Gene Cloning Analysis Research Articles

Microbial Community Structures in Petroleum Contaminated Soils at an Oil Field, Hebei, China

Petroleum hydrocarbons consisting of complicated mixtures of non‐aqueous and hydrophobic components have raised a widespread environmental problem and influenced the subsurface microbial ecology. Three subsurface soil samples were collected at an oil field (Hebei, China) to investigate the microbial diversity patterns and their responds to different petroleum hydrocarbon contaminations by using 16S rRNA gene cloning analysis combined with multivariate statistical analysis. The results showed that bacterial clone libraries fell into 16 phylogenetic divisions and 184 operational taxonomic units. Gammaproteobacteria ubiquitously dominated in all samples, while the relative abundance was lowest in the sample with the highest total petroleum hydrocarbon concentration. Known petroleum degrading bacterial genera Alcanivorax, Stenotrophomonas, Pseudomonas, Pseudoxanthomonas, Sphingomonas, and Acidobacterium were abundantly observed, whereas their relative abundances differed greatly under a selection pressure by the complex hydrocarbons. For example, Alcanivorax spp. were the most abundant genera in polycyclic aromatic hydrocarbons and chlorinated hydrocarbons contaminated soils, while both Alcanivorax spp. and Stenotrophomonas spp. dominated in benzene, toluene, ethylbenzene, and xylenes contaminated soils. Additionally, a large fraction of bacterial clones were expectedly affiliated to some uncultured bacteria originated from several similar polluted ecosystems, and bacteria without certain petroleum degrading capacities. These observations indicated that microbial diversity, abundance and community were highly influenced by the concentrations of petroleum constituents in the contaminated soil ecosystems. Understanding the underlying factors that control the abundance of these heterotrophic microorganisms may be advantageous in the bioremediation of petroleum contaminated environments.

Bacterial Diversity and Biogeochemical Processes of Oil-Contaminated Groundwater, Baoding, North China

ABSTRACTA total of 43 groundwater samples were collected from 9 multimonitoring wells at a petrochemical site, Baoding City, North China, from June 2008 to December 2009 to investigate the biogeochemical processes and/or bacterial conmmunity using both culture-dependent and -independent methods. The results showed that aromatic hydrocarbons and chlorinated hydrocarbons were the major pollutants in the groundwater. Denitrification and iron reduction might be the main biogeochemical processes in the aquifers at this site, which seemed to transform from denitrification-dominated to iron reduction-dominated in some sections. Denaturing gradient gel electrophoresis (DGGE) revealed that the dominant bacterial groups of the groundwater were related to some oil-degrading bacteria, which can grow under denitrifying, iron-reducing and sulfate-reducing anaerobic conditions. In some serious contaminated groundwater niches, there might be sulfur cycles, as sulfur oxidizer was also abundant, which was further confirmed by 16S rRNA gene cloning analysis. The operational taxonomic units (OTUs) that highly related to Pseudomonas sp., Hydrogenophaga sp., Sphingomonas sp., Ferribacterium sp. and Sulfuricurvum Kujiense etc. were predominant in the groundwater contaminated by chlorinated hydrocarbons (CHCs), benzene, toluene, ethylbenzene, and xylenes (BTEX) and/or polycyclic aromatic hydrocarbons (PAHs), respectively. Biodiversity seemed to be undermined by oil contamination, and varied with seasons. The bacterial community in the contaminated groundwater was largely determined by the groundwater geochemistry.

Protocols for Cloning, Expression, and Functional Analysis of Sirtuin2 (SIRT2).

SIRT2 is a NAD(+)-dependent deacetylase that belongs to the sirtuin family, which is comprised of seven members (SIRT1-SIRT7) in humans. Furthermore, recent study shows that the Sirt2 gene has three transcript variants in mice. Several diverse proteins have been identified as SIRT2 substrates. SIRT2 activity involves multiple cell processes including growth, differentiation, and energy metabolism. However, little is known of SIRT2's role in oligodendrocytes or in the myelin sheath, where it is an important component. Here we describe procedures that detail Sirt2 gene cloning, identification, expression, and biological analysis in cultured cells.

Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.

