Nucleotide Sequence Of Region Research Articles

Identification and Pathogenicity of the Soybean Root Rot Pathogen in Arid Conditions

Soy is an important oilseed crop in Kazakhstan. However, its yield is significantly reduced due to an increase in the development of fungal diseases with soil infection. This study aims to identify the root rot pathogen in soybean varieties in Western Kazakhstan and assess its pathogenicity. Samples of soybean roots to determine the root rot pathogen were selected at the experimental plots of the Aktobe agricultural experimental station in 2021. Genetic analysis of isolates observed on potato dextrose agar medium from soybean root slices was carried out using the sequencing reaction of internal transcribed spacer region nucleotide sequences using the Big Dye Terminator protocol (applied biosystems) with a further comparison of data with the Gen bank database. A test for the pathogenicity of the identified isolates of the fungus was carried out. Statistical data processing was carried out using the R-studio software according to the nonparametric Kruskal Wallis test. Phylogenetic analysis of samples of soybean affected by root rot showed that the main biotic agent causing root rot was the imperfect fungus Fusarium equiseti. Sequencing of the highly informative internal transcribed spacer region of fungal isolates allowed identifying strains 1 and 2 belonging to the species Fusarium equiseti. The pathogenicity test established that isolates of the Fusarium equiseti fungus caused discoloration of seedlings and plant roots and necrosis in soybean seedlings.

Molecular Epidemiology Reveals the Co-Circulation of Two Genotypes of Coxsackievirus B5 in China.

Coxsackievirus B5 (CVB5) is an important enterovirus B species (EV-Bs) type. We used the full-length genomic sequences of 53 viral sequences from the national hand, foot, and mouth disease surveillance network in the Chinese mainland (2001-2021). Among them, 69 entire VP1 coding region nucleotide sequences were used for CVB5 genotyping and genetic evolution analysis. Phylogenetic analysis based on a data set of 448 complete VP1 sequences showed that CVB5 could be divided into four genotypes (A-D) worldwide. Sequences from this study belonged to genotypes B and D, which dominated transmission in the Chinese mainland. Two transmission lineages of CVB5 have been discovered in the Chinese mainland, lineage 2 was predominant. Markov chain Monte Carlo analysis indicated that the tMRCA of CVB5 in the Chinese mainland could be traced to 1955, while the global trend could be traced to 1862, 93 years earlier than China. The evolution rate of CVB5 was higher in the Chinese mainland than worldwide. The spatiotemporal dynamics analysis of CVB5 assessed that virus transportation events were relatively active in Central, Northeast, North and Northwest China. Recombination analysis revealed frequent intertypic recombination in the non-structural region of CVB5 genotypes B and D with the other EV-Bs, revealing eight recombination lineages. Our study showed the molecular evolution and phylogeography of CVB5 that could provide valuable information for disease prevention.

Outbreak of Parasitic Dinoflagellate Piscinoodinium sp. Infection in an Endangered Fish from India: Arulius Barb (Dawkinsia arulius).

Freshwater velvet disease is caused by the dinoflagellate parasite, Piscinoodinium sp. This parasite has been reported in tropical and subtropical fishes, and it can cause devastating losses. Moreover, Piscinoodinium sp. is identified as one of the least studied finfish parasites, and the available molecular information about this parasite is meager. Recently, Piscinoodinium sp. was responsible for the 100% cumulative mortality of the captive-bred F1 generation of Arulius barb (Dawkinsia arulius), an endangered freshwater fish native to India. The trophont stages of the parasite were observed in the skin and gills of the affected fish. The total DNA was extracted from the trophonts collected from the affected Arulius barb and the partial nucleotide sequence of the rDNA complex region (2334 bp) was amplified using PCR. The amplified PCR product exhibited a high sequence identity (97.61%) with Piscinoodinium sp. In the phylogenetic analysis of the SSU rDNA, Piscinoodinium sp. emerged as a separate clade from other dinoflagellate species. This is the first report of the infection of Piscinoodinium sp. in Arulius barb and the molecular information generated from this study can serve as a baseline to study the diversity of the parasite in India. Furthermore, the impact of this parasite among wild fish stock is not known, and this parasite needs further research focus to generate more molecular information and to understand the host-pathogen interaction.

40P The detection and quantification of different sequence-variable NPM1 mutations using RNase H-dependent PCR (rhPCR)

Molecular diagnostics for the detection and characterization of genetic variants is based in the majority of cases on PCR technology. Since the primers and probes are designed according to the nucleotide sequence of the DNA region for the specific mutation, PCR technology can only be applied if the mutation is well known. In cases where the mutated region of a gene may have variable sequences, different between patients, a set of primers for each sequence variant is necessary, which involves a large volume of work.

