Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish

Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82 % similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.     

Molecular cloning and sequence analysis of a mannitol dehydrogenase gene and isolation of mdh promoter from Candida magnoliae

The mdh gene encodes mannitol dehydrogenase (MDH), which catalyzes the conversion of fructose into mannitol. The putative mdh gene of Candida magnoliae was isolated by PCR using the primers deduced from the N-terminal amino acid sequences of an intact MDH and its tryptic peptides, cloned in E. coli, and sequenced. The mdh gene consisted of 852 bp encoding for 283 amino acids. Analysis of the amino acid sequence revealed that MDH consisted of typical NADPH-dependent short chain dehydrogenases/reductases (SDRs). To develop a strong promoter to induce expression of the foreign genes in C. magnolia, the putative promoter was isolated. The reporter protein, GFP, was well-expressed under the control of the putative mdh promoter of 153 bp in C. magnoliae.     

克氏原螯虾一种诱导型HSP70基因克隆及分析

克氏原螯虾是我国淡水虾类养殖的重要品种,具有很强的抵御各种环境胁迫和各种刺激的能力。本文以该虾为对象,通过基因克隆以及从基因水平探讨HSP70s与环境应激之间的关系,为深入研究水生无脊椎动物HSP70s功能提供基础。采用RT-PCR和RACE方法从克氏原螯虾（Procambarus clarkii）心脏组织中克隆得到一种HSP70 cDNA（scHSP70）,其全长为2271bp,包括1902bp的完整编码序列、142bp的5′及221bp的3′端非翻译区, GenBank登陆号DQ301506。基因组DNA扩增表明该基因仅由一个外显子组成。根据cDNA序列推导出scHSP70由635个氨基酸组成,分子量为69.6kD,理论等电点为5.34。该序列存在真核细胞HSP70家族的三个特征标签。SWISS-MODEL蛋白三维结构预测显示scHSP70在N端形成ATP酶结构域,在近C端形成底物肽结合结构域。克氏原螯虾在系统发生树上的进化地位与传统分类学相一致。半定量RT-PCR实验表明,scHSP70有广泛的组织分布,在心脏中表达量最高,在血液中最少。热激后该基因大量表达,说明该基因是一种诱导型HSP70。这为从蛋白水平研究克氏原螯虾HSP70与环境应激之间的关系提供基础。     

Characterization and regulation of the gamma-glutamyl transpeptidase gene from the fission yeast Schizosaccharomyces pombe

The structural gene for the putative gamma-glutamyl transpeptidase (GGT) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The determined sequence contained 3324 bp and encoded the predicted 630 amino acid sequence of GGT, which resembles counterparts in Homo sapiens, Rattus norvegicus, Saccharomyces cerevisiae, and Escherichia coli. The S. pombe cells harboring the cloned GGT gene showed about twofold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGT gene was functional. To monitor the expression of the S. pombe GGT gene, we fused the fragment 1085 bp upstream of the cloned GGT gene into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pGT98. The synthesis of beta-galactosidase from the fusion plasmid in S. pombe cells was enhanced by treatments with NO-generating sodium nitroprusside (SN), L-buthionine-(S,R)-sulfoximine (BSO), and glycerol. The GGT mRNA level in the S. pombe cells was increased by SN and BSO. Involvement of Pap1 in the induction of the GGT gene by SN and BSO was observed.     

Molecular cloning,sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.     

Molecular cloning and characterization of the mitogen-activated protein kinase kinase gene (MKK4) and its promoter sequence from oilseed rape (Brassica campestris L.)

Mitogen-activated protein kinase (MAPK) cascades are involved in various processes, including plant growth and development as well as biotic and abiotic stress responses. MAPK kinases (MKKs), which link MPKs and MAPKK kinases (MKKKs), are crucial in MAPK cascades because these kinases mediate various stress responses in plants. However, only few MKKs in Brassica campestris (rape) have been functionally characterized. In this study, a novel gene, MKK4 that belongs to a C MKK group, was isolated and characterized from rape. Bioinformatics analysis revealed that the length of cDNA was 1,317 bp with an open reading frame of 993 bp, which encodes a polypeptide containing 330 amino acids, including a putative signal peptide with 27 amino acid residues and a mature protein with 303 amino acids. The obtained MKK4 exhibited a predicted molecular mass of 36.5 kDa and an isoelectric point of 9.01. Quantitative real-time polymerase chain reaction analysis revealed that MKK4 expression could be induced by cold and salt. We also found that the MKK4 protein is localized in the nucleus. In addition, a 999 bp promoter fragment of MKK4 was cloned. Sequence analysis revealed that several putative regulatory elements were found in the MKK4 promoter. Transient expression assay showed that the MKK4 promoter fragments exhibited promoter activity and stimulated GFP expression. The effects of GFP gene expression at different temperatures and in different onion epidermis culture patterns were compared. Results showed that the MKK4 promoter could respond to low temperature and salt stress. These results suggested that MKK4 is possibly important for the regulation of cold- and salt-stress responses in plants.     

