Preparation of a novel metallothionein-AuNP composite material by genetic modification and AuS covalent combination.

Metallothionein (MTs) can be used in the prevention and treatment of tumors and diabetes due to its antioxidant properties. However, it is necessary to solve its non-transmembrane properties and further improve its antioxidant activity, increase its fluorescence visualization and enhance its stability to meet practical applications in the biomedical field. Here, we report the preparation of a novel metallothionein-AuNP composite material with high transmembrane ability, fluorescence visualization, antioxidant activity, and stability by genetic modification (introducing transduction peptide TAT, fluorescence tag GFP and increasing sulfydryl groups) and immobilization technology (covalently bonding with AuNPs). The transmembrane activity of modified proteins was verified by immunofluorescence. Increasing the sulfhydryl content within a certain range can enhance the antioxidant activity of the protein. In addition, GFP were used to further simplify the imaging of the metallothionein-AuNP composite in cells. XPS results indicated that AuNPs can immobilize metallothionein through AuS covalent bonds. TGA characterization and degradation experiments showed that thermal and degradation stability of the immobilized material was significantly improved. This work provides new ideas to construct metallothionein composites with high transmembrane ability, antioxidant activity, fluorescence visualization and stability to meet novel applications in the biomedical field.

Detoxification Gene Families at the Genome-Wide Level of Rhus Gall Aphid Schlechtendalia chinensis.

The Rhus gall aphid Schlechtendalia chinensis uses the species Rhus chinensis as its primary host plant, on which galls are produced. The galls have medicinal properties and can be used in various situations due to their high tannin content. Detoxification enzymes play significant roles in the insect lifecycle. In this study, we focused on five detoxification gene families, i.e., glutathione-S-transferase (GST), ABC transporter (ABC), Carboxylesterase (CCE), cyto-chrome P450 (CYP), and UDP-glycosyltransferase (UDP), and manually annotated 144 detoxification genes of S. chinensis using genome-wide techniques. The detoxification genes appeared mostly on chromosome 1, where a total of two pair genes were identified to show tandem duplications. There were 38 gene pairs between genomes of S. chinensis and Acyrthosiphon pisum in the detoxification gene families by collinear comparison. Ka/Ks ratios showed that detoxification genes of S. chinensis were mainly affected by purification selection during evolution. The gene expression numbers of P450s and ABCs by transcriptome sequencing data were greater, while gene expression of CCEs was the highest, suggesting they might be important in the detoxification process. Our study has firstly identified the genes of the different detoxification gene families in the S. chinensis genome, and then analyzed their general features and expression, demonstrating the importance of the detoxification genes in the aphid and providing new information for further research.

Genetically modified metallothionein/cellulose composite material as an efficient and environmentally friendly biosorbent for Cd2+ removal.

Metallothioneins (MTs) are a class of cysteine-rich metal-binding proteins. Cadmium (Cd) is one of the toxic heavy metal pollutants. In our previous research, the full-length cDNA of MT (Cd specificity) from freshwater crab (Sinopotamon henanense) (ShMT) was cloned and genetically modified to ShMT3 by site-directed mutagenesis to enhance the tolerance for Cd2+, however, it was limited in actual Cd2+ adsorption due to instability. Here, ShMT3-CBM, a novel recombinant fusion protein, was prepared. CBM is a carbohydrate binding module that can specifically bind cellulose while ShMT3 can effectively chelate Cd2+. The biosorbent Cellulose1-ShMT3-CBM was obtained by screening suitable cellulose materials. The selective adsorption experiments showed that Cellulose1-ShMT3-CBM had a preference for Cd2+. In low-concentration Cd2+ solutions, the removal efficiency was >99 %, and the adsorption equilibrium was reached within 15 min. The saturated adsorption capacity of Cellulose1-ShMT3-CBM for Cd2+ is 180.35 ± 4.67 mg/g (Dry Weight). Regeneration experiments showed that adsorption efficiency was maintained after six cycles. The MTT experiment showed that Cellulose1-ShMT3-CBM had low cytotoxicity. Meanwhile, Cellulose1-ShMT3-CBM can preferentially remove Cd2+ in actual water samples and boiler sewage. In this study, an environmentally friendly biosorbent which can adsorb Cd2+ efficiently and quickly was prepared for actual water treatment.

