Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

As an activator of adenylate cyclase, the neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) impacts levels of cyclic AMP, a key second messenger available in brain cells. PACAP is involved in certain adult behaviors. To elucidate PACAP interactions, a compendium of microarrays representing mRNA expression in the adult mouse whole brain was pooled from the Phenogen database for analysis. A regulatory network was computed based on mutual information between gene pairs using gene expression data across the compendium. Clusters among genes directly linked to PACAP, and probable interactions between corresponding proteins were computed. Database “experts” affirmed some of the inferred relationships. The findings suggest ADCY7 is probably the adenylate cyclase isoform most relevant to PACAP's action. They also support intervening roles for kinases including GSK3B, PI 3-kinase, SGK3 and AMPK. Other high-confidence interactions are hypothesized for future testing. This new information has implications for certain behavioral and other disorders.     

Molecular cloning and expression in vitro of a carboxylesterase gene from the Glanville fritillary butterfly (Melitaea cinxia)

Carboxylesterase (EC 3.1.1.1) is a member of the carboxyl/cholinesterase (CCE) superfamily, which is widely distributed in animals, plants and microorganisms. This enzyme has been known to be associated with insecticide resistance and detoxification. Although CCEs have been extensively studied in insects, including lepidopterans, the research on butterflies, a major subgroup in Lepidoptera, is still poor. In the present study, we cloned a CCE gene (McCCE1) from the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The full-length cDNA encoding McCCE1 was 1786 bp, containing a 1641 bp open reading frame encoding 546 amino acids, a 38 bp 5′-untranslated region (5′-UTR), and a 107 bp 3′-UTR with a poly(A) tail. The functionally conserved amino acids in McCCE1 shared the 55% identity with the cytoplasmic esterase CCE017a in Helicoverpa armigera (Lepidoptera: Noctuidae), which has been associated with detoxification. Assays in vitro showed that the recombinant McCCE1 could hydrolyze α- and β-naphthyl acetate. Thus, the present study adds to the body of knowledge concerning the detoxification of pesticides by lepidopterans.     

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from ‘Wuzishatangju’ (self-incompatible, SI) and ‘Shatangju’ (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between ‘Wuzishatangju’ and ‘Shatangju’, respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of ‘Wuzishatangju’ and ‘Shatangju’. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of ‘Shatangju’ was approximately 10-fold higher than in anthers of ‘Wuzishatangju’. The highest expression level of CrUBE1 was detected in pistils at 7 days after self-pollination of ‘Wuzishatangju’, which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from ‘Wuzishatangju’ and ‘Shatangju’ were successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was significantly inhibited with increasing of CrUBE1 protein concentrations from ‘Wuzishatangju’.     

Molecular cloning,characterization and expression of myoglobin in Tibetan antelope (Pantholops hodgsonii), a species with hypoxic tolerance

The Tibetan antelope (Pantholops hodgsonii) is a hypoxia-tolerant species that lives at an altitude of 4000–5000 m above sea level on the Qinghai–Tibetan plateau. Myoglobin is an oxygen-binding cytoplasmic hemoprotein that is abundantly expressed in oxidative skeletal and cardiac myocytes. Numerous studies have implicated that hypoxia regulates myoglobin expression to allow adaptation to conditions of hypoxic stress. Few studies have yet looked at the effect of myoglobin on the adaptation to severe environmental stress on TA. To investigate how the Tibetan antelope (TA) has adapted to a high altitude environment at the molecular level, we cloned and analyzed the myoglobin gene from TA, compared the expression of myoglobin mRNA and protein in cardiac and skeletal muscle between TA and low altitude sheep. The results indicated that the full-length myoglobin cDNA is composed of 1154 bp with a 111 bp 5′ untranslated region (UTR), a 578 bp 3′ UTR and a 465 bp open reading frame (ORF) encoding a polypeptide of 154 amino acid residues with a predicted molecular weight of 17.05 kD. The TA myoglobin cDNA sequence and the deduced amino acid sequence were highly homologous with that of other species. When comparing the myoglobin sequence from TA with the Ovis aries myoglobin sequence, variations were observed at codons 21 (GGT → GAT) and 78 (GAA → AAG), and these variations lead to changes in the corresponding amino acids, i.e., Gly → Asp and Glu → Lys, respectively. But these amino acid substitutions are unlikely to effect the ability of binding oxygen because their location is less important, which is revealed by the secondary structure and 3D structure of TA myoglobin elaborated by homology modeling. However, the results of myoglobin expression in cardiac and skeletal muscles showed that they were both significantly higher than that in plain sheep not only in mRNA but also protein level. We speculated that the higher expression of myoglobin in TA cardiac and skeletal muscles improves their ability to obtain and store oxygen under hypoxic conditions. This study indicated that TA didn't improve the ability of carrying oxygen by changing the molecular structure of myoglobin, but through increasing the expression of myoglobin in cardiac and skeletal muscles.     

Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica

The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response. In this study, the MiRab5 gene was isolated from stress-induced Mangifera indica. The full-length cDNA sequence was 984 bp and contained an open reading frame of 600 bp, which encoded a 200 amino acid protein with a molecular weight of 21.83 kDa and a theoretical isoelectric point of 6.99. The deduced amino acid sequence exhibited high homology with tomato (91% similarity) and contains all five characteristic Rab motifs. Real-time quantitative RT-PCR analysis demonstrated that MiRab5 was ubiquitously expressed in various mango tree tissues at different levels. The expression of MiRab5 was up-regulated during later stages of fruit ripening. Moreover, MiRab5 was generally up-regulated in response to various abiotic stresses (cold, salinity, and PEG treatments). Recombinant MiRab5 protein was successfully expressed and purified. SDS-PAGE and western blot analysis indicated that the expressed protein was recognized by the anti-6-His antibody. These results provide insights into the role of the MiRab5 gene family in fruit ripening and stress responses in the mango plant.     

Molecular cloning,characterization and expression analysis of ATG1 in the silkworm, Bombyx mori

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process.     

Molecular cloning and functional analysis of two FAD2 genes from American grape (Vitis labrusca L.)

The synthesis of polyunsaturated fatty acids (PUFAs), the most abundant fatty acids in plants, begins with a reaction catalyzed by fatty acid desaturase 2 (FAD2; EC 1.3.1.35), also called microsomal oleate Δ12-desaturase. Since the FAD2 gene was first identified in Arabidopsis thaliana, FAD2 research has gained wide interest as the essential enzyme for synthesizing PUFA. Grapes are one of the most frequently cultivated fruits in the world, with most commercial growers cultivating Vitis vinifera and V. labrusca. Grapeseed oil contains a high proportion, 60–70% of linoleic acid (18:2). We cloned two putative FAD2 genes from V. labrusca cv. Campbell Early based on V. vinifera genome sequences. Deduced amino acid sequences of two putative genes showed that VlFAD2s show high similarity to Arabidopsis FAD2 and commonly contain six transmembrane domain, three histidine boxes and endoplasmic reticulum (ER) retrieval motif representing the characteristics of fatty acid desaturase. Phylogenetic analyses of various plant FAD2s showed that VlFAD2-1 and VlFAD2-2 are separately grouped with constitutive and seed-type FAD2s, respectively. Southern blot showed that one or two bands are found in each lane. Because Campbell Early is a hybrid cultivar, FAD2-1 and FAD2-2 genes may exist as one copy in V. labrusca. Expression analysis in different tissues indicated that VlFAD2-1 is a constitutive gene but VlFAD2-2 is a seed-type gene. Complementation experiments of fad2-1 mutant Arabidopsis with VlFAD2-1 or VlFAD2-2 demonstrated that VlFAD2-1 and VlFAD2-2 can restore low PUFA proportion of fad2 to normal PUFA proportion.     

Molecular and expression characterization of a nanos1 homologue in Chinese sturgeon, Acipenser sinensis

The nanos gene family was essential for germ line development in diverse organisms. In the present study, the full-length cDNA of a nanos1 homologue in A. sinensis, Asnanos1, was isolated and characterized. The cDNA sequence of Asnanos1 was 1489 base pairs (bp) in length and encoded a peptide of 228 amino acid residues. Multiple sequence alignment showed that the zinc-finger motifs of Nanos1 were highly conserved in vertebrates. By RT-PCR analysis, Asnanos1 mRNAs were ubiquitously detected in all tissues examined except for the fat, including liver, spleen, heart, ovary, kidney, muscle, intestines, pituitary, hypothalamus, telencephalon, midbrain, cerebellum, and medulla oblongata. Moreover, a specific polyclonal antibody was prepared from the in vitro expressed partial AsNanos1 protein. Western blot analysis revealed that the tissue expression pattern of AsNanos1 was not completely coincided with that of its mRNAs, which was not found in fat, muscle and intestines. Additionally, by immunofluoresence localization, it was observed that AsNanos1 protein was in the cytoplasm of primary oocytes and spermatocytes. The presented results indicated that the expression pattern of Asnanos1 was differential conservation and divergence among diverse species.     

Molecular cloning and catalytic characterization of a recombinant tropine biosynthetic tropinone reductase from Withania coagulans leaf

Tropinone reductases (TRs) are small proteins belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes. TR-I and TR-II catalyze the conversion of tropinone into tropane alcohols (tropine and pseudotropine, respectively). The steps are intermediary enroute to biosynthesis of tropane esters of medicinal importance, hyoscyamine/scopolamine, and calystegins, respectively. Biosynthesis of tropane alkaloids has been proposed to occur in roots. However, in the present report, a tropine forming tropinone reductase (TR-I) cDNA was isolated from the aerial tissue (leaf) of a medicinal plant, Withania coagulans. The ORF was deduced to encode a polypeptide of 29.34 kDa. The complete cDNA (WcTRI) was expressed in E. coli and the recombinant His-tagged protein was purified for functional characterization. The enzyme had a narrow pH range of substantial activity with maxima at 6.6. Relatively superior thermostability of the enzyme (30% retention of activity at 60 °C) was catalytic novelty in consonance with the desert area restricted habitat of the plant. The in vitro reaction kinetics predominantly favoured the forward reaction. The enzyme had wide substrate specificity but did not cover the substrates of other well-known plant SDR related to menthol metabolism. To our knowledge, this pertains to be the first report on any gene and enzyme of secondary metabolism from the commercially and medicinally important vegetable rennet species.     

Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.