Molecular characterization and expression of type-I interferon gene in Labeo rohita

Genes coding for type-I interferon (I-IFN) has been cloned from Labeo rohita, a commercially important and widely cultured fish in India and South East Asia. In the present study, full-length gene of I-IFN was amplified and sequenced. The sequence analysis revealed that I-IFN consists of 1,786 bp genomic sequence with four introns and five exons and an ORF of 546 bp encoding for a putative protein of 181 amino acids. The mature protein has a molecular weight of 18.97 kDa and consists of 158 amino acids and a signal peptide of 23 amino acids at the N terminus. The sequence carries I-IFN signature motif, one glycosylation site, two conserved cystine amino acids and other conserved amino acids. The sequence showed highest similarity to that of Cyprinus carpio (84 %). In silico analysis of the rohu I-IFN protein was done using various bioinformatic tools. The constitutive expression of I-IFN gene was found to be more in spleen compared to gill and kidney in real time PCR assay. Expression of I-IFN increased about 20-fold in cultured kidney cell 2 h after induction with poly I:C and showed maximum expression at 8 h post-induction.     

Molecular cloning and characterization of the lipopolysaccharide and β-1,3-glucan binding protein from oriental river prawn,Macrobrachium nipponense

The lipopolysaccharide and β-1,3-glucan binding protein (LGBP), one of the pattern recognition proteins, plays an important role in the innate immune response of invertebrates. A 1,506 bp full-length cDNA of a LGBP gene was cloned and characterized from the oriental river prawn Macrobrachium nipponense (named as MnLGBP). Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1,119 bp, encoding a protein of 372 amino acids including a 21-aa signal peptide. The calculated molecular mass of the mature protein (351 aa) was 39.9 kDa with an estimated pI of 4.63. The MnLGBP sequence contains: (1) two putative integrin-binding motifs, (2) a glucanase motif, (3) two putative N-glycosylation sites, (4) one protein kinase C phosphorylation site, and (5) a putative recognition motif for β-1,3-linkage of polysaccharides. Sequence comparison based on the deduced amino acid sequence of MnLGBP showed varied identity of 89, 76 and 74 % with those of Macrobrachium rosenbergii LGBP, Marsupenaeus japonicus β-1,3-glucan binding proteins, and Fenneropenaeus chinensis LGBP, respectively. Quantitative RT-PCR results showed that MnLGBP was expressed in nerve, intestine, muscle, gill, heart, haemocytes and at the highest level in hepatopancreas. After challenge with the pathogen, Aeromonas hydrophila and Vibrio parahaemolyticus, the expression of MnLGBP mRNA was significantly upregulated in the hepatopancreas compared to the control group. At the same time, the mRNA level of MnproPO increased dramatically at 48 h after injection of bacteria. These data should be helpful to better understand the function of MnLGBP in the prawn immune system.     

Molecular Characterization and Expression Analysis of Heat Shock Cognate 70 After Heat Stress and Lipopolysaccharide Challenge in Sea Cucumber (Apostichopus japonicus)

A gene encoding hsc70 was cloned from the sea cucumber Apostichopus japonicus and named AjHsc70. The full-length cDNA sequence was 2,508 bp, containing a 5′-UTR of 77 bp, an ORF of 2,010 bp encoding 670 amino acids, and a 3′-UTR of 421 bp. Quantitative RT-PCR analysis revealed that AjHsc70 was expressed constitutively in all of the tested tissues with respiratory tree tissue showing the highest expression level. AjHsc70 expression was significantly induced by lipopolysaccharide, and the expression levels peaked at different sampling times in the body wall (24 h), coelomocytes (12 h), and intestine and respiratory tree tissues (6 h). After heat stress, AjHsc70 expression in intestine, coelomocytes, and body wall decreased acutely at first and then increased slightly. AjHsc70 expression patterns indicated that hsc70 plays an important role in mediating the responses of A. japonicus to bacterial challenge and heat stress.     

Cloning and functional characterization of a putative sodium channel auxiliary subunit gene from the house fly (Musca domestica)

The functional expression of cloned Drosophila melanogaster and house fly (Musca domestica) voltage-sensitive sodium channels in Xenopus oocytes is enhanced, and the inactivation kinetics of the expressed channels are accelerated, by coexpression with the tipE protein, a putative sodium channel auxiliary subunit encoded by the tipE gene of D. melanogaster. These results predict the existence of a tipE ortholog in the house fly. Using a PCR-based homology probing approach, we isolated cDNA clones encoding an ortholog of tipE (designated Vssc beta) from adult house fly heads. Clones comprising 3444 bp of cDNA sequence contained a 1317 bp open-reading frame encoding a 438 amino acid protein. The predicted Vssc beta protein exhibited 72% amino acid sequence identity to the entire D. melanogaster tipE protein sequence and 97% identity within the two hydrophobic segments identified as probable transmembrane domains. Coexpression of Vssc beta with the house fly sodium channel alpha subunit (Vssc1) in oocytes enhanced the level of sodium current expression five-fold and accelerated the rate of sodium current inactivation 2.2-fold. Both of these effects were significantly larger in magnitude than the corresponding effects of the D. melanogaster tipE protein on the expression and kinetics of Vssc1 sodium channels. These results identify a second example of a putative sodium channel auxiliary subunit from an insect having functional but not structural homology to vertebrate sodium channel beta subunits.     