Genetic modifications of metallothionein enhance the tolerance and bioaccumulation of heavy metals in Escherichia coli.

Metallothioneins (MTs) are low molecular weight cysteine-rich proteins that bind to metals. Owing to their high cysteine (Cys) content, MTs are effective mediators of heavy metal detoxification. To enhance the heavy metal binding ability of MT from the freshwater crab Sinopotamon henanense (ShMT), sequence-based multiple sequence alignment (MSA) and structure-based molecular docking simulation (MDS) were conducted in order to identify amino acid residues that could be mutated to bolster such metal-binding activity. Site-directed mutagenesis was then used to modify the primary structure of ShMT, and the recombinant proteins were further enhanced using the SUMO fusion expression system to yield SUMO-ShMT1, SUMO-ShMT2, and SUMO-ShMT3 harboring one-, two-, and three- point mutations, respectively. The resultant modified proteins were primarily expressed in a soluble form and exhibited the ability to readily bind to heavy metals. Importantly, these modified proteins exhibited significantly enhanced heavy metal binding capacities, and they improved Cd2+, Cu2+ and Zn2+ tolerance and bioaccumulation in Escherichia coli (E. coli) in a manner dependent upon the number of introduced point mutations (SUMO-ShMT3 > SUMO-ShMT2 > SUMO-ShMT1 > SUMO-ShMT > control). Indeed, E. coli cells harboring the pET28a-SUMO-ShMT3 expression vector exhibited maximal Cd2+, Cu2+, and Zn2+ bioaccumulation that was increased by 1.86 ± 0.02-, 1.71 ± 0.03-, and 2.13 ± 0.02-fold relative to that in E. coli harboring the pET28a-SUMO-ShMT vector. The present study offers a basis for the preparation of genetically engineered bacteria that are better able to bioaccumulate and tolerate heavy metals, thus providing a foundation for biological heavy metal water pollution treatment.

[Cloning,expression and characterization of chalcone isomerase from medicinal plant Chinese sumac (Rhus chinensis)].

Flavonoids are a group of secondary metabolites found in plants. They have many pharmacological functions and play an important role in Chinese sumac( Rhus chinensis),which is a well-known traditional Chinese medicinal plant. Chalcone isomerase( CHI,EC 5. 5. 1. 6) is one of the key enzymes in the flavonoids biosynthesis pathway. In this paper,the full-length c DNA sequence encoding the chalcone isomerase from R. chinensis( designated as Rc CHI) was cloned by RT-PCR and rapid-amplification of c DNA Ends( RACE). The Rc CHI c DNA sequence was 1 058 bp and the open reading frame( ORF) was 738 bp. The ORF predicted to encode a 245-amino acid polypeptide. Rc CHI gene contained an intron and two exons. The sequence alignments revealed Rc CHI shared47. 1%-71. 6% identity with the homologues in other plants. Real-time PCR analysis showed that the total flavonoid levels were positively correlated with tissue-specific expressions of Rc CHI mRNA in different tissues. The recombinant protein was successfully expressed in an Escherichia coli strain with the p GEX-6 P-1 vector. In this paper,the CHI gene was cloned and characterized in the family of Anacardiaceae and will help us to obtain better knowledge of the flavonoids biosynthesis of the flavonoid compounds in R. chinensis.

Tandem oligomeric expression of metallothionein enhance heavy metal tolerance and bioaccumulation in Escherichia coli.

