Molecular Characterization, Gene Evolution, and Expression Analysis of the Fructose-1, 6-bisphosphate Aldolase (FBA) Gene Family in Wheat (Triticum aestivum L.).

Fructose-1, 6-bisphosphate aldolase (FBA) is a key plant enzyme that is involved in glycolysis, gluconeogenesis, and the Calvin cycle. It plays significant roles in biotic and abiotic stress responses, as well as in regulating growth and development processes. In the present paper, 21 genes encoding TaFBA isoenzymes were identified, characterized, and categorized into three groups: class I chloroplast/plastid FBA (CpFBA), class I cytosol FBA (cFBA), and class II chloroplast/plastid FBA. By using a prediction online database and genomic PCR analysis of Chinese Spring nulli-tetrasomic lines, we have confirmed the chromosomal location of these genes in 12 chromosomes of four homologous groups. Sequence and genomic structure analysis revealed the high identity of the allelic TaFBA genes and the origin of different TaFBA genes. Numerous putative environment stimulus-responsive cis-elements have been identified in 1,500-bp regions of TaFBA gene promoters, of which the most abundant are the light-regulated elements (LREs). Phylogenetic reconstruction using the deduced protein sequence of 245 FBA genes indicated an independent evolutionary pathway for the class I and class II groups. Although, earlier studies have indicated that class II FBA only occurs in prokaryote and fungi, our results have demonstrated that a few class II CpFBAs exist in wheat and other closely related species. Class I TaFBA was predicted to be tetramers and class II to be dimers. Gene expression analysis based on microarray and transcriptome databases suggested the distinct role of TaFBAs in different tissues and developmental stages. The TaFBA 4-9 genes were highly expressed in leaves and might play important roles in wheat development. The differential expression patterns of the TaFBA genes in light/dark and a few abiotic stress conditions were also analyzed. The results suggested that LRE cis-elements of TaFBA gene promoters were not directly related to light responses. Most TaFBA genes had higher expression levels in the roots than in the shoots when under various stresses. Class I cytosol TaFBA genes, particularly TaFBA10/12/18 and TaFBA13/16, and three class II TaFBA genes are involved in responses to various abiotic stresses. Class I CpFBA genes in wheat are apparently sensitive to different stress conditions.

Effect of low temperature on chlorophyll biosynthesis in albinism line of wheat (Triticum aestivum) FA85.

The 'stage albinism line of winter wheat' FA85 exhibits a severe block in chlorophyll (Chl) biosynthesis with prolonged low-temperature treatment. The correlations between leaf color and low temperature provide more comprehensive understanding of low temperature as an environmental signal that regulate the metabolic changes in the entire Chl-synthesizing pathway. In this study, we investigated differences in Chl biosynthesis between leaves of Aibian1 and FA85 by measuring their Chl precursors and heme content, transcripts for key genes of Chl biosynthesis and key enzyme activities. With prolonged low-temperature treatment, the Chl content gradually decreased, but Chl precursors, including protoporphyrin IX, Mg-protoporphyrin IX and protochlorophyllide (Pchlide), simultaneously accumulated. Parallel to the decline in Chl content, the protoporphyrin IX distribution toward Chl synthesis was less than that in heme synthesis in the leaves of FA85. Corresponding to the change of protoporphyrin IX distribution, the relative changes in magnesium chelatase (EC 6.6.1.1) and ferrochelatase (EC 4.99.1.1) activities in the leaves of FA85 also indirectly reflected channeling of the metabolic flow into heme rather than Chl. A drastic loss in the transcripts for Pchlide oxidoreductase (EC 1.3.1.33) and Chl synthase (EC 2.5.1.62) accounted for a decrease in the metabolic flux and the re-direction of metabolites. The high-level accumulations of Chl precursors and traces of Chl in the leaves of FA85 suggest that a severe block between the steps from Pchlide to Chl formation during Chl biosynthesis is partially derived from the transcriptional downregulation of Pchlide oxidoreductase and Chl synthase.

Targets for human encoded microRNAs in HBV genes.

