, as well as transport and unloading of sucrose into phloem. The changes in traits result from their heterologous expression in Arabidopsis indicates their potential in improving the yield of other plants or crops. The results obtained in this study provide important information for revealing the functions of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in starch and glucose metabolism and formation mechanism of important quality traits in sweet potato.
Ipomoea batatas/metabolism* ; Arabidopsis/metabolism* ; Sucrose/metabolism* ; Saccharomyces cerevisiae/metabolism* ; DNA, Complementary ; Phylogeny ; Plants, Genetically Modified/genetics* ; Membrane Transport Proteins/metabolism* ; Starch/metabolism* ; Plant Proteins/metabolism* ; Gene Expression Regulation, Plant

With prominent medicinal value, Gelsemium elegans has been overexploited, resulting in the reduction of the wild resource. As a result, artificial cultivation turns out to be a solution. However, this medicinal species is intolerant to low temperature, and thus genes responding to the low temperature are important for the cultivation of this species. Based on the transcriptome database of G. elegans at 4 ℃, 29 differentially expressed GeERF genes were identified. Bioinformatics analysis of 21 GeERF gene sequences with intact open reading frames showed that 12 and 9 of the GeERF proteins respectively clustered in DREB subgroup and ERF subgroup. GeDREB1 A-1-GeERF6 B-1, with molecular weight of 23.78-50.96 kDa and length of 212-459 aa, were all predicted to be hydrophilic and in nucleus. Furthermore, the full-length cDNA sequence of GeERF2B-1 was cloned from the leaves of G. elegans. Subcellular localization suggested that GeERF2B-1 was located in the nucleus. According to the quantitative reverse-transcription PCR(qRT-PCR), GeERF2B-1 showed constitutive expression in roots, stems, and leaves of G. elegans, and the expression was the highest in roots. In terms of the response to 4 ℃ treatment, the expression of GeERF2B-1 was significantly higher than that in the control and peaked at 12 h, suggesting a positive response to low temperature. This study lays a scientific basis for the functional study of GeERF transcription factors and provides gene resources for the improvement of stress resistance of G. elegans.
DNA, Complementary ; Gene Expression Regulation, Plant ; Phylogeny ; Plant Proteins/metabolism* ; Temperature ; Transcription Factors/metabolism*

The aim of this study was to construct a recombinant adenovirus expressing extracellular domain gene of human epidermal growth factor receptor variant Ⅲ (EGFRvIII ECD), and to prepare single domain antibody targeting EGFRvIII ECD by immunizing camels and constructing phage display antibody library. Total RNA was extracted from human prostate cancer cell line PC-3 cells and reversely transcribed into cDNA. EGFRvIII ECD gene was amplified using cDNA as template, and ligated into pAdTrack-CMV plasmid vector and transformed into E. coli BJ5183 competent cells containing pAdEasy-1 plasmid for homologous recombination. The recombinant adenovirus expressing EGFRvIII ECD was obtained through transfecting the plasmid into HEK293A cells. The recombinant adenovirus was used to immunize Bactrian camel to construct EGFRvIII ECD specific single domain antibody library. The single domain antibody was obtained by screening the library with EGFRvIII protein and the antibody was expressed, purified and identified. The results showed that recombinant adenovirus expressing EGFRvIII ECD was obtained. The capacity of EGFRvIII specific phage single domain antibody library was 1.4×10⁹. After three rounds of enrichment and screening, thirty-one positive clones binding to EGFRvIII ECD were obtained by phage-ELISA, and the recombinant single domain antibody E14 with highest OD₄₅₀ value was expressed and purified. The recombinant E14 antibody can react with EGFRvIII ECD with high affinity in ELISA assessment. The results indicated that the EGFRvIII specific single domain antibody library with high capacity and diversity was constructed and the single domain antibody with binding activity to EGFRvIII was obtained by screening the library. This study may facilitate the diagnosis and treatment of EGFRvIII targeted malignant tumors in the future.
Adenoviridae/genetics* ; DNA, Complementary ; ErbB Receptors ; Escherichia coli/genetics* ; Genetic Vectors/genetics* ; Humans ; RNA ; Recombinant Proteins/metabolism* ; Single-Domain Antibodies

