Differences in cold tolerance and expression of two fatty acid desaturase genes in the leaves between fingered citron and its dwarf mutant

Fingered citron (Citrus medica L. var. sarcodactylis Swingle), a precious fruit ornamental plant, is sensitive to low temperature. Cold tolerance, evaluated by semi-lethal temperature, was lower in wild-type ‘Qingpi’ than in its mutant ‘Aihua’ trees obtained by γ-radiation. The full-length cDNAs of two genes encoding fatty acid desaturases involved in unsaturated fatty acid biosynthesis were isolated from the fingered citron leaves. The CmsFAD2 open reading frame (ORF) had 1,152?bp and was uninterrupted, encoding a polypeptide of 384 amino acids that showing 82% homology with the microsomal ω-6 desaturase CiFAD2 in Davidia involucrate. The CmsFAD8 ORF contained 1,373?bp and 7 introns, encoding a polypeptide of 458 amino acids showing 76% homology with the plastidial ω-3 desaturase BpFAD8 in Betula pendula. CmsFAD2 was expressed highly in leaves but low in roots and flowers, while CmsFAD8 was obviously expressed in three tissues. Compared with control group (28°C), the expression of CmsFAD2 and CmsFAD8 in leaves of two genotypes was significantly induced at 6°C. The increase of CmsFAD2 and CmsFAD8 was earlier and larger in cold-tolerant ‘Aihua’ than in cold-sensitive ‘Qingpi’. The linolenic acid content increased significantly in leaves of mutant ‘Aihua’ plants exposed to low temperature of 6°C. The results showed that a positive relationship between CmsFAD expression and genotype tolerance to cold may exist.     

Molecular cloning and characterization of a jasmonate biosynthetic pathway gene for allene oxide cyclase from <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Herein, we cloned a full-length cDNA encoding allene oxide cyclase (AOC, EC 5.3.99.6) that is a key enzyme in jasmonates (JAs) biosynthetic pathway from Jatropha curcas L., an important plant species as its seed is the raw material for biodiesels, named as JcAOC (GenBank accession no. FJ874630). The cDNA was 924 bp in length with a complete open reading frame of 750 bp, which encoded a polypeptide of 250 amino acids including a putative signal peptide of 65 amino acid residues and a mature protein of 185 amino acids with a predicted molecular mass of 20.7 kDa and a isoelectric point of 6.24. Phylogenetic analysis indicated that JcAOC belonged to the AOC superfamily. Semi-quantitative RT-PCR analysis revealed that JcAOC mRNA was expressed in roots, stems, leaves, young seeds, endosperms, and flowers, but that the expression level was highest in leaves and lowest in seeds, and mRNA expression of JcAOC could be induced by salt stress (300 mM NaCl) and low temperature (4°C). Furthermore, the full-length coding region of JcAOC excluding signal peptide sequence was inserted into pET-30a and was successfully expressed in Escherichia coli. Overexpression of JcAOC in E. coli conferred its resistance to salt stress and low temperature.     

Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (<Emphasis Type="Italic">Penaeus monodon</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5′ untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 3′ UTR of 288 bp. The sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence. The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37°C. In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage.     

Efficient expression of an alkaline pectate lyase gene from Bacillus subtilis and the characterization of the recombinant protein

The gene encoding a novel alkaline pectate lyase (Apel) from Bacillus subtilis was cloned and expressed in B. subtilis WB600. Apel contained an ORF of 1,260 bp, encoding a signal peptide of 21 amino acids and a mature protein of 399 amino acids with a calculated molecular mass of 45497.9 Da. The mature Apel was structurally related to the enzymes in the polysaccharide lyase family 1. After purification, the recombinant Apel had a specific activity of 445 U mg⁻¹. The enzyme was optimally active at 50°C and pH 9.     

Molecular analysis of the QM gene from <Emphasis Type="Italic">Penaeus monodon</Emphasis> and its expression on the different ovarian stages of development

In present study, a QM gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of black tiger prawn (Penaeus monodon). The full-length black tiger prawn QM (PmQM) cDNA contained a 5′-UTR of 41 bp, an ORF of 663 bp encoding a polypeptide of 220 amino acids with molecular weight 25.5 kDa, and a 3′-UTR of 54 bp. Homology analysis of the deduced amino acid sequence of the PmQM with other known QM sequences by MatGAT software revealed that the PmQM was high homology with other invertebrates. A conserved signature sequence of the QM family was found in the PmQM deduced amino acid sequence. Analysis of the tissue expression pattern of the PmQM gene showed that the PmQM mRNA was expressed in all tissues tested, with highest levels in ovary. Furthermore, the PmQM expression was found to be different in three important ovarian stages of development. The results indicated PmQM might play an important role in ovarian development.     

