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

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 successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent.Here,we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme pu-trescine N-methyltransferase(PMT)and the downstream enzyme hyoscyamine6β-hydroxylase(H6H)of scopolamine biosynthesis in transgenic henbane(Hyoscyamus niger)hairy root cultures.Transgenic hairy root lines expressing both pmt and h6h produced significantly higher(P<0.05)levels of scopolamine compared with the wild-type and transgenic lines har-boring a single gene(pmt or h6h).The best line(T 3 )produced411mg/liter scopolamine,which was over nine times more than that in the wild type(43mg/liter)and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H 11 (184mg/liter).To our knowledge,this is the highest scopolamine content achieved through genetic engi-neering of a plant.We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield,which was more efficient than plants harboring only one of the two genes.It seems that the pulling force of the downstream enzyme(the faucet enzyme)H6H plays a more important role in stimulating scopolamine accumulation in H.niger whereas the functioning of the upstream enzyme PMT is increased propor-tionally.This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.

Insulin expression in livers of diabetic mice mediated by hydrodynamics-based administration

AIM:Transfer and expression of insulin gene in vivo are an alternative strategy to improve glycemic control in type 1 diabetes. Hydrodynamics-based procedure has been proved to be very efficient to transfer naked DNA to mouse livers. The basal hepatic insulin production mediated by this rapid tail vein injection was studied to determine its effect on the resumption of glycemic control in type 1 diabetic mice.METHODS: Engineered insulin cDNA was inserted into plasmid vectors under a CMV promoter, and transferred into STZ induced diabetic mice by hydrodynamic procedure.Glucose levels, body weight of treated mice, insulin levels,immunohistology of the liver, and quantity of insulin mRNA in the liver were assayed to identify the improvement of hyperglycemic complication after plasmid administration.Sleeping Beauty, a transposon system, was also used to prolong the insulin expression in the liver.RESULTS: After plasmid administration, Plasma insulin was significantly increased in the diabetic mice and the livers were insulin-positive by immunostaining. At the same time the hyperglycemic complication was improved. The blood glucose levels of mice were reduced to normal. Glucose tolerance of the treated diabetic mice was improved. Body weight loss was also ameliorated. The rapid tail vein injection did not cause any fatal result.CONCLUSION: Our results suggested that insulin gene could be efficiently transferred into the livers of diabetic mice via rapid tail vein injection and it resulted in high level of insulin expression. The basal hepatic insulin production mediated by hydrodynamics-based administration improved the glycemic control in type 1 diabetes dramatically and ameliorated diabetic syndromes. Hydrodynamics-based administration offers a simple and efficient way in the study of gene therapy for type 1 diabetes.

Transfection of mEpo gene to intestinal epithelium in vivo mediated by oral delivery of chitosan-DNA nanoparticles

AIM: To prepare the chitosan-pmEpo nanoparticles and to study their ability for transcellular and paracellular transport across intestinal epithelia by oral administration. METHODS: ICR mice were fed with recombinant plasmid AAV-tetO-CMV-mEpo (containing mEpo gene) or pCMVβ(containing LacZ gene), whether it was wrapped by chitosan or no. Its size and shape were observed by transmission electron microscopy. Agarose gel electrophoresis was used to assess the efficiency of encapsulation and stability against nuclease digestion. Before and after oral treatmant, blood samples were collected by retro-orbital puncture, and hematocrits were used to show the physiological effect of mEpo. RESULTS: Chitosan was able to successfully wrap the plasmid and to protect it from DNase degradation. Transmission electron microscopy showed that freshly prepared particles were approximately 70-150 nm in size and fairly spherical.Three days after fed the chitosan-pCMVβ complex was fed,the mice were killed and most of the stomach and 30% of the small intestine were stained. Hematocrit was not modified in naive and ‘naked' mEpo-fed mice, a rapid increase of hematocrit was observed during the first 4 days of treatment in chitosan-mEpo-fed animals, reaching 60.9±1.2% (P<0.01),and sustained for a week. The second feed (6 days after the first feed) was still able to promote a second hematocrit increase in chitosan-mEpo-fed animals, reaching 65.9±1.4%(P<0.01), while the second hematocrit increase did not appearin the ‘naked' mEpo-second-fed mice. CONCLUSION: Oral chitosan-DNA nanoparticles can efficiently deliver genes to enterocytes, and may be used as a useful tool for gene transfer.

Construction, expression and characterization of human interferon α2b-(G4S)n-thymosin α1 fusion proteins in Pichia pastoris

AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Tα1 linked by different lengths of (G4S)n (n = 1-3) were constructed and expressed in Pichia pastoris.METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S)n-Tα1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and SuperdexTM 75 gel filtration and analyzed by SDSPAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins.RESULTS: DNA sequencing confirmed that the fusion genes of IFNα2b-(G4 S)n-Tα1 (n = 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b(G4 S)n-Tα1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and SuperdexTM 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNα2b monoclonal antibody and Tα1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay.CONCLUSION: The recombinant IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.

