Molecular Cloning, Transcriptional Profiling, Subcellular Localization, and miRNA-Binding Site Analysis of Six SCL9 Genes in Poplar

The SCL9 subfamily is a key member of the GRAS family that regulates plant development and stress responses. Nevertheless, the functional role of these genes in the growth and development of poplar still unclear. Here, we reported the six SCL9 genes, which were found to be differentially expressed during poplar adventitious root formation. The full-length sequences of PeSCL9 genes of ‘Nanlin895’ poplar (Populus deltoids × Populus euramericana) were cloned by the RACE technique All PeSCL9 genes lacked introns. RT-qPCR revealed that PeSCL9 genes displayed a dynamic expression pattern in the adventitious root of poplar, according to RT-qPCR data. A series of comprehensive genes characteristics analysis were carried out for six genes by bioinformation. Meanwhile, transient expression analysis of the Populus protoplasts showed that all the PeSCL9 proteins were localized in the nucleus. In addition, the degradome and sRNA of ‘Nanlin895’ poplar in combination were used to predict miRNAs that regulate PeSCL9. It was found that miR396a and miR396c may affect PeSCL9 expression via cleavage, which was further verified by a transient expression experiment in Populus protoplasts. Overall, the development of poplar adventitious root and other tissues was closely related to these six SCL9 genes, and they serve as a starting point for further research into the mechanisms regulating poplar growth and development.

Identification and in silico structural and functional analysis of a trypsin-like protease from shrimp Macrobrachium carcinus

Macrobrachium carcinus (Linnaeus, 1758) is a species of freshwater shrimp widely distributed from Florida southwards to southern Brazil, including southeast of Mexico. In the present work, we identified a putative trypsin-like protease cDNA fragment of 736 nucleotides from M. carcinus hepatopancreas tissue by the 3′RACE technique and compared the deduced amino acid sequence to other trypsin-related proteases to describe its structure and function relationship. The bioinformatics analyses showed that the deduced amino acid sequence likely corresponds to a trypsin-like protease closely related to brachyurins, which comprise a subset of serine proteases with collagenolytic activity found in crabs and other crustacea. The M. carcinus trypsin-like protease sequence showed a global sequence identity of 94% with an unpublished trypsin from Macrobrachium rosenbergii (GenBank accession no. AMQ98968), and only 57% with Penaeus vannamei trypsin (GenBank accession no. CAA60129). A detailed analysis of the amino acid sequence revealed specific differences with crustacean trypsins, such as the sequence motif at the beginning of the mature protein, activation mechanism of the corresponding zymogen, amino acid residues of the catalytic triad and residues responsible for substrate specificity.

Description of a Zostera marina catalase gene involved in responses to temperature stress

Catalase (CAT) is an antioxidant enzyme that plays a significant role in cellular protection against oxidative damage by degradation of hydrogen peroxide to oxygen and water. In the present study, the complete CAT cDNA sequence of Zostera marina was identified through expressed sequence tags (EST) analysis and the rapid amplification of cDNA ends (RACE) technique. The nucleotide sequence of ZmCAT cDNA consisted of 1,816 bp with a 1,434 bp open reading frame (ORF), encoding a polypeptide of 477 amino acid residues, which possessed significant homology to other known plant CATs. The molecular mass of the predicted protein was 55.3 kDa with an estimated isoelectric point of 6.40. Phylogenetic analysis showed that ZmCAT was closely related to CAT from gramineous species. In response to temperature stress, H2O2 and MDA contents in Z. marina increased significantly with cold stress (<10 °C) and heat stress (>25 °C). ZmCAT expression was significantly upregulated at temperatures from 5 to 10 °C and then gradually downregulated, reaching its lowest expression at 30 °C. Recombinant ZmCAT protein exhibited strong antioxidant activity over a wide temperature range, with the highest rZmCAT activity observed at 25 °C and a higher relative activity retained even with heat stress. All these results indicated that ZmCAT was a member of the plant CAT family and involved in minimizing oxidative damage effects in Z. marina under temperature stress.

