Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosiue agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate （LK-4） was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as ＂Streptomyces espinosus strain LK4 （KF806735）＂. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinasc enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.     

Isolation and Identification of Canine Parvovirus Serotype 2a and Its VP2 Protein Expression in Transgenic Tobacco

A strain of canine parvovirus (CPV) was isolated from feces of an ill puppy in an animal hospital in Wuhan, China. It was designated as CPV/WH02/06. This isolate was identified as serotype CPV-2a by the hemagglutination test, CPV Ag detection strip, electron microscopy, and PCR. The vp2 gene was cloned and sequenced and assigned GenBank accession number EU377537. A 1242 bp segment of the 5' region of the vp2 gene was cloned and inserted into the binary vector pBI121 and used for Agrobacterium-mediated tobacco transformation. Transgenic tobacco plants were selected on MS medium supplemented with 100 μg/mL kanamycin and 100 μg/mL timentin. Integration of the vp2 gene into the tobacco genome was confirmed by PCR using T1 progeny plants, and the expression of the VP2 protein was confirmed by Western blotting.     

Phylogeny of Chinese Allium (Liliaceae) using PCR-RFLP analysis

Eighteen representative species were selected from all the nine sections of Chinese Allium on the basis of the classification of morphology and cytotaxonomy. The trnK and rpL16 gene fragments of chloroplast DNA were amplified from 18 species by PCR method. The two cpDNA fragments were digested by 26 restriction enzymes, and 303 polymorphic restriction sites were found, of which 163 were informative. The restriction site data were analyzed with PAUP (version 3.1.1) and MEGA (version 1.01) as well as PHYLIP. As a result, the genus Allium could be classified into six subgenera. The recognition of Sect. Anguinum in the Flora of China is reasonable, Sect. Rhizirideum, Sect. Haplostemon and Sect. Cepa are not monophyletic. The infrageneric system of this genus was also discussed.     

Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The resuits showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfeetion efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatoeytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.     

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi （Brassica campestris L. ssp. chinensis Makino）

For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only fulllength cDNA from the differential fragment BcMF-A 18T 16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and largesized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed ofa 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2gene on microspore development is discussed in the present paper.     

Protein trans-splicing based dual-vector delivery of the coagulation factor Ⅷ gene

A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.     

Effects of different land use patterns on nifH genetic diversity of soil nitrogen-fixing microbial communities in Leymus Chinensis steppe

Biological nitrogen fixation through prokaryotic microbe is an important source of nitrogen been input into many natural ecosystems. In this study the active diazotrophic community was investigated in the three treatments of mowed, grazed and enclosed Leymus chinensis steppes in Hulunbeier grassland of Inner Mongolia by using approaches of polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) and sequence analysis. The community structure and diversity of the bacterial groups from the different samples was further analyzed by using different techniques, such as statistical analysis and diversity index evaluation of the band patterns etc. The results showed that grazing activity significantly reduced the number of species and quantities of nitrogen-fixing microorganisms, as well as the nifH gene diversity. However, enclosed plots had the lowest diversity of nifH gene. While the highest one found in mowing plots. A total of 30 sequences representing 25 different sequence types were recovered from the DGGE gels after phylogenetic constructions. The results also revealed that most sequences were coming from Alphaproteobacteria of Proteobacteria, and characterized by sequences of members of Rhodobacter, Bradyrhizobium, Mesorhizobium, Rhodopseudomonas, Xanthomonas, Azospirillum, Gluconacetobacter, Methylobacterium and Methylocystis. Symbiotic nitrogen-fixers existed in grazing, mowing and enclosed plots accounted for 21.4%, 47.3% and 31.3% respectively in their dominant nitrogen-fixing bacteria. The result of principal components analysis showed that the influence of different land use patterns on nitrogen-fixing microbial communities composition can be ordered as grazing plots > enclosed plots > mowing plots. Nitrogen-fixing microbial communities in L. chinensis steppe were significantly (P < 0.05) influenced by the levels of nitrate nitrogen, total phosphorus, available phosphorus contents and pH value when canonical correspondence analysis was employed to identify relationship between nifH gene and soil physicochemical factors under different land use patterns. The result obtained from correlation analysis showed that there was a significant (P < 0.05) negative relationship between the nitrate nitrogen and the total phosphorus content, furthermore, the available phosphorus content was strongly correlated (P < 0.01) with the pH value.     