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins, which play important roles in metal homeostasis and heavy metal detoxification. In our previous study, a novel full length MT cDNA was successfully cloned from the freshwater crab (Sinopotamon henanense). In the present study, tandem repeats of two and three copies of the crab MT gene were integrated by overlap extension PCR (SOE-PCR) and expressed in Escherichia coli. The SUMO fusion expression system was adopted to increase the stability and solubility of the recombinant MT proteins. The recombinant proteins were purified and their metal-binding abilities were further analyzed by the ultraviolet absorption spectral scan. Furthermore, the metal tolerance and bioaccumulation of E. coli cells expressing oligomeric MTs were determined. Results showed that the recombinant plasmids pET28a-SUMO-2MT and pET28a-SUMO-3MT were successfully constructed. SDS-PAGE analysis showed that the SUMO-2MT and SUMO-3MT were expressed mainly in the soluble forms. Oligomeric MTs expression significantly enhanced Cu, Cd or Zn tolerance and accumulation in E. coli in the order: SUMO-3MT˃SUMO-2MT˃SUMO-MT˃control. Cells harboring pET28a-SUMO -3MT exhibited the highest Cu, Cd or Zn bioaccumulation at 5.8-fold, 3.1-fold or 6.7-fold higher than that of the control cells. Our research could lay a foundation for large-scale preparation of MTs and provide a scientific basis for bioremediation of heavy metal pollution by oligomeric MTs.

Historical biogeography of Eastern Asian-Eastern North American disjunct Melaphidina aphids (Hemiptera: Aphididae: Eriosomatinae) on Rhus hosts (Anacardiaceae).

Intercontinental biotic disjunctions have been documented and analyzed in numerous Holarctic taxa. Patterns previously synthesized for animals compared to plants suggest that the timing of animal disjunctions are mostly Early Tertiary and were generated by migration and vicariance events occurring in the North Atlantic, while plant disjunctions are mostly Mid-Late Tertiary and imply migration and vicariance over Beringia. Melaphidina aphids (Hemiptera: Aphididae: Fordini) exhibit host-alternating life cycles comprising an obligate seasonal shift between Rhus subgenus Rhus species (Anacardiaceae) and mosses (Bryophyta). Similar to their Rhus hosts, melaphidines are distributed disjunctly between Eastern Asia and Eastern North America. We examined evolutionary relationships within Melaphidina to determine the position of the North American lineage, date its divergence from Asian relatives, and compare these results to a previous historical biogeographic study of Rhus. We sampled nine species and three subspecies representing all six genera of Melaphidina. Data included sequences of mitochondrial cytochrome c oxidase subunits I and II+leucine tRNA, cytochrome b, and nuclear elongation factor 1α genes. Phylogenetic analyses (Bayesian, maximum-likelihood, parsimony) of the combined data (3282 bp) supported the monophyly of all genera except Nurudea and Schlechtendalia, due to the position of N. ibofushi. While the exact position of the North American Melaphis was not well resolved, there was high support for a derived position within Asian taxa. The divergence of Melaphis from Asian relatives centered on the Eocene-Oligocene boundary (~33-35Ma), which coincides with closure of Beringian Land Bridge I. This also corresponded to the Asian-North American disjunction previously estimated for subgenus Rhus spp. We suggest the late-Eocene Bering Land Bridge as the most likely migration route for Melaphis ancestors, as was also hypothesized for North American Rhus ancestors. Results for the Melaphidina disjunction depart from the modal pattern in animal lineages, and present a case where insect and host-plant taxa apparently responded similarly to Tertiary climate change.

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. 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 Phe(126) 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.

High altitude adaptation of the schizothoracine fishes (Cyprinidae) revealed by the mitochondrial genome analyses.