MicroRNAs (miRNAs) are increasingly being shown to play vital roles in development, apoptosis, and oncogenesis by interfering with gene expression at the post-transcriptional level. miRNAs, in principle, can contribute to the repertoire of host-pathogen interactions during infection by the Hepatitis B virus (HBV). Using a consensus-scoring approach, high-scoring miRNA-target pairs were selected, which were identified by four well-established target-prediction softwares. The miRNAs miR-7, miR196b, miR433, and miR511 target the polymerase or S gene of HBV, miR205 targets the X gene, and miR345 targets the preC gene. The minimum free-energy values for the bound complexes were the lowest, and the rules so far observed for miRNA-target pairing, namely, (1) pairing at a continuous stretch of 6-7 bases toward the 5'-end of the miRNA and (2) incomplete complementarity with the target sequence, were found to be valid. The target regions were highly conserved across the various clades of HBV. miRNA expression profiles from previously reported Solexa-sequencing based experiments showed that the four human miRNAs are expressed in the liver. This is the first report of human miRNAs that can target crucial HBV genes.

Cloning and identification of a novel P-II class snake venom metalloproteinase from Gloydius halys.

Ahpfibrase was a new snake venom metalloproteinase (SVMP) which was cloned from Gloydius halys. The cDNA sequence with 1,891 base pairs encodes an open reading frame of 477 amino acids which includes a 17 amino acid signal peptide, plus a 171 amino acid segment of zymogen-like propeptide, a metalloproteinase domain of 200 amino acids, a spacer of 16 amino acids, and a disintegrin-like peptide of 73 amino acids. The metalloproteinase domain contained a conserved signature zinc-binding motif HEXXHXXGXXH in the catalytic region and a methionine-turn CIM. To determine the activity of ahpfibrase, the coding region including both the metalloproteinase domain and disintegrin region was amplified by PCR, inserted into the pET25b(+) vector, and expressed in Escherichia coli. The recombinant protein was recovered from inclusion bodies with 8 M urea and refolding was performed by fed-batch dilution method, and purified recombinant ahpfibrase showed the fibrinolytic activity and platelet aggregation-inhibition ability.

Proteome analysis of chloroplast proteins in stage albinism line of winter wheat (triticum aestivum) FA85.

The "stage albinism line of winter wheat" FA85 was a specific natural mutant strain on leaf color. This physiological mutation was controlled by cytogene. In order to reveal the genetic and biochemical mechanism of albinism, 2-DE was used to investigate the difference of chloroplast protein expression pattern between FA85 and its parent wheat Aibian 1. From the results of 2-DE gels analysis, approximately 683 spots were detected on each gel, and 57 spots were expressed differently at least two-fold. Using MALDI-TOF/TOF MS, 14 of 57 spots were identified, which could be categorized into four classes: carbon metabolism, energy metabolism, defense/stress response and signal transduction. Compared with the parent wheat, the expression of ATPase-gamma and GP1-alpha was up-regulated in FA85, and of other proteins was down-regulated. Together, we concluded that the expression of chloroplast proteins had changed obviously in FA85, which might be related to the leaf color mutant.

[Prokaryotic expression, purification and preparation of polyclonal antibody for human SUMO-2 gene].

AIM: To construct a prokaryotic expression vector of human SUMO-2, purify GST-SUMO2-SUMO2 fusion protein produced by the expression system, and prepare its antiserum. METHODS: The human SUMO-2 gene was amplified by PCR. The target fragment digested by the enzyme was cloned into a pET41a(+) expression vector and then transfected into E.coli. BL21 (DE3) pLysS, in which GST-SUMO2-SUMO2 fusion protein was induced by IPTG. After the soluble protein was purified by GST affinity chromatography and by identified by SDS-PAGE, the rabbits were immunized with the fusion protein and the antiserum was obtained. RESULTS: DNA sequence analysis showed the cloned SUMO-2 gene sequence was completely corresponding to GenBank data. SDS-PAGE and Western blot showed that the GST-SUMO2-SUMO2 fusion protein was about 52 kDa, which was mainly the soluble protein of E.coli and could be purified by GST affinity chromatography. The result of ELISA was positive and Western blot confirmed the antiserum reacted specifically to SUMO-2 protein. CONCLUSION: SUMO-2 protein and its specific polyclonal antibody have been prepared, which provides a basis for the establishment of immunoassays of human SUMO-2.

