Bemisia tabaci (Gennadius)’de İki Nikotinik Asetilkolin Reseptör Genin Klonlanması

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect nervous system. Neonicotinoid insecticides exhibit insecticidal activities by targeting these receptors. The aim of this study was to isolate cDNA clones of nAChR subunit genes of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). RACEs (Rapid Amplification of cDNA Ends) were applied to obtain partial-length cDNA sequences. We identified two partial cDNA clones encoding β1 and α8 subunit genes (Btβ1-1168 bp and Btα8-755 bp), respectively, from Bemisia tabaci (Bt). This is the first report of isolation of α8 from B. tabaci by cloning. Btβ1 and Btα8 possess characteristics that are typical of nAChR subunits. Phylogenetic analysis showed that Btβ1 and Btα8 clustered with the orthologous genes of other insect species.

A Highly Sensitive and Label-Free Microbead-Based ‘Turn-On’ Colorimetric Sensor for the Detection of Mercury(II) in Urine Using a Peroxidase-Like Split G-Quadruplex–Hemin DNAzyme

A highly sensitive and label-free microbead-based ‘turn-on’ assay was developed for the detection of Hg2+ in urine based on the Hg2+-mediated formation of intermolecular split G-quadruplex–hemin DNAzymes. In the presence of Hg2+, T–T mismatches between the two partial cDNA strands were stabilized by a T–Hg2+–T base pair, and can cause the G-rich sequences of the two oligonucleotides to associate to form a split G-quadruplex which is able to bind hemin to form the catalytically active G-quadruplex–hemin DNAzyme. This microbead-based ‘turn-on’ process allows the detection of Hg2+ in urine samples at concentrations as low as 0.5 pM. The relative standard deviation and recovery are 1.2–3.9 and 98.7–103.2%, respectively. The remarkable sensitivity for Hg2+ is mainly attributed to the enhanced mass transport ability that is inherent in homogeneous microbead-based assays. Compared with previous developments of intermolecular split G-quardruplex–hemin DNAzymes for the homogeneous detection of Hg2+ (the limit of detection was 19nM), a signal enhancement of ~1000 times is obtained when such an assay is performed on the surface of microbeads.

Expression of the histone chaperone SET/TAF-Iβ during the strobilation process of Mesocestoides corti (Platyhelminthes, Cestoda)

SUMMARYThe histone chaperone SET/TAF-Iβ is implicated in processes of chromatin remodelling and gene expression regulation. It has been associated with the control of developmental processes, but little is known about its function in helminth parasites. In Mesocestoides corti, a partial cDNA sequence related to SET/TAF-Iβ was isolated in a screening for genes differentially expressed in larvae (tetrathyridia) and adult worms. Here, the full-length coding sequence of the M. corti SET/TAF-Iβ gene was analysed and the encoded protein (McSET/TAF) was compared with orthologous sequences, showing that McSET/TAF can be regarded as a SET/TAF-Iβ family member, with a typical nucleosome-assembly protein (NAP) domain and an acidic tail. The expression patterns of the McSET/TAF gene and protein were investigated during the strobilation process by RT-qPCR, using a set of five reference genes, and by immunoblot and immunofluorescence, using monospecific polyclonal antibodies. A gradual increase in McSET/TAF transcripts and McSET/TAF protein was observed upon development induction by trypsin, demonstrating McSET/TAF differential expression during strobilation. These results provided the first evidence for the involvement of a protein from the NAP family of epigenetic effectors in the regulation of cestode development.

Cloning and expression of apyrase gene from Ancylostoma caninum in Escherechia coli

Apyrase encoding metal-ions activated plasma membrane protease is present in animal and plant tissues. This enzyme can hydrolyze ADP and ATP pyrophosphate bond, resulting in AMP and free phosphate groups, and plays an important role for insects and parasites to evade host immune system. However localization and function of apyrase in the canine hookworm, Ancylostoma caninum, remains unknown. To analyze apyrase gene in A. caninum (a eukaryotic parasitic hookworm), a pair of primers was designed according to the previous EST data. The full-length cDNA of apyrase gene was amplified from A. caninum by RT-PCR. The partial cDNA of apyrase encodes 249 amino acid protein was expressed in Escherechia coli. The recombinant protein was induced to express under proper conditions and the molecular size was as expected. The recombinant protein was purified. The transcripts of apyrase in different stages of A. caninum were analyzed by the Real-time PCR assay, and Immuno-localization assays were used to research the protein expression in different stages of A. caninum

Lack of evidence for the presence of an interferon in invertebrate

In vertebrates, the interferon (IFN) response is the primary form of innate antiviral defense. Previously (2005), a partial cDNA which could encode an interferon-like protein (IntlP) is reported in shrimp, later Rosa et al. (2008) argue that this partial cDNA should encode a portion of insect mitochondrial ATP synthase (MAS) B-chain. Recently (2009), it is demonstrated IntlP also possess antibacterial activity beside antiviral activity reported before. Lacking of a consensus opinion to the question of whether this gene encodes IntlP or MAS, we try to provide more evidences to identify this gene exactly. Here we obtain the full length cDNAs of IntlP/ MAS in Litopenaeus vannamei, and perform the tissue distribution and induced expression analysis. Our results confirm that IntlP is coded by a mistaken ORF and the actual protein indeed is a L. vannamei mitochondrial ATP synthase (LvMAS) whose function is unknown in antiviral responses.

Molecular Cloning and Characterization of PI3K-p85α Gene from Inner Mongolia Cashmere Goat (Capra hircus)

PI3K, phosphatidylinositol-3-kinase, is a specific lipid kinase family. It is associated with cell proliferation and survival..Class-IA PI3K is heterodimers composed of catalytic subunit (p110) and regulatory subunit (p85). p85α is the most abundant regulatory subunit of PI3K family. PI3K-p85α partial cDNA was amplified by RT-PCR from Inner Mongolia Cashmere Goat (Capra hircus) and sequenced. The molecular characterization of the PI3K-p85α gene was described. The amplified fragment is 1152bp in length, corresponding to a polypeptide of 384 amino acids residues with 45.4 kDa predicted molecular mass. The cDNA nucleotide sequence has 98% identity with regulatory subunit alpha (PIK3R1) of bovine and 93% identity with human. The goat PI3K-p85α patial cDNA is cloned.