Molecular cloning and expression analysis of a new putative gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

A new putative gene encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase (designated as SmHMGS, GenBank Accession No. FJ785326), which catalyses the condensation of acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the mevalonic acid pathway, was isolated from young leaves of Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of the putative SmHMGS was 1,655 bp containing a 1,381 bp open reading frame (ORF) encoding a polypeptide of 460 amino acids. Comparative and bioinformatic analyses revealed that SmHMGS showed extensive homology with HMGSs from other plant species. Phylogenetic tree analysis indicated that SmHMGS belonged to the plant HMGS super family and had the closest relationship with HMGS from Hevea brasiliensis. Tissue expression pattern analysis revealed that the putative SmHMGS was constitutively expressed in all the tested tissues and strong in leaf, moderate in stem, weak in root, which was in contrast to SmHMGR reported before. The putative SmHMGS was found to be an elicitor-responsive gene, which could be induced by exogenous elicitors, including salicylic acid (SA) and methyl jasmonate (MJ). These results will help in understanding the role of HMGS in tanshinones biosynthesis in S. miltiorrhiza.     

Expression and characterization of two new alkane-inducible cytochrome P450s from <Emphasis Type="Italic">Trichoderma harzianum</Emphasis>

n-Dodecane and fatty acids were good inducers of cytochrome P450 (CYP) and the ω-hydroxylase of lauric acid, which is a marker for ω-hydroxylation of n-alkanes, in Trichoderma harzianum. A cDNA, containing an ORF of 1520 bp, encoding a CYP52 of 520 amino acids, was isolated by RACE. Another n-alkane-inducible CYP was identified by LLC-MS/MS analysis of a microsomal protein band induced by n-dodecane in a library of T. harzianum. This suggests that T. harzianum has a CYP-dependent conversion of alkanes to fatty acids allowing their incorporation into lipids.     

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.     

Isolation and characterization of microsomal omega-6-desaturase gene (fad2-1) from soybean

A genomic DNA sequence (fad2-1) encoding seed specific microsomal 0-6 desaturase was isolated from soybean (Glycine max. L cv. Pusa-9702). A positive genomic clone of 1852 nucleotides containing a single uninterrupted 3' end exonic region with an ORF of 1140 bp encoding a peptide of 379 amino acids, a complete 3' UTR of 206 bp and 86 bp of 5' UTR interrupted by a single intron of 420 bp was obtained on screening the sub-genomic library of soybean. Southern blots revealed at least two copies of the gene per haploid genome. Analysis of the translated product showed the presence of three histidine boxes, with the general sequence HXXXH and five probable transmembrane segments reported to be involved in substrate specificity.     

Isolation of a novel C18-Δ9 polyunsaturated fatty acid specific elongase gene from DHA-producing <Emphasis Type="Italic">Isochrysis galbana</Emphasis> H29 and its use for the reconstitution of the alternative Δ8 pathway in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A novel gene (IgASE2) encoding a C18-Δ9 polyunsaturated fatty acids specific (C18-Δ9-PUFAs-specific) elongase was isolated and characterized from DHA-rich microalga, Isochrysis galbana H29. The IgASE2 gene was 1,653 bp in length, contained a 786 bp ORF encoding a protein of 261 amino acids that shared 87% identity with Δ9 elongase, IgASE1, and possessed a 44 bp 5′-untranslated region (5′-UTR) and a 823 bp 3′-untranslated region (3′-UTR). IgASE2, by its heterologous expression in Saccharomyces cerevisiae, elongated linoleic acid (LA, 18:2n−6) and α-linolenic (ALA, 18:3n−3) to eicosadienoic acid (EDA, 20:2n−6) and eicosatrienoic acid (ETrA, 20:3n−3), respectively. The conversions of LA to EDA and ALA to ETrA were 57.6 and 56.1%, respectively. Co-expression of this elongase with Δ8 desaturase required for the synthesis of C20-polyunsaturated fatty acids resulted in the accumulation of dihomo-γ-linolenic acid (20:3n−6) from LA and eicosatetraenoic acid (20:4n−6) from ALA. These results demonstrated that IgASE2 exhibited C18-Δ9-PUFAs-specific elongase activity and the alternative Δ8 pathway was reconstituted.     