cDNA cloning and functional characterisation of four antimicrobial peptides from Paa spinosa

Antimicrobial peptides (AMPs) are small peptides found in many organisms defending themselves against pathogens. AMPs form the first line of host defence against pathogenic infections and are key components of the innate immune system of amphibians. In the current study, cDNAs of precursors of four novel antimicrobial peptides in the skin of Paa spinosa were cloned and sequenced using the 3′-RACE technique. Mature peptides, named spinosan A–D, encoded by the cDNAs were chemically synthesized and their chemical properties were predicted. The antimicrobial, antioxidative, cyotoxic and haemolytic activities of these four AMPs were determined. While the synthesised spinosans A–C exhibited no activity towards any of the bacterial strains tested, spinosan-D exhibited weak but broad-spectrum antimicrobial activities against Gram-positive and Gram-negative bacteria. All peptides were weakly haemolytic towards rabbit erythrocytes, had a strong antioxidative activity, and a low cytotoxic activity against HeLa cells. These findings provide helpful insights that may be useful in the future design of anti-infective peptide agents.

A New 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase Gene Encoding the Committed-Step Enzyme in the MEP Pathway from Rauvolfia verticillata

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes a committed step of the methylerythritol phosphate (MEP) pathway for the biosynthesis of pharmaceutical terpenoid indole alkaloid (TIA) precursors. The full-length cDNA sequence was cloned and characterized from a TIA-producing species, Rauvolfia verticillata, using rapid amplification of cDNA ends (RACE) technique. The new cDNA was named as RvDXR and submitted to GenBank® to be assigned with an accession number (DQ779286). The fulllength cDNA of RvDXR was 1804 bp containing a 1425 bp open reading frame (ORF) encoding a polypeptide of 474 amino acids with a calculated molecular mass of 51.3 kDa and an isoelectric point of 5.88. Comparative and bioinformatic analyses revealed that RvDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPHbinding motif in its N-terminal region owned by all plant DXRs. The phylogenetic analysis revealed that DXRs had two groups including a plant and bacterial group; RvDXR belonged to angiosperm DXRs that were obtained from Synechocystis through gene transfer according to the phylogenetic analysis. The structural modeling of RvDXR showed that RvDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that RvDXR expressed in all tissues including roots, stems, leaves, fruits and followers but at different levels. The lowest transcription level was observed in followers and the highest transcription was found in fruits of R. verticillata; the transcription level of RvDXR was a little higher in roots and stems than in leaves. The cloning and characterization of RvDXR will be helpful to understand more about the role of DXR involved in R. verticillata TIA biosynthesis at the molecular level and provides a candidate gene for metabolic engineering of the TIAs pathway in R. verticillata.

Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase

Phosphate-activated glutaminase (GA) is overexpressed in certain types of tumour but its exact role in tumour cell growth and proliferation is unknown. Here we describe the isolation of a full-length cDNA clone of human breast cancer ZR75 cells, by a combination of λgt10 cDNA library screening and the rapid amplification of cDNA ends (‘RACE’) technique. The cDNA of human GA is 2408 nt with a 1806-base open reading frame encoding a 602-residue protein with a predicted molecular mass of 66309 Da. The deduced amino acid sequence contains a putative mitochondrial import presequence of 14 residues at the N-terminal end. Heterologous expression and purification in Escherichia coli yielded a product of the expected molecular size that was recognized by using antibodies against the recombinant human GA. Sequence analyses showed that human GA was highly similar to the rat liver enzyme. Northern gel analysis revealed that the gene is present in human liver, brain and pancreas, in which a major transcript of 2.4 kb was demonstrated, but not in kidney, heart, skeletal muscle, lung or placenta. These results strongly suggest that the first human GA cloned, the GA from ZR-75 breast cancer cells, and presumably those from human liver and brain, are liver-type isoenzymes, in sharp contrast with the present view that considers the kidney type as the isoform expressed in all tissues with GA activity, with the exception of postnatal liver.