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).     

Cloning,characterization and expression of the extracellular lipase gene from <Emphasis Type="Italic">Aureobasidium </Emphasis><Emphasis Type="Italic">pullulans</Emphasis> HN2-3 isolated from sea saltern

The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.     

Molecular characterization and association analysis of porcine adipose triglyceride lipase (<Emphasis Type="Italic">PNPLA2</Emphasis>) gene

The adipose triglyceride lipase (PNPLA2, also known as ATGL) is a novel triacylglycerol (TG) lipase which specifically removes the first fatty acid from the triglyceride molecule generating free fatty acid and diglyceride (DG) in mammalian cells. Here we describe the molecular characterization of the porcine ATGL gene. The full-length cDNA sequence contains a 1,461 bp open reading frame encoding a protein of 486 amino acids with a calculated molecular mass of 53.2 kDa and an isoelectric point of 7.90. The porcine ATGL protein shares high identity with other mammalian ATGL. The ATGL gene contains 9 coding exons, spans approximately 6 kb. The porcine ATGL mRNA was expressed predominantly in backfat, mildly in muscle, small intestine and heart, and almost absent in liver, spleen, lung, stomach, kidney and ovary. Statistical analysis showed the ATGL gene polymorphism (G/A³⁹²) was different between Chinese indigenous and introduced commercial western pig breeds, and was highly associated with almost all the fat deposition and carcass traits, including subcutaneous fat thickness, viscera adipose tissue, lean percentage, loin eye traits and even rib numbers.     

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 and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

Identification and expression analysis of <Emphasis Type="Italic">CjLTI</Emphasis>, a novel low temperature responsive gene from <Emphasis Type="Italic">Caragana jubata</Emphasis>

Using rapid amplification of cDNA ends, a full length cDNA (CjLTI) was cloned from apical buds of Caragana jubata, a plant species that grows under extreme cold. The cDNA obtained was 573 bp long consisting of an open reading frame of 351 bp encoding 116 amino acids. Homology analysis did not exhibit significant similarity with any sequence at NCBI database, therefore it was deduced as a novel gene. Secondary structure analysis suggested that the deduced CjLTI contained 25.86% α-helices, 4.31% β-turns, 6.90% extended strands, and 62.93% random coils. The hydropathy profile suggested CjLTI to be a hydrophobic protein having characteristic features of signal peptides at N-terminus. The gene exhibited down-regulation at 5 min of exposure to low temperature (LT, 4 ± 3°C) followed by a strong up-regulation after 15 min and onwards. Methyl jasmonate (MJ) lead to up-regulation of CjLTI starting at 5 min onwards. The gene exhibited up- and down-regulation of expression pattern in response to abscisic acid (ABA) and salicylic acid (SA). Mild drought stress slightly up-regulated gene expression and at severe drought (up to 115% reduction in leaf water potential) slight down-regulation of gene expression was observed. These results suggested CjLTI to be a LT responsive gene wherein MJ, ABA and SA pathways might be involved in regulating the gene expression.     

<Emphasis Type="Italic">Arthroderma benhamiae</Emphasis> (The Teleomorph of <Emphasis Type="Italic">Trichophyton mentagrophytes</Emphasis>) Mating Type-Specific Genes

This study first report to identify the mating type (−)-specific gene of alpha-box and the mating type (+)-specific gene of the high-mobility-group (HMG) DNA-binding domain in zoophilic dermatophytes of Arthroderma benhamiae in an effort to understand the epidemiological characteristics of Trichophyton mentagrophytes. The sequence of the alpha-box gene (1,387 bp) was found to contain two exons, from 184 to 475 bp and from 525 to 1,387 bp, coding a protein of 384 amino acids, beginning with a putative initiating methionine (ATG). The sequence of the HMG gene (1,910 bp) contained two exons, from 234 to 415 bp and from 479 to 1,457 bp, coding a protein of 386 amino acids, beginning with a putative initiating methionine (ATG).     