The schizothoracine fishes, also known as "mountain carps" are widely distributed in the Qinghai-Tibetan Plateau and its peripheral regions. Although they provide a prime example of high altitude adaptation, the phylogenetic relationships and the divergence times among these carp lineages are still controversial. Moreover, the genetic basis for high altitude adaptation is also poorly understood. In this study, we determined the mitochondrial genomes from two species of the schizothoracine fishes, representing a "morphologically primitive" clade and "morphologically specialized" clade, respectively. The phylogenetic tree and the divergence times were estimated within the evolutionary framework of the entire order Cypriniformes. Our results indicate a polyphylyetic relationship of the schizothoracine fishes and suggest two independent migration events into the Qinghai-Tibetan Plateau: one by the "morphologically primitive" clade in the Late Miocene and another by the "morphologically specialized" clade in the Eocene. Rapid speciation events of each clade from the Late Miocene to the Pliocene correspond to the timing of the geologic acceleration of the Qinghai-Tibetan Plateau. Interestingly, we found evidence for positive selection acting on the protein coding genes in the mitochondrial genomes of the "morphologically specialized" clade, implying a possible genetic basis for high altitude adaptation in this derived lineage of cypriniform fishes.

A molecular phylogeny of Oxya (Orthoptera: Acridoidea) in China inferred from partial cytochrome b gene sequences.

The grasshoppers of the genus Oxya are well known to damage rice, sugar cane, and other crops, yet their phylogenetic relationships have not been examined with molecular data. In this study, we obtained the 432 bp DNA sequences of the mitochondrial cytochrome b gene from 91 individuals of nine Oxya species and two outgroups (Gesonula punctifrons and Acrida cinerea). Phylogenetic analyses for the molecular data set were then carried out using the maximum parsimony and neighbor-joining methods. The results showed that the nine Oxya species form four well-supported clades, which include (1) O. intricata and O. flavefemura; (2) O. japonica and O. bicingula; (3) O. agavisa; and (4) O. chinensis, O. brachyptera, O. adentata, and O. hainanensis, respectively. In particular, the monophyly of O. hainanensis and O. agavisa is strongly supported, respectively. However, O. flavefemura and O. intricata, O. bicingula, and O. japonica form paraphyletic groups, respectively, and O. chinensis, O. adentata, and O. brachyptera form a polyphyletic group, suggesting that they should be merged as few as three species.

Chromosome aberration assays for the study of cyclophosphamide and Bacillus thuringiensis in Oxya chinensis (Orthoptera: Acrididae).

Chromosome aberrations induced by an anti-neoplastic drug, cyclophosphamide (CP) and a bioinsecticide, Bacillus thuringiensis (B.t.) were examined using grasshoppers as an animal model, with injection as the route of exposure. Oxya chinensis (Thunberg), having a small number (2n male symbol =23) of large-sized chromosomes in males, was used for this purpose. The fifth instar nymphs were treated with various concentrations of CP (2, 5 and 10 mg/ml) and B.t. (0.55, 1.83 and 5.50 IU/ml) by injection into the abdomen, using physiological saline and distilled water as negative controls, respectively. The chromosomal preparations were made from the spermatogonia of the specimen testis at different intervals after dosing (24 and 48 h). The effect of the high dose of CP (10 mg/ml) in O. chinensis was also analyzed at the 42-h time point. The chromosome aberrations observed were mainly chromatid and chromosome breaks. CP induced a dose- and time-dependents increase in the number of chromosome aberrations (CAs) per cell and in the percentage of aberrant cells. The strongest effect was seen when grasshoppers were injected with the highest dose and cells were analyzed at the 48-h time point. The results show that CP induced a significant increase in the frequency of CAs in testicular cells of O. chinensis with the three doses employed, compared to the negative control. Our results suggest that there exists in the grasshopper an enzyme system analogous to liver-S9 fraction, and that CP may be used as a positive control in genotoxicity test in this species. In addition, the evaluation of the chromosome aberrations induced by B.t. in the grasshoppers' testicular cells showed that B.t. may induce chromosome aberrations, mainly chromatid and chromosome breaks, in spermatogonia. By statistical analysis, B.t. showed significant dose-effect relationships and it may be mutagenic in this species. Recent research has focused on the development of biological insecticides to protect cereal crops against damage by insect species, such as beetles and grasshoppers. The present studies may contribute to our knowledge of entomological genotoxicity in grasshoppers and provide reference for the research on the mechanism of B.t. toxicity.