Leptin receptor overlapping transcript (LepROT) plays multiple roles in the regulation of immune systems. However, very little information is available about the anti-infectious mechanisms of amphibians LepROT. In this study, the cDNA sequence of the Rana dybowskii LepROT gene was determined by using RT-PCR and bioinformatics analysis. Then, the Aeromonas hydrophila (Ah) and lipopolysaccharides (LPS) infected models of R. dybowskii was constructed to obtain histopathological characteristics. Constitutive expression of LepROT mRNA and NF-κB signaling pathway were detected by real-time quantitative PCR. The full-length cDNA of LepROT gene was 396 bp and encoded 131 amino acids. Amino acid sequence analysis revealed LepROT shares 93.74% and 86.39% identity with homologues from other amphibians and mammals respectively, and the LepROT gene was quite conserved among different species. After infection, the relative expression levels of LepROT, NF-κB, IKKα and IKKβ mRNA were all significantly upregulated (P < 0.01), but showed a diverse temporal pattern of up-regulation in different tissues. Therefore, it was proposed that the LepROT gene of R. dybowskii might activate the NF-κB signaling pathway to exert anti-infectious effects, thus providing evidence for further extending the biological function of LepROT.
Animals ; Cloning, Molecular ; DNA, Complementary ; Gene Expression Profiling ; Gene Expression Regulation ; Mammals/metabolism* ; NF-kappa B/genetics* ; Phylogeny ; RNA, Messenger/genetics* ; Ranidae/genetics*

Soybean mosaic virus (SMV), one of the major viral diseases of Pinellia ternata (Thunb.) Breit., has had a serious impact on its yield and quality. The construction of viral infectious clones is a powerful tool for reverse genetics research on viral gene function and interaction between virus and host. To clarify the molecular mechanism of SMV infection in Pinellia ternata, it is particularly important to construct the SMV full-length cDNA infectious clone. Therefore, the infectious clone of Soybean mosaic virus Shanxi Pinellia ternata isolate (SMV-SXBX) was constructed in this study by Gibson in vitro recombination system, and the healthy Pinellia ternata leaves were inoculated by Agrobacterium infiltration, further through mechanical passage and RT-PCR, confirming that the 3' end of the SMV-SXBX infectious clone had a stable infectivity when it contained 56-nt of poly(A) tail. This method is not only convenient and efficient, but also avoids the instability of SMV infectious clones in Escherichia coli. The construction of SMV full-length infectious cDNA clones laid the foundation for further study on the molecular mechanism of SMV replication and pathogenesis.
DNA, Complementary ; Pinellia ; virology ; Plant Diseases ; virology ; Potyvirus ; isolation & purification ; metabolism

We investigated the microRNA172 (miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA (cDNA) of gloxinia (Sinningia speciosa) APETALA2-like (SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up- or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172a, whereas the expression pattern of miR172a was negatively correlated with that of miR156a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.
Arabidopsis/genetics* ; Arabidopsis Proteins/metabolism* ; Cloning, Molecular ; DNA, Complementary/metabolism* ; Flowers/physiology* ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Homeodomain Proteins/metabolism* ; Lamiales/physiology* ; MicroRNAs/metabolism* ; Nuclear Proteins/metabolism* ; Plants, Genetically Modified/physiology* ; Plasmids/metabolism* ; Polymerase Chain Reaction ; Transgenes

Objective: To investigate the characteristics of the expression of the RIKEN cDNA 1700008O03 (1700008O03Rik) gene in the testis of the mouse from birth to sexual maturity and its potential role in regulating spermatogenesis. METHODS: Using mouse gene expression profile microarray, we screened the testis-specific gene 1700008O03Rik from the mouse. We studied the expression characteristics of the gene in the development of the mouse testis by reverse transcription PCR, quantitative real-time PCR, Western-blot, immunohistochemistry and immunofluorescence, and analyzed the structure of the 1700008O03Rik protein and its homology with other species using the bioinformatic software. RESULTS: 1700008O03Rik gene was highly expressed in the testis of the mouse, increasing in an age-dependent manner, and mainly in the endochylema of oblong spermatozoa. Bioinformatic analysis revealed a high homology of the 1700008O03Rik protein between human and mice, and phylogenetic tree analysis showed it to be highly conserved in mammalian evolution. CONCLUSIONS 1700008O03Rik is a highly expressed gene in the mouse testis, mainly in the endochylema of oblong spermatozoa, which may be involved in the regulation of spermatogenesis in mice.
Age Factors ; Animals ; Blotting, Western ; Computational Biology ; DNA, Complementary ; Gene Expression Regulation, Developmental ; Humans ; Male ; Mice ; Proteins ; genetics ; Spermatogenesis ; genetics ; Spermatozoa ; metabolism ; Testis ; metabolism

