Introduction to Molecular Analysis of Ectomycorrhizal Communities

A number of methods are available for those researchers considering the addition of molecular analyses of ectomycorrhizal (EcM) fungi to their research projects and weighing the various approaches they might take. Analyzing natural EcM fungal communities has traditionally been a highly skilled, time-consuming process relying heavily on exacting morphological characterization of EcM root tips. Increasingly powerful molecular methods for analyzing EcM communities make this area of research available to a much wider range of researchers. Ecologists can gain from the body of work characterizing EcM while avoiding the requirement for exceptional expertise by carefully combining elements of traditional methods with the more recent molecular approaches. A cursory morphological analysis can yield a traditional quantification of EcM fungi based on tip numbers, a unit with functional and historical significance. Ectomycorrhizal root DNA extracts may then be analyzed with molecular methods widely used for characterizing microbiota. These range from methods applicable only to the simple mixes resulting from careful morphotyping, to community-oriented methods that identify many types in mixed samples as well as provide an estimate of their relative abundances. Extramatrical hyphae in bulk soil can also be more effectively studied, extending characterization of EcM fungal communities beyond the rhizoplane. The trend toward techniques permitting larger sample sets without prohibitive labor and time requirements will also permit us to more frequently address the issues of spatial and temporal variability and better characterize the roles of EcM fungi at multiple scales.

Early Embryonic Exposure to Polychlorinated Biphenyls Disrupts Heat-Shock Protein 70 Cognate Expression in Zebrafish

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that have documented neurological effects in children exposed in utero. To better define neuronally linked molecular targets during early development, zebrafish embryos were exposed to Aroclor 1254, a mixture of PCB congeners that are common environmental contaminants. Microarray analysis of the zebrafish genome revealed consistent significant changes in 38 genes. Of these genes, 55% (21) are neuronally related. One gene that showed a consistent 50% reduction in expression in PCB-treated embryos was heat-shock protein 70 cognate (Hsc70). The reduction in Hsc70 expression was confirmed by real-time polymerase chain reaction (PCR), revealing a consistent 30% reduction in expression in PCB-treated embryos. Early embryonic exposure to PCBs also induced structural changes in the ventro-rostral cluster as detected by immunocytochemistry. In addition, there was a significant reduction in dorso-rostral neurite outgrowth emanating from the RoL1 cell cluster following PCB exposure. The serotonergic neurons in the developing diencephalon showed a 34% reduction in fluorescence when labeled with a serotonin antibody following PCB exposure, corresponding to a reduction in serotonin concentration in the neurons. The total size of the labeled neurons was not significantly different between treated and control embryos, indicating that the development of the neurons was not affected, only the production of serotonin within the neurons. The structural and biochemical changes in the developing central nervous system following early embryonic exposure to Aroclor 1254 may lead to alterations in the function of the affected regions.

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods nand Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.

Sex-linked microsatellite marker detected in the female gametophytes of Undaria pinnatifida (Phaeophyta)

P>In our microsatellite analysis of three male and three female gametophytes of Undaria pinnatifida (Harv.) Suringar, a microsatellite marker (part of the locus Up-AC-2A8, GenBank accession no. AY738602.1) was only polymerase chain reaction-amplified in three female gametophytes. This putative female-specific marker was further tested by the use of 32 male and 21 female gametophytes maintained in the Marine Biological Culture Collection Centre, China. In addition, three sporophytes were included for confirmation. Results showed that the marker was present in all of the female gametophytes and sporophyte cultures, but absent in all of the male gametophytes. To our knowledge, this is the first sex-related marker ever reported in U. pinnatifida. The discovery of this marker will accelerate gender identification and shed light on our understanding of the mechanisms of sex determination at a molecular level in this commercially important seaweed.