Expression, purification, and activity identification of Alfimeprase in Pichia pastoris.

Alfimeprase (ALF) is a truncated form of non-hemorrhagic zinc metalloproteinase fibrolase. In order to achieve a high level secretion and full activity expression of ALF, the Pichia pastoris (P. pastoris) expression system was used. ALF coding sequence fused with a 6 *histidine tag and an enterokinase recognition site at the N-terminus was cloned into the expression vector pPIC9K and then expressed in P. pastoris strains of GS115 and KM71 by methanol induction. SDS-PAGE and Western blotting analysis showed that the secreted recombinant ALF (rALF) had a molecular weight of 23.8 kDa and was bound specifically to mouse anti-His. tag monoclonal antibody. Under the optimized culture parameters of pH value, initial A(600) value, methanol daily addition concentration and induction time length, the production of rALF reached up to 510 mg/L and 465 mg/L of the GS115 and KM71 transformants, respectively. It also appeared that KM71 was producing a more pure protein than GS115 while GS115 was producing more rALF per unit volume. Through one-step affinity chromatography, the purity of rALF was as high as 96%. The fibrinolytic activity of rALF revealed by the modified fibrin plate method indicated that the protein was efficiently secreted and functionally expressed, and thrombolysis of rALF was demonstrated to be dose-dependent and time-relative. The improved expression system will facilitate further studies and industrial production of ALF.

HBV-encoded microRNA candidate and its target.

MicroRNAs (miRNAs) are a group of short (approximately 22 nt) noncoding RNAs that specifically regulate cellular gene expression at the post-transcriptional level. miRNA precursors (pre-miRNAs), which are imperfect stem loop structures of approximately 70 nt, are processed into mature miRNAs by cellular RNases III. To date, hundreds of miRNAs and their corresponding targets have been reported in kinds of species. Although only a few of these miRNA/target pairs have been functionally verified, some do play important roles in regulating normal development and physiology. Several viruses (e.g. the Epstein-Barr virus and human herpesvirus Kaposi's sarcoma-associated herpesvirus) has been reported to encode miRNAs. Here, we extend the analysis of miRNA-encoding potential to the Hepatitis B virus (HBV). Using computational approaches, we found that HBV putatively encodes only one candidate pre-miRNA. We then matched deduced mature miRNA sequence from this pre-miRNA against a database of 3' untranslated sequences (UTR) from the human genome. Surprisingly, none of cellular transcripts could potentially be targeted by the viral miRNA (vmiRNA) sequence. However, one viral mRNA was found to be targeted by the vmiRNA when we searched the target from viral mRNAs. We propose that HBV has evolved to use vmiRNAs as a means to regulate its own gene expression for its benefit.

Cytological and molecular characterization of a novel monogenic dominant GMS in Brassica napus L.

A novel genic male sterile (GMS) line in Brassica napus L., which was identified in 1999, was found to be controlled by a monogenic dominant gene, which we have designated as MDGMS. The microspores of the MDGMS abort before the degradation of the tapetal cell layer. The F1 fertility from any fertile lines crossed with MDGMS segregated and the ratio was close to 1:1. Bulked segregation analysis (BSA) was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the Ms gene in MDGMS. Among 880 random 10-mer oligonucleotide primers screened against the bulk DNA of sterile and fertile, one primer S243 (5'-CTATGCCGAC-3') gave a repeatable 1500-bp DNA polymorphic segment S243(1500) between the two bulks. Analysis of individual plants of each bulks and other types of GMS and cytoplasmic male sterility (CMS) lines suggest that the RAPD marker S243(1500) is closely linked to the MDGMS locus in rapeseed. This RAPD marker has been converted into sequence characterized amplified region (SCAR) marker to aid identification of male-fertility genotypes in segregating progenies of MDGMS in marker-assisted selection (MAS) breeding programs.

[Molecular markers linked to mono-dominant genic male sterile gene in rapeseed (Brassica napus L.)].