Germacrene A synthase in yarrow (Achillea millefolium) is an enzyme with mixed substrate specificity: gene cloning, functional characterization and expression analysis.

Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5) residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS) that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS). The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3), functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP), while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP) and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP). Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes.

Gene cloning and bioinformatics analysis of new gene for chlorogenic acid biosynthesis of Lonicera hypoglauca

To obtain the key genes for chlorogenic acid biosynthesis of Lonicera hypoglauca, four new genes ware obtained from the our dataset of L. hypoglauca. And we also predicted the structure and function of LHPAL4, LHHCT1 , LHHCT2 and LHHCT3 proteins. The phylogenetic tree showed that LHPAL4 was closely related with LHPAL1, LHHCT1 was closely related with LHHCT3, LHHCT2 clustered into a single group. By Real-time PCR to detect the gene expressed level in different organs of L. hypoglauca, we found that the transcripted level of LHPAL4, LHHCT1 and LHHCT3 was the highest in defeat flowers, and the transcripted level of LHHCT2 was the highest in leaves. These result provided a basis to further analysis the mechanism of active ingredients in different organs, as well as the element for in vitro biosynthesis of active ingredients.

Composition, variation, expression and evolution of low-molecular-weight glutenin subunit genes in Triticum urartu.

BackgroundWheat (AABBDD, 2n = 6x = 42) is a major dietary component for many populations across the world. Bread-making quality of wheat is mainly determined by glutenin subunits, but it remains challenging to elucidate the composition and variation of low-molecular-weight glutenin subunits (LMW-GS) genes, the major components for glutenin subunits in hexaploid wheat. This problem, however, can be greatly simplified by characterizing the LMW-GS genes in Triticum urartu, the A-genome donor of hexaploid wheat. In the present study, we exploited the high-throughput molecular marker system, gene cloning, proteomic methods and molecular evolutionary genetic analysis to reveal the composition, variation, expression and evolution of LMW-GS genes in a T. urartu population from the Fertile Crescent region.ResultsEight LMW-GS genes, including four m-type, one s-type and three i-type, were characterized in the T. urartu population. Six or seven genes, the highest number at the Glu-A3 locus, were detected in each accession. Three i-type genes, each containing more than six allelic variants, were tightly linked because of their co-segregation in every accession. Only 2-3 allelic variants were detected for each m- and s-type gene. The m-type gene, TuA3-385, for which homologs were previously characterized only at Glu-D3 locus in common wheat and Aegilops tauschii, was detected at Glu-A3 locus in T. urartu. TuA3-460 was the first s-type gene identified at Glu-A3 locus. Proteomic analysis showed 1-4 genes, mainly i-type, expressed in individual accessions. About 62% accessions had three active i-type genes, rather than one or two in common wheat. Southeastern Turkey might be the center of origin and diversity for T. urartu due to its abundance of LMW-GS genes/genotypes. Phylogenetic reconstruction demonstrated that the characterized T. urartu might be the direct donor of the Glu-A3 locus in common wheat varieties.ConclusionsCompared with the Glu-A3 locus in common wheat, a large number of highly diverse LMW-GS genes and active genes were characterized in T. urartu, demonstrating that this progenitor might provide valuable genetic resources for LMW-GS genes to improve the quality of common wheat. The phylogenetic analysis provided molecular evidence and confirmed that T. urartu was the A-genome donor of hexaploid wheat.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0322-3) contains supplementary material, which is available to authorized users.

Gene cloning and expression analysis of AhR and CYP4 from Pinctada martensii after exposed to pyrene.