Molecular Cloning and Characterization of Enolase from Oilseed Rape （Brassica napus）

An enolase-encoding cDNA clone in oilseed rape (Brassica napus L.) was isolated. This gene (accession number: AY307449) had a total length of 1 624 bp with an open reading frame of 1 335 bp, and encoded a predicted polypeptide of 444 amino acids with a molecular weight of 47.38 kD. The deduced amino acid sequence shared identity with a number of enolases ranging from Bacillus subtilis to human beings and had much higher identity with other plant enolases than with enolases from Bacillus, yeast and human beings. Comparison of its primary structure with those of other enolases revealed the presence of an insertion of five amino acids in enolase of B. napus. Southern blotting analysis of genomic DNA indicated that enolase was likely to be a low-copy gene in the oilseed rape genome. Expression of the cloned enolase gene increased under salt stress, but decreased in response to low temperature. Our studies suggested that the cloned gene was a new member of plant enolase gene family, which contributed to the energy supply in stress-treated tissues.

Generation and characterization of transgenic mice expressing tamoxifen-inducible cre-fusion protein specifically in mouse liver

AIM: To establish transgenic mice expressing tamoxifeninducible Cre-ERt recombinase specifically in the liver and to provide an efficient animal model for studying gene function in the liver and creating various mouse models mimicking human diseases.METHODS: Alb-Cre-ERt transgenic mice were produced by microinjecting the construct with Cre-ERt fusion gene of DNA fragments into fertilized eggs derived from inbred C57BL/6strain. Transgenic mice were identified by using PCR and Southern blotting. Expression of Cre-ERt fusion gene was analyzed in the liver, kidney, brain and lung from F1generation transgenic mice at 8 weeks of age by reverse transcription (RT)-PCR.RESULTS: Four hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant AlbCre-ERt DNA fragments, and 312 survival eggs injected were transferred to the oviducts of 12 pseudopregnant recipient mice, 6 of 12 recipient mice became pregnant and gave birth to 44 offsprings. Of the 44 offsprings, two males and one female carried the hybrid Cre-ERt fusion gene. Three mice were determined as founders, and were back crossed to set up F1 generations with other inbred C57BL/6 mice.Transmission of Cre-ERt fusion gene in F1 offspring followed Mendelian rules. The expression of Cre-ERt mRNA was detected only in the liver of F1 offspring from two of three founder mice.CONCLUSION: Transgenic mice expressing tamoxifeninducible Cre-ERt recombinase under control of the liverspecific promoter are preliminary established.

Cluster Analysis and Significance of Novel Genes Related to Molecular Classification of Glioma

OBUECTIVE To screen differentially expressed genes in the development of human glioma and establish a primary molecular classification of glioma based on gene expression using cDNA microarrays.METHODS Brain specimens were obtained from 18 patients with glioma, 10 males and 8 females, ages 14-62 with an average age of 44.4. The total RNAs of these glioma specimens and two specimens of donated brain of normal adults were extracted. BioStarH140S microarrays (including 8,347 old genes and 5,592 novel genes) were adopted and hybridized with probes which were prepared from the total RNAs. Differentially expressed genes between normal tissues and glioma tissues were assayed after scanning cDNA microarrays with ScanArray4000. Northern hybridization and in situ hybridization (ISH) were used to identify functions of novel genes. Those differentially expressed genes were studied with a Hierarchical method and molecular classification of glioma was preliminary carried out.RESULTS Among the 13,939 target genes, there were 1,200 (8.61%) differentially expressed genes, of which 395 (2.83%) were novel genes. A total of 348 genes were up-regulated and 852 genes were down-regulated in the gliomas. The results of bioinformatical analysis, Northern hybridization and ISH revealed that those novel genes were highly associated with gliomas. There were multiple genes, such as the MAP gene,cytoskeleton & matrix motility genes, etc, which were of relevance to classification by the Hierarchical method. Molecular classification of glioma using a Hierarchical cluster was in accordance with pathology and suggested a molecular process of tumorigenesis and development.CONCLUSION Multiple genes play important roles in development of glioma, cDNA microarray technology is a powerful technique in screening for differentially expressed genes between two different kinds of tissues. Further analysis of gene expression and novel genes would be helpful to understand the molecular mechanism of glioma development.