Identification of vhhP2, a novel genetic marker of Vibrio harveyi, and its application in the quick detection of V. harveyi from animal specimens and environmental samples

Aims: To investigate the species-specific prevalence of vhhP2 among Vibrio harveyi isolates and the applicability of vhhP2 in the specific detection of V. harveyi from crude samples of animal and environmental origins. Methods and Results: A gene (vhhP2) encoding an outer membrane protein of unknown function was identified from a pathogenic V. harveyi isolate. vhhP2 is present in 24 V. harveyi strains isolated from different geographical locations but is absent in 24 strains representing 17 different non-V. harveyi species, including V. parahaemolyticus and V. alginolyticus. A simple polymerase chain reaction method for the identification of V. harveyi was developed based on the conserved sequence of vhhP2. This method was demonstrated to be applicable to the quick detection of V. harveyi from crude animal specimens and environmental samples. The specificity of this method was tested by applying it to the examination of two strains of V. campbellii, which is most closely related to V. harveyi. One of the V. campbellii strains was falsely identified as V. harveyi. Conclusions: vhhP2 is ubiquitously present in the V. harveyi species and is absent in most of the non-V. harveyi species; this feature enables vhhP2 to serve as a genetic marker for the rapid identification of V. harveyi. However, this method can not distinguish some V. campbellii strains from V. harveyi. Significance and Impact of the Study: the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples.

Comparison of photobioreactors and optimal light regime for rapid vegetative propagation of transgenic Undaria pinnatifida gametophytes

BACKGROUND: Previously, tachyplesin gene (tac) has been successfully transferred into Undaria pinnatifida gametophytes using the method of microprojectile bombardment transformation. The objectives of this study were to compare and evaluate the performance of bubble-column and airlift bioreactors to determine a preferred configuration of bioreactor for vegetative propagation of transgenic U. pinnatifida gametophytes, and to then investigate the influence of light on vegetative propagation of these gametophytes, including incident light intensity, photoperiod and light quality to resolve the problems of rapid vegetative propagation within the selected bioreactor. RESULTS: Experimental results showed that final dry cell density in the airlift bioreactor was 12.7% higher than that in the bubble-column bioreactor under the optimal aeration rate of 1.2 L air min(-1) L-1 culture. And a maximum final dry cell density of 2830 mg L-1 was obtained within the airlift bioreactor using blue light at 40 mu mol m(-2) s(-1) with a light/dark cycle of 14/10 (h). Polymerase chain reaction (PCR) analysis indicated that genes (bar and tac) were not lost during rapid vegetative propagation within the airlift bioreactor. CONCLUSION: The airlift bioreactor was shown to be much more suitable for rapid vegetative propagation of transgenic U. pinnatifida gametophytes than the bubble-column bioreactor in the laboratory. The use of blue light allows improvement of vegetative propagation of transgenic U. pinnatifida gametophytes. (C) 2009 Society of Chemical Industry

Molecular Cloning and Expression Analysis of a Cytosolic Hsp70 gene from Laminaria japonica (Laminariaceae, Phaeophyta)

In this study, a full-length cytosolic heat shock protein 70 complementary DNA (cDNA) of Laminaria japonica (designated as LJHsp70) was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends. The full length of LJHsp70 cDNA was 2,918 bp, with a 5' untranslated region of 248 bp, a 3' untranslated region of 696 bp, and an open reading frame of 1,974 bp encoding a polypeptide of 657 amino acids with an estimated molecular mass of 72.03 kDa and an estimated isoelectric point of 4.97. There was highly repeated sequence of CAA in 5' untranslated region of LJHsp70. The result of phylogenetic tree of Hsp70s, the BLAST program, analysis and cytosolic Hsp70-specific motif of LJHsp70 verified that the cloned LJHsp70 belonged to cytosolic Hsp70 family. Three typical Hsp70 signature motifs were detected in LJHsp70 by InterPro analysis. Under different stress conditions, messenger RNA (mRNA) expression levels of LJHsp70 were quantified by quantitative RT-PCR. To L. japonica sporophytes kept in different temperatures for 1 h, the expression level of LJHsp70 at 30A degrees C was highest and twofold higher than that at 10A degrees C. To L. japonica sporophytes kept at 25A degrees C for different times, the mRNA expression level of LJHsp70 reached a maximum level after 7 h and then dropped progressively. The expression level of LJHsp70 at 0 or 5aEuro degrees salt concentration for 2 h was twofold higher than that at 30aEuro degrees salt concentration for 2 h. The results showed that LJHsp70 may be a kind of potential biomarker used to monitor environment conditions.