Pyrene, a typical polycyclic aromatic hydrocarbon, is a common pollutant in the marine environment. Polycyclic aromatic hydrocarbons initiate cellular detoxification in an exposed organism via the activation of the aryl hydrocarbon receptor (AhR). Subsequent metabolism of these xenobiotics is mainly by the cytochrome P450 enzymes of the phase I detoxification system. Full-length complementary DNA sequences from the pearl oyster Pinctada martensii (pm) encoding AhR and cytochrome P4 were cloned. The P. martensii AhR complementary DNA sequence constitutes an open reading frame that encodes for 848 amino acids. Sequence analysis indicated PmAhR showed high similarity with its homologues of other bivalve species. The cytochrome P(CYP)4 complementary DNA sequence of P. martensii constitutes an open reading frame that encodes for 489 amino acids. Quantitative real-time analysis detected both PmAhR and PmCYP4 messenger RNA expressions in the mantle, gill, hepatapancreas and adductor muscle of P. martensii exposed to pyrene. The highest transcript-band intensities of PmAhR and PmCYP4 were observed in the gill. Temporal expression of PmAhR and PmCYP4 messenger RNAs induction was observed in gills and increased between 3 and 5 days post exposure; then returned to control level. These results suggest that messenger RNAs of PmAhR and PmCYP4 in pearl oysters might be useful parameters for monitoring marine environment pyrene pollution.

Surveillance of hepatitis E virus contamination in shellfish in China.

Background: Hepatitis E virus (HEV) has been confirmed to be a zoonotic virus of worldwide distribution. HEV contamination in the water environment has not been well examined in China. The objective of this study was to evaluate HEV contamination in shellfish in a coastal area of China. Such contamination would be significant for evaluating public health risks. Methods: samples of three species shellfish were collected from thirteen points of estuarine tidal flats around the Bohai Gulf and screened for HEV RNA using an in-house nested RT-PCR assay. The detected HEV-positive samples were further verified by gene cloning and sequencing analysis. Results: the overall HEV-positive detection rate is approximately 17.5% per kilogram of shellfish. HEV was more common among S. subcrenata (28.2%), followed by A. granosa (14.3%) and R. philippinarum (11.5%). The phylogenetic analysis of the 13 HEV strains detected revealed that gene fragments fell into two known 4 sub-genotypes (4b/4d) groups and another unknown group. Conclusions: 13 different sub-genotype 4 HEVs were found in contaminated shellfish in the Bohai Gulf rim. The findings suggest that a health risk may exist for users of waters in the Bonhai area and to consumers of shellfish. Further research is needed to assess the sources and infectivity of HEV in these settings, and to evaluate additional shellfish harvesting areas.

Keys to Cultivating Uncultured Microbes: Elaborate Enrichment Strategies and Resuscitation of Dormant Cells

Keys to Cultivating Uncultured Microbes: Elaborate Enrichment Strategies and Resuscitation of Dormant Cells

De novo assembly and transcriptome analysis of the Mediterranean fruit fly Ceratitis capitata early embryos.

The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, belongs to the Tephritidae family, which includes a large number of other damaging pest species. The Medfly has been the first non-drosophilid fly species which has been genetically transformed paving the way for designing genetic-based pest control strategies. Furthermore, it is an experimentally tractable model, in which transient and transgene-mediated RNAi have been successfully used. We applied Illumina sequencing to total RNA preparations of 8–10 hours old embryos of C. capitata, This developmental window corresponds to the blastoderm cellularization stage. In summary, we assembled 42,614 transcripts which cluster in 26,319 unique transcripts of which 11,045 correspond to protein coding genes; we identified several hundreds of long ncRNAs; we found an enrichment of transcripts encoding RNA binding proteins among the highly expressed transcripts, such as CcTRA-2, known to be necessary to establish and, most likely, to maintain female sex of C. capitata. Our study is the first de novo assembly performed for Ceratitis capitata based on Illumina NGS technology during embryogenesis and it adds novel data to the previously published C. capitata EST databases. We expect that it will be useful for a variety of applications such as gene cloning and phylogenetic analyses, as well as to advance genetic research and biotechnological applications in the Medfly and other related Tephritidae.

Environmentally determined differences in the murine lung microbiota and their relation to alveolar architecture.

Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect on tissue homeostasis and host defense. In contrast to the upper respiratory tract, the lower respiratory tract of healthy individuals has largely been considered free of microorganisms. To understand airway micro-ecology we studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non-specific-pathogen-free (SPF) conditions as well as inbred mice maintained in non-SPF, SPF or germ-free (GF) facilities. High-throughput pyrosequencing of reverse transcribed 16S rRNA revealed metabolically active murine lung microbiota in all but GF mice. The overall composition across samples was similar at the phylum and family level. However, species richness was significantly different between lung microbiota from SPF and non-SPF mice. Non-cultivatable Betaproteobacteria such as Ralstonia spp. made up the major constituents and were also confirmed by 16S rRNA gene cloning analysis. Additionally, Pasteurellaceae, Enterobacteria and Firmicutes were isolated from lungs of non-SPF mice. Bacterial communities were detectable by fluorescent in situ hybridization (FISH) at alveolar epithelia in the absence of inflammation. Notably, higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae, which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. Our data indicate a common microbial composition of murine lungs, which is diversified through different environmental conditions and affects lung architecture. Identification of the microbiota of murine lungs will pave the path to study their influence on pulmonary immunity to infection and allergens using mouse models.

Simultaneous heterotrophic nitrification and aerobic denitrification by the marine origin bacterium Pseudomonas sp. ADN-42.

Recent research has highlighted the existence of some bacteria that are capable of performing heterotrophic nitrification and have a phenomenal ability to denitrify their nitrification products under aerobic conditions. A high-salinity-tolerant strain ADN-42 was isolated from Hymeniacidon perleve and found to display high heterotrophic ammonium removal capability. This strain was identified as Pseudomonas sp. via 16S rRNA gene sequence analysis. Gene cloning and sequencing analysis indicated that the bacterial genome contains N2O reductase function (nosZ) gene. NH3-N removal rate of ADN-42 was very high. And the highest removal rate was 6.52 mg/L · h in the presence of 40 g/L NaCl. Under the condition of pure oxygen (DO >8 mg/L), NH3-N removal efficiency was 56.9 %. Moreover, 38.4 % of oxygen remained in the upper gas space during 72 h without greenhouse gas N2O production. Keeping continuous and low level of dissolved oxygen (DO <3 mg/L) was helpful for better denitrification performance. All these results indicated that the strain has heterotrophic nitrification and aerobic denitrification abilities, which guarantee future application in wastewater treatment.

Gene cloning, expression, and function analysis of SpL14-3-3ζ in Spodoptera litura and its response to the entomopathogenic fungus Nomuraea rileyi.

The 14-3-3 proteins, a highly evolutionarily conserved and ubiquitous protein family in eukaryotic cells, have a range of biological functions including regulation of signal transduction, stress response, apoptosis, and control of the cell cycle. To investigate the function of 14-3-3 in Spodoptera litura, the full length of 14-3-3ζ was cloned from S. litura on the basis of an expressed sequence tag of 14-3-3ζ from the S. litura fat body suppression subtractive hybridization library, and named SpL14-3-3ζ. SpL14-3-3ζ cDNA was 1196 bp with an open reading frame of 744 bp, encoding 247 amino acids. Multiple alignment analysis revealed the putative amino acids shared >80% homology with 14-3-3ζ from other organisms and shared typical conservative structures. Phylogenetic analysis confirmed SpL14-3-3ζ was closely related to other available Lepidoptera 14-3-3ζ. Real-time PCR analysis indicated SpL14-3-3ζ was expressed throughout the developmental stages of S. litura, with a relatively high expression level in pre-pupa, and was expressed constitutively in all examined tissues with relatively high levels in hemocytes and midgut. Moreover, the transcription level of SpL14-3-3ζ could be induced by Nomuraea rileyi infection, up-regulated in hemocytes, followed by head, fat body and midgut. Knocking down SpL14-3-3ζ transcripts by RNAi significantly increased S. litura sensitivity to fungal infection, and resulted in higher mortality of S. litura during the larval development. These results provide novel insights into the 14-3-3ζ signal regulation which may be related to host defense as well as larval development in S. litura.