Bulked segregant analysis (BSA) was used to identify randomly amplified polymorphic DNA (RAPD) markers linked to the MS gene in mono-dominant GMS of rapeseed (Brassica napus L.), which was bred by Hybrid Rapeseed Research Center of Shaanxi Province. A total of 300 random 10-mer oligonucleotide primers were screened on the DNA from fertile and sterile bulks. Primer S(243) (5'CTATGCCGAC3') gave identical 1.5 kb DNA polymorphic segment OPU-03(1500) in the bulk S, but not in the bulk F (Fig.2). The DNAs from individual plants of each bulk and from their sister lines, which were generated from the same original crossing, were then screened with the primer S(243), and the same results were obtained (Figs.3,4). Other types of GMS and CMS were analyzed using primer S(243), and the specific 1.5 kb DNA segment was not found (Fig.5). Therefore, the RAPD marker OPU-03(1500) is linked to the mono-dominant GMS trait in rapeseed. This RAPD marker OPU-03(1500) was cloned into a T-easy vector and sequenced. The sequence here obtained was highly homologous to one of the Arabidopsis DNA sequences. According to this DNA conserved region in different species, we designed a pair of specific primers P1 (5'ATGTCGCTGAGGCCG-AGCAC3') and P2 (5'GGCACACTGTCACG-ATCCTTGG3') and amplified only one specific 2.3 kb DNA fragment in each bulk. There are two mutant loci between the two DNA fragments after sequencing. We designed another pair of specific primers P3 (5'CTCCAGCAGCAGCAGC-AGCCT3') and P4 (5'GCAGGAATGAGAA-CCGTAGG3') according to the DNA sequence at the mutant loci. A specific DNA segment was amplified only in the fertile line but not in the sterile line using the primers P3 and P4 (Fig.6). Therefore the RAPD marker were converted into SCAR marker. Moreover, the SCAR marker detection method was improved (Fig.7).

Expression of soluble and functional snake venom fibrinolytic enzyme fibrolase via the co-expression of DsbC in Escherichia coli.

Fibrolase is a non-hemorrhagic zinc metalloproteinase found in southern copperhead snake venom (Agkistrodon contortrix contortrix). It is capable of degrading fibrin clots that result from purified fibrinogen or blood plasma. The DNA of fibrolase was amplified by recursive PCR, and cloned into the pET25b(+) expression vector. The effect of co-expression of signalless versions of catalysts or molecular chaperones FkpA, Skp and DsbC in cytoplasm was examined. When co-expressed with DsbC, compared to the totally insoluble inclusion bodies of fibrolase expressed separately, more than 90 % of recombinant fibrolase was soluble, according to denaturing polyacrylamide gel electrophoresis analysis. We also determined that FkpA and Skp had no effects on the solubility of target protein when co-expressed with fibrolase in Escherichia coli. Fibrolase was successfully purified using metal ion affinity chromatography and hydrophobic chromatography, and a maximum yield of 20 mg/L fibrolase was obtained. Fibrinolytic activity of recombinant fibrolase was demonstrated using fibrin plate assays and fibrinogen hydrolysis.

[Construction and activity analysis of a recombinant immunotoxin composed of PE38 and a disulfide stable single-chain antibody].

AIM: To construct the expression vector of a recombinant toxin composed of a disulfide stable single-chain antibody from mAb B3 and PE38 and examine the binding ability and cytotoxicity of the purified renatured products against the B3 positive carcinoma cells. METHODS: The V(H) and V(L) fragments of the mAb B3 were ligated by overlaping PCR and the resulting product was cloned to the pET22b expression vector. The PE38 fragment was inserted into the B3dsscFv-pET22b expression vector which was digested by EcoR I and Hind III. The identified expression plasmid was tansformed into E.coli BL21(DE3) followed by IPTG induction. The inclusion body was purified through Q-Sepharose anion exchange column after denaturing and refolding. The binding and cytotoxic ability of the purified products were examined by cell-ELISA and Non-Radioactive Cell Proliferation Assay seperately. The stability assay was performed by incubating the protein sample at 37degrees Celsius. RESULTS: The expression vector B3dsscFv-PE38-pET was constructed successfully and the expression product existed mainly in the form of inclusion body, accounting for 45% of the totle protein. The refolding product remained the binding ability of the single-chain antibody and exerted cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37degrees Celsius. CONCLUSION: This genetically engineered B3dsscFv-PE38 fusion protein was stable and bifunctional, tumor targeting and tumor cell killing, supporting that it would be a promising candidate for tumor targeted immunotherapy.