Effects of different land use patterns on nifH genetic diversity of soil nitrogen-fixing microbial communities in Leymus Chinensis steppe

Biological nitrogen fixation through prokaryotic microbe is an important source of nitrogen been input into many natural ecosystems. In this study the active diazotrophic community was investigated in the three treatments of mowed, grazed and enclosed Leymus chinensis steppes in Hulunbeier grassland of Inner Mongolia by using approaches of polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) and sequence analysis. The community structure and diversity of the bacterial groups from the different samples was further analyzed by using different techniques, such as statistical analysis and diversity index evaluation of the band patterns etc. The results showed that grazing activity significantly reduced the number of species and quantities of nitrogen-fixing microorganisms, as well as the nifH gene diversity. However, enclosed plots had the lowest diversity of nifH gene. While the highest one found in mowing plots. A total of 30 sequences representing 25 different sequence types were recovered from the DGGE gels after phylogenetic constructions. The results also revealed that most sequences were coming from Alphaproteobacteria of Proteobacteria, and characterized by sequences of members of Rhodobacter, Bradyrhizobium, Mesorhizobium, Rhodopseudomonas, Xanthomonas, Azospirillum, Gluconacetobacter, Methylobacterium and Methylocystis. Symbiotic nitrogen-fixers existed in grazing, mowing and enclosed plots accounted for 21.4%, 47.3% and 31.3% respectively in their dominant nitrogen-fixing bacteria. The result of principal components analysis showed that the influence of different land use patterns on nitrogen-fixing microbial communities composition can be ordered as grazing plots > enclosed plots > mowing plots. Nitrogen-fixing microbial communities in L. chinensis steppe were significantly (P < 0.05) influenced by the levels of nitrate nitrogen, total phosphorus, available phosphorus contents and pH value when canonical correspondence analysis was employed to identify relationship between nifH gene and soil physicochemical factors under different land use patterns. The result obtained from correlation analysis showed that there was a significant (P < 0.05) negative relationship between the nitrate nitrogen and the total phosphorus content, furthermore, the available phosphorus content was strongly correlated (P < 0.01) with the pH value.     

cDNA sequence analysis of ribosomal protein S13 gene in Plutella xylostella （Lepidoptera： Plutellidae）

Ribosomal protein S 13 gene has been cloned and analyzed in many organisms,but there are few documents relating to insects. In this communication, the full-length cDNA sequence of ribosomal protein S 13 gene in the diamondback moth, Plutella xylostella(Lepidoptera: Plutellidae), was determined by using PCR amplification technique. The features of the ribosomal protein S 13 gene sequence were analyzed and the deduced amino acids sequence was compared with those from other insects. The results of multi-alignment of the amino acid sequences between the diamondback moth and other insect species revealed that this gene sequence is highly conserved in insects. Based on maximum likelihood method, a phylogenetic tree was constructed from 10 different species using PHYLIP software. It showed that nematode is one separate lineage and the five insect speciesbe long to another lineage, whereas those species higher than insects form the third one. The pattern of this phylogenetic tree evidently represented the evolution of different species.     

In vitro flowering of Dendrobium candidum

Dendrobium candidum, a wild orchid species from China, normally requires three to four years of cultivation before it can produce flowers. The effects of plant hormones and polyamines on flower initiation of this species in tissue culture were investigated. The addition of spermidine, or BA, or the combination of NAA and BA to the culture medium can induce protocorms or shoots to flower within three to six months with a frequency of 31.6% -45.8%. The flowering frequency can be further increased to 82.8 % on the average by pre-treatment of protocorms in an ABA-containing medium followed by transfer onto MS medium with BA. The induction of precocious flowering de-pends on the developmental stage of the experimental materials (protocorms, shoots and plantlets) used , and usually occurs only when root formation is inhibited.     

Comparing two Bayesian methods for gene tree/species tree reconstruction: simulations with incomplete lineage sorting and horizontal gene transfer