Nocvel potential targets and related genes of transcription factor Caplp in Candida albicans 1

AIM Capl p, encoded by CAP1 in Candida albicans, is highly homologous to Saccharomyces cerevisiae transcription factor Yapl p. It has been associated with tolerance to oxidative stress and resistance to a variety of toxicants previously. We used homemade microarray to reveal Capl p related genes in a broad spectrum as well as to lucubrate the functions of Capl p. METHODS Microarray analysis was used to identify differentially expressed genes between CAP1 deletion strain CJD21 and its parental strain CAI4. CAP1 over-expression strain was constructed to confirm the relationship between CAP1 and some differentially expressedgenes. Bioinformatics was applied to reveal promoters with Capl p binding site as well as the clusters of differentially expressed genes. RT-PCR and drug efflux analysis were used to lucubrate the functions of Caplp in Candida albicans.

cDNA microarray in isolation of novel differentially expressed genes related to human glioma and clone of a novel full- length gene

Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5’RACE and bioinformatics. Results Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b).Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.

Enhanced Resistance of Snowdrop Lectin （Galanthus nivalis L. agglutinin） Expressing Maize to Asian Corn Borer （Ostriniafurnacalis Guenée）

In order to enhance the resistance to pests, transgenic maize (Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin (Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through the Agrobacterium tumefaciens-mediated also studied. Thirty-six independently derived plants were subjected to molecular analyses. The level of GNA expression at 0.13%-0.28% of total soluble protein was observed in different transgenic plants. The progeny of three GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to ACB. These plants synthesized GNA at levels above 0.24% total soluble protein and enhanced resistance to ACB was demonstrated by exposing the plants to insects under greenhouse conditions. Semi-artificial diet bioassays also showed the toxic effect of GNA on ACB. Field evaluation of the transgenic plants supported the results from the artificial trial. In the present study, we have obtained new insect-resistant maize material for further breeding work and have found that GNA-expressing plants not only gained significant resistance to homopterans, but also showed toxicity to ACB, which is a type of Lepidoptera.

Expression of human clotting factor Ⅸ mediated by recom- binant lentiviral vector in cultured cells and hemophilia B mice

To explore the expression of human clotting factor Ⅸ (hFⅨ) cDNA in vitro and the feasibility of gene therapy for hemophilia B mice mediated by recombinant lentiviral vector, a recombinant hFⅨ lentiviral vector driven by ubiquitin-C promoter, FUXW, and by ABP liver specific promoter, FAXW, was constructed respectively. Recombinant lentivirus was harvested from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids (transgene vector, CMV腞8.2, VSV-G). hFⅨ expression was detected in supernatant of 293T, BHK and L-02 cells infected with FUXW virus, whereas higher expression of hFⅨ levels (630 ng/106 cells/48 h) was detected only in L-02 cells infected with FAXW virus. Serum hFⅨ antigen was detected in all hemophilia B mice treated with FAXW virus by tail vein injection, an efficiency level of hFⅨ was observed (45 ng/mL, approximately 1% of normal human levels), the expression lasted for more than 60 d. The results indicated that HIV-based lentiviral vectors offer a promising approach to the gene therapy of hemophilia B.

Preparation of rAAV/hFⅨ by HSV/AAV hybrid helper virusand evaluation of its safety

The recombinant adeno-associated viral vector with human coagulation Factor Ⅸ minigene which wasregulated by CMV promoter was constructed. Largequantity of recombinant adeno-associated viral particles (rAAV/ hFⅨ) was prepared by the HSV/AAV hybrid helper virus method. Southern dot blot assay and QC-PCR indicated that the titer of the virus was 3.6×1012 v.g./mL. It demonstrated that this method can effectively overcome the hurdles of mass production of AAV vector. Followed by anintramuscular injection of viral vectors (7.5×1011 v.g./mouse) in the quadriceps femoris, an elevation of human Factor Ⅸexpression in the plasma of hemophilia B mice was detected (387 ng/mL) and persisted more than 12 weeks. The level of anti-virus antibody in plasma aligned with the Factor Ⅸexpression curve. The QC-PCR method is easier and moreaccurate than traditional dot hybridization fordetermination of the titer of recombinant adeno-associated virus. Moreover, there are no HSV particles existing inproduced AAV assayed by RT-PCR. AAV is the only virus that has been amplified from AAV-injected muscle by PCR.

Efficient expression of human factor Ⅸ cDNA in liver mediated by hydrodynamics-based plasmid administration

Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor Ⅸ (hFⅨ) cDNA in 2.2 mL Ringers solution into mice within 7 s. The peak level of expression of hFⅨ was 2921 ng/mL in mouse plasma. The hFⅨ cDNA expression in-creased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFⅨ cDNA was detected in various organs, but the highest level of gene ex-pression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver dam-age, which was rapidly repaired within 3—10 d. These re-sults suggested that high-level expression of hFⅨ cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is now further used for gene therapy and gene function study in our lab.