cDNA,genomic sequence cloning and overexpression of glyceraldehyde-3-phosphate dehydrogenase gene (<Emphasis Type="Italic">GAPDH</Emphasis>) from the Giant Panda

GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a key enzyme of the glycolytic pathway and it is related to the occurrence of some diseases. The cDNA and the genomic sequence of GAPDH were cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using the RT-PCR technology and Touchdown-PCR, respectively. Both sequences were analyzed preliminarily. The cDNA of GAPDH cloned from the Giant Panda is 1191 bp in size, contains an open reading frame of 1002 bp encoding 333 amino acids. The genomic sequence is 3941 bp in length and was found to possess 10 exons and 9 introns. Alignment analysis indicates that the nucleotide sequence and the deduced amino acid sequence are highly conserved in some mammalian species, including Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupus familiaris and Bos taurus. The homologies for the nucleotide sequences of the Giant Panda GAPDH to that of these species are 90.67, 90.92, 90.62, 95.01 and 92.32% respectively, while the homologies for the amino acid sequences are 94.93, 95.5, 95.8, 98.8 and 97.0%. Primary structure analysis revealed that the molecular weight of the putative GAPDH protein is 35.7899 kDa with a theoretical pI of 8.21. Topology prediction showed that there is one Glyceraldehyde 3-phosphate dehydrogenase active site, two N-glycosylation sites, four Casein kinase II phosphorylation sites, seven Protein kinase C phosphorylation sites and eight N-myristoylation sites in the GAPDH protein of the Giant Panda. The GAPDH gene was overexpressed in E. coli BL21. The results indicated that the fusion of GAPDH with the N-terminally His-tagged form gave rise to the accumulation of an expected 43 kDa polypeptide. The SDS-PAGE analysis also showed that the recombinant GAPDH was soluble and thus could be used for further functional studies.     

Identification of a novel UDP-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine enolpyruvyl transferase (MurA) from <Emphasis Type="Italic">Vibrio fischeri</Emphasis> that confers high fosfomycin resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 μg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.     

Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

Molecular cloning and characterization of caprine <Emphasis Type="Italic">calpastatin</Emphasis> gene

Calpastatin (CAST) is an important gene for meat quality traits in livestock and poultry. The cDNA of caprine CAST gene was amplified for the first time using RACE-PCR. Results showed the full-length cDNA of caprine CAST gene (Accession no. GU944861) was 2435 base pair (bp) and contained a 2187 bp open reading frame encoding a protein with 728 amino acid residues. Bioinformatic analysis indicated that caprine CAST cDNA was 89.8–95.4, 83.5–92.2, 72.8–81.8 and 69.8–73.5% identical to sheep, cattle, pig and human CAST cDNA. It was predicted that caprine CAST contained four conserved domains with 42 serine phosphorylation loci, 18 threonine phosphorylation loci, 1 tyrosine phosphorylation locus and 5 specific PKC phosphorylation loci. This work provided an important experimental basis for further research on the function of CAST in goat.     

Cloning and expression of <Emphasis Type="Italic">Tenebrio molitor</Emphasis> antifreeze protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded β-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.     

Partial molecular characterization,expression pattern,polymorphism and association analysis of porcine <Emphasis Type="Italic">SKP2</Emphasis> gene

As a component of E3 ubiquitin protein ligases called SCFs, SKP2 protein belongs to a member of FBLs protein which is the biggest eukaryotic subfamily of F-BOX proteins with 12 members. In this study, we cloned and sequenced partial cDNA, intron 1 and intron 6 of porcine SKP2 gene. The partial cDNA is 1,402 bp long and has an open reading frame of 1,272 bp which encodes 424 putative amino acids. The deduced protein comprises a conserved F-BOX domain at position from the 90th to 140th amino acid. The phylogenetic tree indicated that porcine SKP2 has the closest genetic relationship with bovine SKP2 than other selected animal species. Quantitative RT-PCR analysis displayed that the tissue expression level of porcine SKP2 fluctuated remarkably in a large range, and it expressed in thymus with the highest level and in longissimus dorsi muscle with the lowest level. Two SNPs were identified, meanwhile, further polymorphism analysis with Cfr42I showed that AA genotype was in dominance absolutely among four kinds of unrelated Chinese indigenous miniature and one introduced Landrace pig breeds. In addition, association analysis with immune traits and blood parameters revealed that the SNP Cfr42I in intron 1 was significantly associated with red cell distribution width of neonate piglets at 0 day (P = 0.027).     

Molecular cloning,characterization and expression analysis of <Emphasis Type="Italic">Cm</Emphasis>APX

The RT PCR and RACE methods were used to obtain the cDNA sequence of an APX gene of muskmelon after the leaves were induced with powdery mildew. The cDNA length of the APX gene is 1,047 bp with a 750 bp ORF encoded a 249 amino acid and the molecular weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns. The identity of the amino acid sequence deduced from the cDNA with the APX family of other homologous members was about 74–97%. A Full-length of ORF was sub-cloned into prokaryotic expression vector pET24a. The recombinant proteins had high expression level in E. coli. Analysis of expression at mRNA level showed that CmAPX exhibited highly tissue-specific patterns of expression. The mRNA level and enzyme activities assays showed that CmAPX might play an important role in the pathogenesis of powdery mildew.     

Molecular Cloning and Expression Analysis of a Terpene Synthase Gene, <Emphasis Type="Italic">HcTPS2</Emphasis>, in <Emphasis Type="Italic">Hedychium coronarium</Emphasis>

The Hedychium coronarium can emit a strong scent which is mainly composed of monoterpenes. A cDNA clone, HcTPS2 (H. coronarium terpene synthases), was cloned from H. coronarium flower. The gene has an open reading frame of 1,788 bp which encodes a protein of 596 amino acids with a calculated molecular mass of 66.7 kDa. The deduced amino acid sequence shows 35–38% identity with known monoterpene synthases in other angiosperm species. HcTPS2 was appreciably expressed in the petals, sepals, and stamens of H. coronarium, whereas no expression signal was detected in those of nonscented species. To the best of our knowledge, this is for the first time to clone the terpene synthase gene from H. coronarium, which provides the basis for biotechnological manipulation of scent composition in H. coronarium.     

Molecular cloning,sequence and expression analysis of <Emphasis Type="Italic">ZmArf2</Emphasis>, a maize ADP-ribosylation factor

A full-length cDNA encoding a maize GTP-binding protein of the ADP-ribosylation factor family was cloned by suppression subtractive hybridization and an in silico cloning approach. The cDNA was 938 bp in length and contained a complete ORF of 612 bp, which encodes a protein of 203 amino acid residues. Its deduced amino acids sequence had an 83% identity with that of a GTP-binding protein in rice. The gene was designated ZmArf2. The ZmArf2 gene consists of G1, G2, G3, G4 and G5 boxes, and Switch I and Switch II regions. Eight nucleotides differed and five amino acids changed between the popcorn inbred N04 and the dent corn inbred Dan232. One changed amino acid was in the G1 box. RT-PCR analysis showed that ZmArf2 expression increased in the early stages of endosperm development and was not tissue-specific.