Gene cloning and expression analysis of IRF1 in half-smooth tongue sole (Cynoglossus semilaevis)

Interferon regulatory factor 1 (IRF1) was known to play key roles in antiviral defense in several species, and some other important biological processes. In this report, full length cDNA of IRF1 from Cynoglossus semilaevis (CsIRF1) was identified. It was of 1,455 bp, containing a 5' UTR of 104 bp, a 3' UTR of 541 bp with a poly (A) tail and an ORF of 810 bp encoding a putative protein of 269 amino acids. The putative CsIRF1protein contained one conserved IRF domain (1-113aa), and two low complexity regions (140-158aa and 230-242aa, respectively). Phylogenetic analysis showed that CsIRF1 was conserved in the teleost evolutionary branch, which was independent of mammalian, birds and amphibians. Additionally, CsIRF1 had the 96% homology with marine fishes, while 66% with freshwater fishes. The expression profiles of CsIRF1was analyzed by quantitative real-time PCR in healthy tissues and in immune tissues challenged with different pathogens [Vibrio anguillarum and Lymphocystis disease virus (LCDV)], respectively. CsIRF1 was widely expressed in healthy tissues of Cynoglossus semilaevis and with the highest expression in blood, as much as 19 times of that in liver. V. anguillarum and LCDV both induced the CsIRF1 gene expression distinctly in liver, with the peak value reached to 98-fold at 6 h and 25-fold at 24 h, respectively. The bacteria induced CsIRF1 suddenly up-expression in each detected tissues. However, at the initial stage of the challenge of virus LCDV, the CsIRF1 expression in blood and spleen were up regulated; on the contrary, its expression in liver and head kidney were down regulated, 0.3 and 0.4-fold 6 h post virus injection, respectively. These results suggested that CsIRF1 gene might involve in not only antiviral activity but also antibacterial procedure, indicating its vital role in Cynoglossus semilaevis innate defense system.

Morphological and physiological analysis of narrow and striped leaf 1 (nsl1) mutant of rice (Oryza sativa L.) and the gene mapping

The shape and color of rice leaves are important agronomic traits that directly influence the proportion of sunlight energy utilization and ultimately affect the yield and quality. A new mutant exhibiting stable inheritance was identified as derived from ethyl methane sulfonate (EMS)-treated restorer Jinhui 10, tentatively named as narrow and striped leaf 1 (nsl1). The nsl1 displayed pale white leaves at the seeding stage and then white striped leaves in parallel to the main vein at the jointing stage. Meanwhile, its leaf blades are significantly narrower than the control group of Jinhui 10. The chloroplast structures of cells in the white striped area of the nsl1 mutant break down, and the photosynthetic pigments are significantly lower than that of the wild type. Moreover, fluorescence parameters, such as F0, Fv/Fm, ΦpsII, qp, and ETR, in the nsl1 mutant are significantly lower than those of the wild type, and the photosynthetic efficiency is also significantly decreased. These changes in leaf color and shape, together with physiological changes in the nsl1, result in smaller plant height and a decrease in the most important agronomic traits, such as the number of grains per panicle, grain weight, etc. Genetic analysis shows that the narrow and striped traits of the nsl1 mutant are controlled by a single recessive nuclear gene, which is located between InDel 16 and InDel 12 in chromosome 3. The physical distance is 204 kb. So far, no similar genes of such leaf color and shape in this area have been reported. This study has laid a solid foundation for the gene cloning and function analysis of NSL1.

Genetic Analysis and Fine Mapping of a Premature Leaf Senescence Mutant in Rice ( Orzya sativa L.)

Leaf senescence induces degradation of chlorophyll and other macromolecules, reducing leaf photosynthetic capacity. This process is accompanied by the accumulation of reactive oxygen species(ROS), the decreasing of cell antioxidant enzyme(SOD, CAT, and APX) activity, and the increasing of aging related gene(SAG) expression, leading in early maturity and yield reduction. Therefore, studies on the genetic mechanism and gene function of premature senescence in rice, has the important effect and significance in genetic improvement of rice. PLS2 from space radiation mutation breeding project showed leaf senility, at booting stage. Compared with the wild type, in PLS2 the photosynthetic capacity decreased, the plant height, internode and panicle length shortened, tiller and effective tiller number reduced, number of grains per ear and seed setting rate were significantly lower, 1000-grain weight decreased, main panicle was stunted and grain-filling was not full. CAT activity decreased significantly in leaves, H2O2 accumulated, and the number of dead cell increased, chloroplast structures in leaves were worse, with more starches grains and osmiophilic granules. Dark treatment accelerated mutant leaf senescence, chloroplast ultrastructure was spheroidized. Using implicit localization population derived from PLS2/Shuhui 527 and PLS2/02428 the pls2 was located between markers RM14704(8 674 283 bp) and SL-I-5(8 758 394 bp) on chromosome 3, with physical distance of 84.11 kb, including 14 genes in the interval. Sequencing result showed that C was replaced with T in the position of 41 base pair of the ninth extron of LOC_Os03g15840 leading to an exchange of Arg(R) is replaced by Cys(C). LOC_Os03g15840 coding a glycosyl transferase(GTs) may be the candidate gene of pls2. These results provide a foundation for the further gene cloning and functional analysis of pls2.