Disulfide-stabilized single-chain antibody-targeted superantigen: construction of a prokaryotic expression system and its functional analysis.

AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines. METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography. RESULTS: The expression vector B3 (scdsFv)-SEA-PET was constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 degrees. CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.

[A simple and visible assay for cre recombinase activity in Escherichia coli by using two incompatible plasmids].

The Cre/loxP system derived from bacteriophage P1 is widely used to carry out complex manipulations of DNA molecules both in vitro and in vivo. In order to further characterize and modify the Cre/loxP system, a convenient method for assaying the recombination efficiency is needed. A simple and visible assay is described, in which two incompatible plasmids, separately carrying the cre gene and loxP-flanked gfp gene, were co-transferred into E. coli. The cre gene was inserted into a kanamycin-resistant bacterial expression vector, designated pET30a-Cre. The gfp gene, flanked by directly repeated loxP sites, was cloned into an ampicillin-resistant expression vector to generate pET23b-loxGFP. E. coli BL21 (DE3) was cotransformed with pET30a-Cre and pET23b-loxGFP, and cultured in the presence of both ampicillin and kanamycin. Under UV illumination, the Cre-mediated recombination events can be easily detected. The fidelity of recombination was verified by SDS-PAGE analysis and restriction analysis followed by DNA sequencing. Thus, this cotransformation method provides a straightforward assay that can be used to modify the Cre/loxP system.

[Integration and expression of sdh gene in Escherichia coli].

The chloramphenicol-resistant cassette with short shared sequences of ptsG gene on both ends was PCR-generated from plasmid pKF3 and ligated to pMD18-T to construct pMD18-PC. The sdh gene for sorbose dehydrogenase was generated from plasmid pQE60-SDH and inserted into pMD18-PC, then pMD18-PC-SDH was constructed. It was digested with Pvu II and the target fragment ptsG1-cat-sdh-ptsG2 was recovered and electroporated into Escherichia coli JM109/pKD46. Homologous-recombination between linear DNA cassettes and Escherichia coli chromosomes took place by Red recombination. The detection result showed that the integron JM109s was of sorbose dehydrogenase activity. The PCR products assay using the upstream and downstream sequences of ptsG gene as primers and JM109s genomic DNA as template, indicated that sdh gene had been integrated at the ptsG gene site in Escherichia coli.

[Soluble expression of recombinant immunotoxin against human bladder carcinoma and its anti-tumor activity].

AIM: To construct soluble expression vector of the recombinant immunotoxin against human bladder carcinoma and express and purify the immunotoxin with high biological activity. METHODS: The gene fragment of the immunotoxin containing the genes encoding the humanized single-chain antibody against human bladder carcinoma and the truncated and modified form of Pseudomonas exotoxin(PE) named PE38/KDEL was digested from the plasmid pABDIT and inserted into the expression vector pTMFK containing the FkpA gene. The recombinant plasmid was used to transform the E.coli strain BL21(DE3)-Star and the immunotoxin was co-expressed with the FkpA which served as the folding assistant factor. The immunotoxin was purified through Ni-NTA agarose and anti-PEAb Sepharose 4B columns. The binding activity of the immunotoxin to the antigen on BIU-87 cells was detected by ELISA. The specific cytotoxic effect of the immunotoxin against BIU-87 cells was assessed by MTT colorimetry. RESULTS: The immunotoxin was over-expressed in soluble form in E.coli. The immunotoxin showed good binding activity to the membrane antigen on BIU-87 cells. The result of MTT assay demonstrated that purified recombinant immunotoxin could specifically kill BIU-87 cells. CONCLUSION: The immunotoxin was obtained with specific cytotoxicity to human bladder cancer cells, which lays the foundation for the further application of the immunotoxin in the target therapy of human bladder carcinoma.