Isolation and characterization of a <Emphasis Type="Italic">Glutamate decarboxylase</Emphasis> (GAD) gene and their differential expression in response to abiotic stresses from <Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

Glutamate decarboxylase (GAD) catalyzes the conversion of l-glutamate to γ-aminobutyric acid (GABA). A full-length cDNA encoding GAD (designated as PgGAD) was isolated and characterized from the root of Panax ginseng C. A. Meyer. The length cDNA of PgGAD was 1881 bp and contained a 1491 bp open reading frame (ORF) encoding a glutamate decarboxylase protein of 496 amino acids, possessing a Ser-X-X-Lys active site, which belongs to the GAD group. The deduced amino acid sequence of the PgGAD was classified in the plant GAD family and has 76–85% high similarity with other plants as like petunia, Arabidopsis, tomato. Secondary structure of PgGAD was predicted by using SOPMA software program. Southern blot analysis of genomic DNA suggests that, there is more than one copy of the PgGAD gene. The organ specific gene expression pattern also studied in P. ginseng seedlings, in which the stem showed elevated expression than root, leaf, bud and rhizomes. Along with this, we also confirmed the gene expression of PgGAD under various abiotic stresses like temperature stress, osmotic stress, anoxia, oxidative stress, and mechanical damage. Temporal analysis of gene expression except exposure of oxidative stress revealed an enhanced expression after each stresses. The enzyme activity of PgGAD was stimulated to 2-fold under cold stress.     

Cloning and characterization of a novel CoA-ligase gene from <Emphasis Type="BoldItalic">Penicillium chrysogenum</Emphasis>

A novel phenylacetic acid (PAA)-induced CoA-ligase-encoding gene, designated as phlC, has been cloned from penicillin-producing fungus Penicillium chrysogenum. The open reading frame of phlC cDNA was 1671 bp and encoded a 556 amino acid residues protein with the consensus AMP binding site and a peroxisomal targeting signal 1 on its C terminus. The deduced amino acid sequence showed 37% and 38% identity with characterized P. chrysogenum Phl and PhlB protein, respectively. Functional recombinant PhlC protein was overexpressed in Escherichia coli. The purified recombinant enzyme was capable to convert PAA into its corresponding CoA ester with a specific activity of 129.5 ± 3.026 pmol/min per mg protein. Similar to Phl and PhlB, PhlC displayed broad substrate spectrum and showed higher activities to medium- and long-chain fatty acids. The catalytic properties of PhlC have been determined and compared to those of Phl and PhlB.     

Identification and characteristics of a novel gene, <Emphasis Type="Italic">EJO1</Emphasis>, in the Chinese mitten crab (<Emphasis Type="Italic">Eriocheir japonica sinensis</Emphasis>) ovary

EJO1, a novel gene presumably involved in the ovary development of the Chinese mitten crab (Eriocheir japonica sinensis), was identified and characterized by suppression subtractive hybridization and cDNA macroarray analysis. EJO1 expression was 2.6-fold higher at stage III than at stage II during the ovary development of the mitten crab. EJO1 is 876 bp in length containing a 759 bp open reading frame which encodes a 252-amino-acid protein. Homology analysis showed that no sequence significantly matching EJO1 was found in SwissProt, so it was deduced as a novel gene (GenBank accession number: AY185917). The EJO1 protein is probably a secretion protein with a signal peptide of 17 amino acids. The pI/Mw deduced from the amino acid sequence was 6.18/28.18 kDa. Expression profile showed that EJO1 mRNA is highly expressed in the heart, intestine, and ovary of the crab, while there is little or no expression in muscle and hepatopancreas. The differential expression of EJO1 at the different developmental stages of the ovary was further confirmed by Northern blot analysis. In conclusion, EJO1 is a novel gene differentially expressed in the ovary of the Chinese mitten crab, which may play an important role in the ovary development.     

Molecular cloning and expression analysis of a F-type lectin gene from Japanese sea perch (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the fucose-binding lectin (F-type lectin) gene from Japanese sea perch (Lateolabrax Japonicus). The full-length cDNA of sea perch F-lectin (JspFL) contained a 5′ untranslated region (UTR) of 39 bp, an ORF of 933 bp encoding a polypeptide of 310 amino acids with an estimated molecular mass of 10.82 kDa and a 3′ UTR of 332 bp. The searches for nucleotides and protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of JspFL was homological to the Fucose-binding lectin in other fish species. In the JspFL deduced amino acid sequence, two tandem domains that exhibit the eel carbohydrate-recognition sequence motif were found. The temporal expressions of gene in the different tissues were measured by real-time PCR. And the mRNA expressions of the gene were constitutively expressed in tissues including spleen, head-kidney, liver, gill, and heart. The JspFL expression in spleen was different during the stimulated time point, 2 h later the expression level became up-regulated, and 6 h later the expression level became down-regulated. The result indicated that JspFL was constitutive and inducible expressed and could play a critical role in the host-pathogen interaction.