Metagenomic Insights into the Microbial Community Diversity between Leaching Heap and Acid Mine Drainage

The leaching heap and acid mine drainage are two key nodes in a bioleaching system. This study aimed to investigate the microbial community structural and functional diversity between the two nodes in bioleaching system from Dexing copper mine in Jiangxi province, China. 16SrRNA gene cloning and metagenomic analysis consistently indicated that there were obvious differences on microbial community structural and functional diversity in the two nodes. In leaching heap, the dominant species was the heterotrophic bacterium Acidiphilium; while the dominant species was the autotrophic bacterium Acidithiobacillus in acid mine drainage. Seven bacteria species were found in both two nodes, while the unique bacteria species in leaching heap and acid mine drainage were eleven and eight, respectively. In relation to the microbial community function aspect, all contigs and singlets were annotated against the non-redundant protein database of NCBI and clustering analyzed with COG database. For the two nodes, the COG clustering results showed that the functional category abundances were different, though the functional categories were similar. And the great majority of ORFs were forecasted as function unknown. All the results meant that the microbial community structural and functional diversity of bioleaching system was not as simple as former thought. This study could provide a new meta-view of theoretical support to bioleaching process.

Gene cloning and bioinformatics analysis of SABATH methyltransferase in Lonicera japonica var. chinensis

To clone SABATH methyltransferase (rLjSABATHMT) gene in Lonicera japonica var. chinensis, and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L. japonica with L. japonica var. chinensis. It provide a basis for gene regulate the formation of L. japonica floral scents. The cDNA and genome sequences of LjSABATHMT from L. japonica var. chinensis were cloned according to the gene fragments in cDNA library. The LjSABATHMT protein was characterized by bioinformatics analysis. SABATH family phylogenetic tree were built by MEGA 5.0. The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L. japonica and L. japonica var. chinensis using RT-PCR analysis. Intron sequences of SABATHMT orthologous were also analyzied. The cDNA of LjSABATHMT was 1 251 bp, had a complete coding frame with 365 amino acids. The protein had the conservative SABATHMT domain, and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase. Higher expression of SABATH methyltransferase orthologous was found in flower. The intron sequence of L. japonica and L. japonica var. chinensis had rich polymorphism, and two SNP are unique genotype of L. japonica var. chinensis. The motif elements in two orthologous genes were significant differences. The intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L. japonica and L. japonica var. chinensis. These results will provide important base on regulating active compounds of L. japonica.

Gene Cloning and Function Analysis Involved in the Biodegradation of Aromatic Compounds by Bacillus NAPZ

A bacterial strain NAPZ, which utilized polycyclic aromatic hydrocarbons (PAHs) as the sole carbon and energy source for growth, was studied on its apparent characteristics, key gene structure and functions. It was preliminarily identified as a Bacillus sp. according to its physiological characteristics and the phylogenetic property of its 16S rRNA gene sequence. Based on the reported RHD gene sequences, a pair of primers was designed to amplify the RHD gene from the genomic DNA of strain NAPZ by PCR. Then, the RHD gene was cloned and sequenced. Based on this sequence and its related sequences in the GenBank database, a multiple alignment was conducted then a phylogenetic tree was constructed with DNAMAN and MEGA software. The analysis revealed that strain NAPZ oxidized PAHs via ring hydroxylating dioxygenase (RHD) pathway. The study provided experimental and theoretical support for revealing the structure and function of key genes as well as the PAHs-degrading pathways in strain NAPZ.