Cloning of cytoplasmic heat shock protein 90 (FcHSP90) from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia

Heat shock protein 90 (HSP90) works as a multi-functional chaperone and is involved in the regulation of many essential cellular pathways. In this study, we have identified a full-length complementary DNA (cDNA) of HSP90 (FcHSP90) from Chinese shrimp Fenneropenaeus chinensis. FcHSP90 full-length cDNA comprised 2,552 bp, including a 2,181-bp open reading frame encoding 726 amino acids. Both homology analyses using alignment with previously identified HSP90 and a phylogeny tree indicated that FcHSP90 was a cytoplasmic HSP90. Real-time reverse transcription polymerase chain reaction analysis revealed that FcHSP90 was ubiquitously expressed in all the examined tissues but with highest levels in ovary of F. chinensis. FcHSP90 mRNA levels were sensitively induced by heat shock (from 25A degrees C to 35A degrees C) and reached the maximum at 6 h during heat shock treatment. Under hypoxia conditions, FcHSP90 mRNA levels, in both hemocytes and gill, were induced at 2 h and depressed at 8 h during hypoxia stress. The assessment of FcHSP90 mRNA levels under heat shock and hypoxia stresses indicated that the transcription of FcHSP90 was very sensitive to heat shock and hypoxia, so we deduced that FcHSP90 might play very important roles for shrimp to cope with environmental stress.

The integrative expression of GUS gene driven by FCP promoter in the seaweed Laminaria japonica (Phaeophyta)

In this study, the background activity of beta-glucuronidase (GUS) was analyzed histochemically and fluorometrically in the negative control of Laminaria japonica (Phaeophyta) thalli, showing low level of activity. GUS gene transformation without selectable gene in L. japonica was performed using four different promoters, i.e., Cauliflower mosaic virus 35S promoter (CaMV35S) from cauliflower mosaic virus, ubiquitin promoter (UBI) from maize, adenine-methyl transfer enzyme gene promoter (AMT) from virus in green alga Chlorella, and fucoxanthin chlorophyll a/c-binding protein gene promoter (FCP) from diatom Phaeodactylum tricornutum. The GUS transient activity was determined fluorometrically after bombarding sliced parthenogenetic sporophytes explants, and it was found that the activity resulting from CaMV35S and FCP promoters (in 114.3 and 80.6 pmol MU min(-1) (mg protein)(-1), respectively) was higher than for the other two promoters. The female gametophytes were bombarded and regenerated parthenogenetic sporophytes. FCP was the only promoter that resulted in detectable GUS chimeric expression activity during histochemical staining and polymerase chain reaction. Results of Southern blot showed that GUS gene was integrated with the L. japonica genome.

The polymorphism of lysozyme gene in Zhikong scallop (Chlamys farreri) and its association with susceptibility/resistance to Listonella anguillarum

Lysozyme functions as a crucial biodefence effector against the infection of bacterial pathogens in innate immunity. The nucleotide sequence polymorphisms in promoter region of a nuclear goose type lysozyme gene from Zhikong scallop Chlamys farreri (designated as CFLysG) were investigated to explore their association with susceptibility/resistance to Listonella anguillarum infection. Eight sites of single nucleotide polymorphisms (SNPs) and two sites of insert-deletion (ins-del) polymorphisms were identified in the promoter region of CFLysG. Two of them, -753 TATCTCGATCAGG ins-del polymorphism and -391 A-G SNP were selected to analyze their distribution in the susceptible and resistant stocks, which were identified according to the survival time after L. anguillarum challenge. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), two genotypes were found at each site, which were ins/del and ins/ins at locus -753, and A/A and A/G at locus -391, respectively. The -753 ins/del genotype was more prevalent in the resistant stock than that in the susceptible stock, 30% vs 16.67% in frequency, but there was no significant difference in the frequency distribution between these two stocks (P=0.15). In contrast, the frequency of -391A/G genotype in the resistant stock was significantly higher (30%) than that in the susceptible stock (7.14%) (P=0.007), indicating a significant association with the resistance of Zhikong scallop to L anguillarum. To confirm the presumption, another independent challenge experiment was performed, in which the cumulative mortality of scallops with -391 A/A genotype (96.8%) was significantly higher than those with -391 A/G genotype (64.5%) (P=0.001), which further validate the association between -391 A/G genotype and the resistance of Zhikong scallop to L anguillarum. These results suggested that the -391 A/G could be a potential marker applied in future selection of Zhikong scallop with enhanced resistance to L anguillarum. (C) 2008 Elsevier Ltd. All rights reserved.