With the increasing interest in recognizing the discordance between gene genealogies, various gene tree/species tree reconciliation methods have been developed. We present here the first attempt to assess and compare two such Bayesian methods, Bayesian estimation of species trees (BEST) and BUCKy (Bayesian untangling of concordance knots), in the presence of several known processes of gene tree discordance. DNA alignments were simulated under the influence of incomplete lineage sorting (ILS) and of horizontal gene transfer (HGT). BEST and BUCKy both account for uncertainty in gene tree estimation but differ substantially in their assumptions of what caused gene tree discordance. BEST estimates a species tree using the coalescent model, assuming that all gene tree discordance is due to ILS. BUCKy does not assume any specific biological process of gene tree discordance through the use of a nonparametric clustering of concordant genes. BUCKy estimates the concordance factor (CF) of a clade, which is defined as the proportion of genes that truly have the clade in their trees. The estimated concordance tree is then built from clades with the highest estimated CFs. Because of their different assumptions, it was expected that BEST would perform better in the presence of ILS and that BUCKy would perform better in the presence of HGT. As expected, the species tree was more accurately reconstructed by BUCKy in the presence of HGT, when the HGT events were unevenly placed across the species tree. BUCKy and BEST performed similarly in most other cases, including in the presence of strong ILS and of HGT events that were evenly placed across the tree. However, BUCKy was shown to underestimate the uncertainty in CF estimation, with short credibility intervals. Despite this, the discordance pattern estimated by BUCKy could be compared with the signature of ILS. The resulting test for the adequacy of the coalescent model proved to have low Type I error. It was powerful when HGT was the major source of discordance and when HGT events were unevenly placed across the species tree.     

How has horizontal gene transfer shaped the evolution of insect genomes?

As the most diverse group of animals on Earth, insects are key organisms in ecosystems. Horizontal gene transfer (HGT) refers to the transfer of genetic material between species by non-reproductive means. HGT is a major evolutionary force in prokaryotic genome evolution, but its importance in different eukaryotic groups, such as insects, has only recently begun to be understood. Genomic data from hundreds of insect species have enabled the detection of large numbers of HGT events and the elucidation of the functions of some of these foreign genes. Although quantification of the extent of HGT in insects broadens our understanding of its role in insect evolution, the scope of its influence and underlying mechanism(s) of its occurrence remain open questions for the field.     

Horizontal gene and chromosome transfer in plant pathogenic fungi affecting host range

Plant pathogenic fungi adapt quickly to changing environments including overcoming plant disease resistance genes. This is usually achieved by mutations in single effector genes of the pathogens, enabling them to avoid recognition by the host plant. In addition, horizontal gene transfer (HGT) and horizontal chromosome transfer (HCT) provide a means for pathogens to broaden their host range. Recently, several reports have appeared in the literature on HGT, HCT and hybridization between plant pathogenic fungi that affect their host range, including species of Stagonospora/Pyrenophora, Fusarium and Alternaria. Evidence is given that HGT of the ToxA gene from Stagonospora nodorum to Pyrenophora tritici-repentis enabled the latter fungus to cause a serious disease in wheat. A nonpathogenic Fusarium species can become pathogenic on tomato by HCT of a pathogenicity chromosome from Fusarium oxysporum f.sp lycopersici, a well-known pathogen of tomato. Similarly, Alternaria species can broaden their host range by HCT of a single chromosome carrying a cluster of genes encoding host-specific toxins that enabled them to become pathogenic on new hosts such as apple, Japanese pear, strawberry and tomato, respectively. The mechanisms HGT and HCT and their impact on potential emergence of fungal plant pathogens adapted to new host plants will be discussed.     

A model of horizontal gene transfer and the bacterial phylogeny problem

How much horizontal gene transfer (HGT) between species influences bacterial phylogenomics is a controversial issue. This debate, however, lacks any quantitative assessment of the impact of HGT on phylogenies and of the ability of tree-building methods to cope with such events. I introduce a Markov model of genome evolution with HGT, accounting for the constraints on time -- an HGT event can only occur between concomitantly living species. This model is used to simulate multigene sequence data sets with or without HGT. The consequences of HGT on phylogenomic inference are analyzed and compared to other well-known phylogenetic artefacts. It is found that supertree methods are quite robust to HGT, keeping high levels of performance even when gene trees are largely incongruent with each other. Gene tree incongruence per se is not indicative of HGT. HGT, however, removes the (otherwise observed) positive relationship between sequence length and gene tree congruence to the estimated species tree. Surprisingly, when applied to a bacterial and a eukaryotic multigene data set, this criterion rejects the HGT hypothesis for the former, but not the latter data set.     

Multilevel populations and the evolution of antibiotic resistance through horizontal gene transfer

Horizontal gene transfer (HGT) can create diversity in the genetic repertoire of a lineage. Successful gene transfer likely occurs more frequently between more closely related organisms, leading to the formation of higher-level exchange groups that in some respects are comparable to single-species populations. Genes that appear fixed in a single species can be replaced through distant homologs or iso-functional analogs acquired through HGT. These genes may originate from other species or they may be acquired by an individual strain from the species pan-genome. Because of their similarity to alleles in a population, we label these gene variants that are exchanged between related species as homeoalleles. In a case study, we show that biased gene transfer plays an important role in the evolution of aminoacyl-tRNA synthetases (aaRS). Many microorganisms make use of these genes against naturally occurring antibiotics. We suggest that the resistance against naturally occurring antibiotics is the likely driving force behind the frequent switching between divergent aaRS types and the reason for the maintenance of these homeoalleles in higher-level exchange groups. Resistance to naturally occurring antibiotics may lead to the maintenance of different types of aminoacyl-tRNA synthetases in Bacteria through gene transfer.     