Cloning and characterization of a functional flavanone-3ß-hydroxylase gene from <Emphasis Type="Italic">Medicago truncatula</Emphasis>

As a key enzyme in the biosynthesis of flavonols, anthocyanidins and proanthocyanidins, flavanone-3ß-hydroxylase (F3H) plays very important roles in plant stress response. A putative flavanone-3ß-hydroxylase gene from Medicago truncatula (MtF3H), a model legume species, was identified from a bio-data analysis platform. It was speculated to be induced by salt stress based on the outcomes of the analysis platform. The complementary DNA (cDNA) consists of 1499 bp with an open reading frame (ORF) of 1098 bp, which encodes a putative protein of 365 amino acids with a molecular weight of about 41.36 kDa and an isoelectric point of 5.60. To measure the catalytic activity of the protein, the MtF3H gene was ligated to pYES2 vector and heterologously expressed in yeast. The recombinant protein converted naringen into dihydrokaempferol and displayed different enzymatic efficiencies with other flavanones, confirming that MtF3H coding a functional flavanone-3ß-hydroxylase. The expression pattern of the MtF3H gene was analyzed by comparative quantitative RT-PCR and a higher level of expression was observed in the roots than was observed in stems and leaves. Furthermore, the expression was induced by salt stress in the roots, and to a greater extent in the stems, but the response of the gene activity to salt stress in the stems was slower in the first 12 h following treatment when compared to the roots.     

Characterization of Squalene synthase Gene from Chlorophytum borivilianum (Sant. and Fernand.)

Saponins are important group of secondary metabolites known for their pharmacological properties. Chlorophytum borivilianum contains high amount of saponins and is thus, recognized as an important medicinal plant with aphrodisiac properties. Though the plant is well known for its pharmaceutical properties, there is meager information available about the genes and enzymes responsible for biosynthesis of saponins from this plant. Squalene synthase (SqS) is the key enzyme of saponin biosynthesis pathway and here, we report cloning and characterization of SqS gene from C. borivilianum. A full-length CbSqS cDNA consisting of 1,760 bp was cloned which contained an open reading frame (ORF) of 1,233 bp, encoding a protein of 411 amino acids. Analysis of deduced amino acid sequence of CbSqS predicted the presence of conserved isoprenoid family domain and catalytic sites. Phylogenetic analysis revealed that CbSqS is closer to Glycine max and monocotyledonous plants. 3D structure prediction using various programs showed CbSqS structure to be similar to SqS from other species. C-terminus truncated recombinant squalene synthase (TruncCbSqS) was expressed in E. coli M15 cells with optimum expression induced with 1 mM IPTG at 37 °C. The gene expression level was analyzed through semi-quantitative RT-PCR and was found to be higher in leaves as compared to the roots.     

Cloning and characterisation of a phenylalanine ammonia-lyase gene from Rhus chinensis

Key message

The gene and cDNA sequence encoding PAL from Chinese medicinal plant Rhus chinensis were cloned and analyzed, furthermore the biochemical properties, kinetic parameters, differential expression and key sites were studied.

Abstract

Rhus chinensis is a well-known Chinese medicinal plant. Phenylalanine ammonia-lyase (PAL) is the first enzyme of phenylpropanoid pathway. Several recent studies suggested that PAL also play an important role in plant–aphid interaction. In this study, both the cDNA and the genomic sequence encoding PAL from Rhus chinensis (designated as RcPAL) were cloned and analyzed. The 3,833 bp gene contained a 1,342 bp intron and two extrons. The ORF was 2,124 bp and predicted to encode a 707-amino acid polypeptide. The results of real-time PCR showed that RcPAL expressed in all tested tissues and followed the order: stems > young leaves > petioles > roots > seeds > mature leaves. RcPAL was successfully expressed in E. coli with the pET-28a-RcPAL recombinant vector. The recombinant protein exhibited a high level of PAL activity. Biochemical properties and kinetic parameters of recombinant RcPAL were further studied. The results showed that the optimal temperature and pH for RcPAL activity were 45 °C and 9.0, and the K _m and K _cat values were 7.90 mM and 52.31 s^?1, respectively. The active sites and substrate selectivity site were also investigated with site-directed mutagenesis methods, suggesting that Phe¹²⁶ is responsible for the substrate selectivity. To our knowledge, this was the first full-length PAL gene cloned and characterized from the family Anacardiaceae so far.