The complete mitochondrial genome sequence of the cutlassfish Trichiurus japonicus (Perciformes: Trichiuridae) : Genome characterization and phylogenetic considerations

Mitochondrial genome sequence and structure analysis has become a powerful tool for studying molecular evolution and phylogenetic relationships. To understand the systematic status of Trichiurus japonicus in suborder Scombroidei, we determined the complete mitochondrial genome (mitogenome) sequence using the long-polymerase chain reaction (long-PCR) and shotgun sequencing method. The entire mitogenome is 16,796 by in length and has three unusual features, including (1) the absence of tRNA(Pro) gene, (2) the possibly nonfunctional light-strand replication origin (O-L) showing a shorter loop in secondary structure and no conserved motif (5'-GCCGG-3'), (3) two sets of the tandem repeats at the 5' and 3' ends of the control region. The three features seem common for Trichiurus mitogenomes, as we have confirmed them in other three T. japonicus individuals and in T nanhaiensis. Phylogenetic analysis does not support the monophyly of Trichiuridae, which is against the morphological result. T. japonicus is most closely related to those species of family Scombridae; they in turn have a sister relationship with Perciformes members including suborders Acanthuroidei, Caproidei, Notothenioidei, Zoarcoidei, Trachinoidei, and some species of Labroidei, based on the current dataset of complete mitogenome. T japonicus together with T. brevis, T lepturus and Aphanopus carbo form a clade distinct from Lepidopus caudatus in terms of the complete Cyt b sequences. T. japonicus mitogenome, as the first discovered complete mitogenome of Trichiuridae, should provide important information on both genomics and phylogenetics of Trichiuridae. (C) 2009 Elsevier B.V. All rights reserved.

Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15A degrees C and 3.9-fold at 4A degrees C compared to the alga at 25A degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.

Cloning, characterization and expression of ferritin subunit from clam Meretrix meretrix in different larval stages

Shell formation is one of the important events during larval development and metamorphosis in bivalves. However, the molecular mechanisms and environmental cues regulating shell initiation and growth are unclear. Here, we report that ferritin, a principal protein for biological iron storage and metabolism, might play a role in larval shell development of the bivalve mollusk Meretrix meretrix. A full-length ferritin subunit cDNA, named as MmeFer, was cloned and characterized. The MmeFer mRNA expression in different developmental stages, from trochophore to post larvae, was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). MmeFer mRNA expression in larvae of later developmental stages increased at least 8-fold following trochophores. Moreover, the temporal and spatial expressions of MmeFer mRNA were examined by whole mount in situ hybridization. In the trochophore stage, MmeFer was detectable where it was supposed to be for shell initiation. In the later developmental stages, MmeFer was found near digestive glands and mantle that secret larval shell. MmeFer expression was also detected in larvae cultured in artificial seawater with different iron concentrations ranging from 0 to 100 mu M. These results suggest that ferritin may play a role in the shell formation of mollusks. (C) 2009 Elsevier Inc. All rights reserved.

Molecular cloning and expression profile of putative antilipopolysaccharide factor in Chinese shrimp (Fenneropenaeus chinensis)

A new antimicrobial protein gene of the anti-lipopolysaccharide factor family (tentatively named as ALFFc) has been cloned from hemocytes of the Chinese shrimp Fenneropenaeus chinensis by rapid amplification of 3' and 5' complementary DNA ends with polymerase chain reaction. The full-length complementary DNA of ALFFc consists of 600 bp with a 369-bp open reading frame, encoding 123 amino acids. The deduced peptide contains a putative signal peptide of 25 amino acids and mature peptide of 98 amino acids. The molecular mass of the deduced mature peptide is 13799.16 Da. It is highly cationic, with a theoretical pI of 10.3. The deduced amino acid sequence of ALFFc showed 56% homology with sequences of Tachypleus tridentatus and L. polyhemus. The tissue expression profile of this gene was studied by Northern blot, and ALFFc transcripts were mainly detected in hemocytes, gill, and intestine. RNA in situ hybridization showed that ALFFc was constitutively expressed in hemocytes. Capillary electrophoresis reverse transcriptase PCR was used to quantify the variation of messenger RNA transcription level during the artificial infection process with Vibrio anguillarum. Significant enhancement of ALFFc transcription appeared during the first 24 hours in response to Vibrio infection. These results provide useful information for understanding the function of ALFFc in shrimp.

Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.