For further research of the apoptosis mechanism of Schistosoma japonicum (S. japonicum). The cDNA encoding Sjcaspase3 of Schistosoma japonicum was amplified by polymerase chain reaction (PCR) technique, which contained 900 nucleotides and encoded 299 amino acids. The theory molecular weight and isoelectric point (PI) of the deduced protein is 33.5 kDa and 6.39, respectively. Real-time PCR was used to analyze the transcription profiles of Sjcaspase3 at different development stages of S. japonicum. The results showed that this gene was expressed in all stages of S. japonicum with the highest expression in 21d worms, and the level of gene transcription in 42 d female worms was higher than that of male worms. The recombinant plasmid pXJ40-FLAG-Sjcaspase3 was constructed and transfection into Hela cells successfully. Real-time PCR and Western blotting analysis showed Sjcaspase3 was successfully expressed in Hela cells. Enzyme activity analysis revealed that recombinant Sjcaspase3 possessed the activity to cut substrate DEVD. Flow cytometry proved that Sjcaspase3 could induce early apoptosis of Hela cells. The results provide the basis for proceeding further study on the biological function of Sjcaspase3 and better understand the apoptosis mechanism of S. japonicum.
Animals ; Apoptosis ; Blotting, Western ; Caspase 3 ; genetics ; metabolism ; Cloning, Molecular ; DNA, Complementary ; Female ; HeLa Cells ; Helminth Proteins ; genetics ; metabolism ; Humans ; Male ; Real-Time Polymerase Chain Reaction ; Recombinant Proteins ; Schistosoma japonicum

Bm04862 is a novel hemocyte-specific gene, which has been identified and cloned through the microarray data in silkworm, Bombyx mori. Initially, we successfully obtained the full-length cDNA sequence of Bm04862 via the rapid amplification of cDNA ends (RACE). The sequence consists of an open reading frame (ORF) with 819 bp, which could encode 273 amino acid residues. The bioinformatics analysis predicted that Bm04862 was a transmembrane protein. Furthermore, qRT-PCR analysis revealed that the expression of Bm04862 was specifically detected in hemocyte and reached a peak at L4M and PP2 stage. Bm04862 was overexpressed in Sf9 insect cells, and the cellular localization indicated that Bm04862 was specifically located in cytoplasm and nuclei membrane. Interestingly, the expression of Bm04862 increas'd dramatically after challenged with Escherichia coli for 24 hours, which predicted its potential role in the innate immune system. Overall, this study could provide the fundamental knowledge for further research.
Animals ; Bombyx ; genetics ; Cloning, Molecular ; Computational Biology ; DNA, Complementary ; Hemocytes ; metabolism ; Insect Proteins ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Open Reading Frames

Multidrug resistant genes are highly expressed in hepatocellular carcinoma that seriousty affects the effect of chemotherapy. Screening of resistant genes from HCC cells and studying its mechanism of drug resistance will be helpful to improve the effecacy of chemotherapy for hepatocellular carcinoma. Here we described an alternative method called cyclical packaging rescue (CPR). First we constructed a retrovirus cDNA library of hepatoma cells and used it to infect fibroblasts. Then we added drugs to screen survival cells. The survival cells, stably integrated helper-free retroviral libraries, were recovered rapidly after transfection with plasmids expressing retroviral gag-pol and env genes. Through this method, retroviral RNAs were directly repackaged into new infectious virions. Recovered retroviral supernatant was then used to reinfect fresh target cells. When performed in concert with selection using functional assays, cDNAs regulating functional responses could be identified by enrichment through multiple rounds of retroviral library recovery and retransmission. Using CPR, we obtained several cDNAs. After a preliminary detection, we found Ribosomal protein S11 (RPS11), Ribosomal protein L6 (RPL6), Ribosomal protein L11 (RPL11), Ribosomal protein L24 (RPL24) possibly had drug resistant function.
Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Line, Tumor ; DNA, Complementary ; Drug Resistance, Neoplasm ; genetics ; Gene Library ; Genetic Vectors ; Humans ; Liver Neoplasms ; genetics ; pathology ; Plasmids ; Retroviridae ; Ribosomal Proteins ; genetics ; metabolism ; Transfection