Molecular cloning,characterization and function analysis of the gene encoding HMG-CoA reductase from <Emphasis Type="Italic">Euphorbia Pekinensis</Emphasis> Rupr

A new full-length cDNA encoding 3-hydroxy-3-methylglutoryl-Coenzyme A reductase (HMGR; EC1.1.1.34), which catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway, was isolated from young leaves of Euphorbia Pekinensis Rupr. by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of HMGR (designated as EpHMGR, GenBank Accession NO. EF062569) was 2,200 bp containing a 1,752 bp ORF encoding 583 amino acids. Bioinformatic analyzes revealed that the deduced EpHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of EpHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that at most two copies of EpHMGR gene existed in E. Pekinensis genome. Tissue expression analysis revealed that EpHMGR expressed strongly in roots, weakly in stems and leaves. The functional colour complementation assay indicated that EpHMGR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that EpHMGR plays an influential role in isoprenoid biosynthesis.     

NADP+-dependent isocitrate dehydrogenase from a psychrophilic bacterium,Psychromonas marina

The gene encoding NADP⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) of a psychrophilic bacterium, Psychromonas marina, was cloned and sequenced. The open reading frame of the gene encoding IDH of P. marina (PmIDH) was 2229 bp in length and corresponded to a polypeptide composed of 742 amino acids. The molecular mass of IDH was calculated as 80,426 Da. The deduced amino acid sequence of PmIDH exhibited high degrees of homology with the monomeric IDH from other bacteria such as Colwellia maris (62% identity) and Azotobacter vinelandii (AvIDH) (64%). His-tagged PmIDH overexpressed in Escherichia coli cells was purified and characterized. The optimum temperature of PmIDH activity was about 35 °C; however, the enzyme lost 74% of the activity after incubation for 10 min at 30 °C, indicating that this enzyme is thermolabile. Chimeric enzymes produced through domain swapping between PmIDH and mesophilic AvIDH were constructed and their optimum temperatures and thermostability were determined. The results suggest that regions 2 and 3, especially region 3, of the two IDHs are involved in their catalytic activities and optimum temperature and thermostability for activity.

     

cDNA cloning of heat shock protein 90 gene and protein expression pattern in response to heavy metal exposure and thermal stress in planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

Heat shock protein 90 (HSP90) is an abundant and highly conserved molecular chaperone, playing important roles in multiple cellular stress responses. The full-length cDNA of planarian Dugesia japonica Hsp90 (designated DjHsp90) was firstly cloned using rapid amplification of cDNA ends (RACE) techniques. It is 2,354 bp, including an open reading frame (ORF) of 2,148 bp encoding a polypeptide of 715 amino acids with all five HSP90 family signatures. We sequenced the ORF sequences from genomic DNA, and found only one intron (48 bp) existed in Djhsp90 gene structure. We used western blot and immunohistochemistry to analyze the expression pattern of DjHsp90 in response to heavy metal exposure and thermal stress at the protein level. Our results show that low doses of heavy metals and elevated culture temperature induced, but high doses of heavy metals and severe heat shock inhibited DjHsp90 expression. In response to heavy metals and thermal stress, DjHsp90-positive cells only appeared in the parenchymal tissue under epidermis cells along the bilateral from head to tail. These positive cells are presumably sensor cells that can detect external environment changes. Our work provides basic data for the study of stress responses in planarians.     

Differential expression patterns of two delta‐9‐acyl‐CoA desaturases in Thitarodes pui (Lepidoptera: Hepialidae) during different seasons and cold exposure

Thitarodes pui larvae have a limited distribution in the Tibetan Plateau and are the host of a parasitic fungus, Ophiocordyceps sinensis. Low temperature is a main environmental stress. However, understanding of T. pui cold adaptation mechanisms is insufficient. Delta‐9‐acyl‐CoA desaturase (D9D) is closely correlated with cold adaptation for many organisms. To further understand the cold adaptation processes in T. pui larvae, two D9Ds, TpdesatA and TpdesatB were sequenced, and expression patterns were investigated during different seasons and cold exposure (under 0°C) in the laboratory. The full lengths of two cDNAs are 1,290 bp and 1,603 bp, and the ORFs encode a polypeptide of 348 and 359 amino acids, respectively. Four transmembrane domains, three conserved histidine residues and five hydrophobic regions exist in these two sequences. The expression level of TpdesatA is up‐regulated in the long‐term cold exposure and negatively correlated with temperature in seasonal patterns. TpdesatB responds to cold temperature in short‐term cold exposure and positively corresponds temporarily in seasonal expression. Two D9Ds may have different substrate specificities, TpdesatA tends to use C16:0 and C18:0 as substrate while TpdesatB prefers C18:0. In conclusion, TpdesatA may play a very important role in T. pui cold tolerance and TpdesatB regulates function in short‐term cold exposure and content change of fatty acids in the body.