Horizontal gene transfer in plants

Horizontal gene transfer (HGT) has played a major role in bacterial evolution and is fairly common in certain unicellular eukaryotes. However, the prevalence and importance of HGT in the evolution of multicellular eukaryotes remain unclear. Recent studies indicate that plant mitochondrial genomes are unusually active in HGT relative to all other organellar and nuclear genomes of multicellular eukaryotes. Although little about the mechanisms of plant HGT is known, several studies have implicated parasitic plants as both donors and recipients of mitochondrial genes. Most cases uncovered thus far have involved a single transferred gene per species; however, recent work has uncovered a case of massive HGT in Amborella trichopoda involving acquisition of at least a few dozen and probably hundreds of foreign mitochondrial genes. These foreign genes came from multiple donors, primarily eudicots and mosses. This review will examine the implications of such massive transfer, the potential mechanisms and consequences of plant-to-plant mitochondrial HGT in general, as well as the limited evidence for HGT in plant chloroplast and nuclear genomes.     

植物中的水平基因转移

水平基因转移是不通过生殖而进行的遗传物质交流, 在原核生物和单细胞真核生物的进化中起着重要作用。然而, 水平基因转移在多细胞真核生物之间的发生频率以及对多细胞真核生物进化的影响尚不明确。近期的一些研究显示, 水平基因转移在高等植物之间以及高等植物和其它生物之间普遍存在。该文将对高等植物中已发现的一些水平基因转移现象进行综述, 并尝试解析植物之间水平基因转移可能的机制及其重要意义。     

What can we learn from tobacco and other Solanaceae about horizontal DNA transfer?

In eukaryotic organisms, horizontal gene transfer (HGT) is regarded as an important though infrequent source of reticulate evolution. Many confirmed instances of natural HGT involving multicellular eukaryotes come from flowering plants. This review intends to provide a synthesis of present knowledge regarding HGT in higher plants, with an emphasis on tobacco and other species in the Solanaceae family because there are numerous detailed reports concerning natural HGT events, involving various donors, in this family. Moreover, in-depth experimental studies using transgenic tobacco are of great importance for understanding this process. Valuable insights are offered concerning the mechanisms of HGT, the adaptive role and regulation of natural transgenes, and new routes for gene trafficking. With an increasing amount of data on HGT, a synthetic view is beginning to emerge.     

Horizontal gene transfer in plants

Horizontal gene transfer (HGT) describes the transmission of genetic material across species boundaries. HGT often occurs in microbic and eukaryotic genomes. However, the pathways by which HGTs occur in multicellular eukaryotes, especially in plants, are not well understood. We systematically summarized more than ten possible pathways for HGT. The intimate contact which frequently occurs in parasitism, symbiosis, pathogen, epiphyte, entophyte, and grafting interactions could promote HGTs between two species. Besides these direct transfer methods, genes can be exchanged with a vector as a bridge: possible vectors include pollen, fungi, bacteria, viruses, viroids, plasmids, transposons, and insects. HGT, especially when involving horizontal transfer of transposable elements, is recognized as a significant force propelling genomic variation and biological innovation, playing an important functional and evolutionary role in both eukaryotic and prokaryotic genomes. We proposed possible mechanisms by which HGTs can occur, which is useful in understanding the genetic information exchange among distant species or distant cellular components.     

Gene transfer from a parasitic flowering plant to a fern

The rattlesnake fern (Botrychium virginianum (L.) Sw.) is obligately mycotrophic and widely distributed across the northern hemisphere. Three mitochondrial gene regions place this species with other ferns in Ophioglossaceae, while two regions place it as a member of the largely parasitic angiosperm order Santalales (sandalwoods and mistletoes). These discordant phylogenetic placements suggest that part of the genome in B. virginianum was acquired by horizontal gene transfer (HGT), perhaps from root-parasitic Loranthaceae. These transgenes are restricted to B. virginianum and occur across the range of the species. Molecular and life-history traits indicate that the transfer preceded the global expansion of B. virginianum, and that the latter may have happened very rapidly. This is the first report of HGT from an angiosperm to a fern, through either direct parasitism or the mediation of interconnecting fungal symbionts.