Molecular Detection and Characterization of Trypanosomes Infecting Traditionally Managed Cattle in the Tropic Warm Sub-Humid Zone of Nigeria

Abstract Traditionally managed cattle constitutes the main source of animal protein to humans in Nigeria. However, seasonal migration in search of pasture exposes them to several vector-borne infections such as the African Animal Trypanosomosis (AAT), which limits their productivity. In this study, blood samples from 130 cattle in Plateau and Nasarawa states collected from May to June, 2021 were examined by the Polymerase Chain Reaction (PCR) and sequencing methods to determine the prevalence of pathogenic trypanosomes. Overall, the DNA of T. vivax was detected in 19 out of the 130 (14.6 %) samples examined by the PCR. However, using the micro-hematocrit centrifugation technique, motile haemoparasites were detected in only six (4.6 %, confidence interval [CI] 0.5—6.9 %) of the samples. The higher prevalence of T. vivax was recorded in samples sourced from the abattoir than in samples submitted from the field in Plateau state (16.7 % versus 11.5 %). However, the reverse was the case in Nasarawa state (2.9 % versus 37.5 %). The difference in prevalence of T. vivax between the abattoir and field samples was significant (P = 0.009) in Nasarawa state, but not in Plateau state (P = 0.55). The mean PCV (Packed Cell Volume) of the trypanosome infected animals was lower than that of the non-infected animals, but the difference was not significant (P = 0.29). The internal transcribe spacer region (ITS) nucleotide sequences of T. vivax generated in this study were 100 % identical to each other and formed a monophyletic cluster with the sequences of T. vivax from different countries in the GenBank. AAT remains a major constraint to profitable cattle production and food security in Nigeria and deserves more attention.

Mechanisms of action of cytoplasmic microRNAs. Part 1. Mechanisms of interaction of microRNA and mRNA molecules. Influence of microRNAs on translation

The scientific review presents the mechanisms of action of cytoplasmic miRNAs, namely the relationship between miRNA and mRNA molecules and the influence of miRNAs on translation. To write the article, information was searched using Scopus, Web of Science, MEDLINE, PubMed, Google Scholar, Embase, Global Health, The Cochrane Library, CyberLeninka databases. The authors state that the interaction of microRNA and mRNA requires the presence in the region of the 3'-end of the mRNA molecule of small nucleotide sequences — miRNA regulatory elements, which are complementary to the sequences of the “seed” region of microRNA. It is known that only six nucleotide matches in the “seed” region (position 2–8) are required to initiate the interaction of microRNA with the mRNA target. It is emphasized that the interaction of miRNA with mRNA depends on the availability of the mRNA binding site. The authors suggest that accessory proteins are involved in the interaction of microRNA and mRNA. It is known that the process of mRNA and miRNA hybridization depends on the presence of SNP. Scientists believe that the main function of cytoplasmic miRNAs is to regulate the activity of protein synthesis. It is presented that microRNAs can repress and activate the mRNA translation process. In addition, some miRNAs are able to both inhibit and enhance the translation of mRNA depending on specific local conditions and the spectrum of microenvironmental factors. Thus, the mechanism of action of cytoplasmic miRNAs is realized due to the interaction of miRNAs and mRNAs, which is due to the presence of complementary nucleotide sequences of special regions. The interaction of miRNAs with mRNAs depends on the availability of the mRNA binding site, the involvement of accessory proteins, and the presence of SNP. Violations of microRNA-mRNA interactions lead to the development of pathological processes. Cytoplasmic miRNAs perform their main function, namely the regulation of protein synthesis activity, due to miRNA-mediated repression and activation of mRNA translation.

Isolation and characterization of Bradyrhizobium elkanii as a root nodule symbiont of red sword bean Canavalia gladiata var. gladiata

ABSTRACT Bacterial strains were isolated from root nodules of red sword bean (Canavalia gladiata var. gladiata) cultivated in Shizuoka, Japan, in order to elucidate the taxonomy of the symbionts. Of the 52 bacterial isolates, 28 strains were identified as the genus Bradyrhizobium and 10 as Rhizobium, based on the nucleotide sequences of 16S rRNA genes. Nine Bradyrhizobium isolates, which were phylogenetically selected from the 28 strains, exhibited nucleotide sequences of ITS regions that were 99.9% or 100% identical with known B. elkanii strains. These nine strains shared more than 70% similarity with B. elkanii USDA 76 T or USDA 94 in DNA-DNA hybridization analysis, indicating that the strains are B. elkanii. In a nodule formation experiment using red sword bean seeds treated with mercury chloride solution, all of the plants which were inoculated with each of the B. elkanii strains (the isolate TI06 or MI08, or the type strain USDA 76 T) formed round-type root nodules (234–664 nodules per plant), while no nodules were observed in control plants that were cultivated without inoculating bacterial strains. The bacterial strains, which were isolated from the obtained nodules, exhibited nucleotide sequences of the ITS regions that were identical to those of the corresponding inoculated strains. The root nodules formed in the experiment exhibited acetylene-reducing activity, suggesting the nitrogen-fixation activity of the nodules. We thus conclude that B. elkanii is a root nodule symbiont of red sword bean.

Complete genome sequence of pueraria virus A, a new member of the genus Caulimovirus.

The complete genome sequence of a new caulimovirus in Pueraria montana was determined using high-throughput sequencing. The 7,572 nucleotide genome of pueraria virus A (PVA) contains genes that encode a movement protein, an aphid transmission factor, a virion-associated protein, a coat protein, a protease + reverse transcriptase + ribonuclease H, and a transactivator/viroplasmin protein, as well as two intergenic regions, which are all common features of members of the genus Caulimovirus. A sequence alignment revealed that the complete genome of PVA shares 66.82% nucleotide sequence identity with strawberry vein banding virus (GenBank accession no. KX249738.1). The results of phylogenetic analysis and the observation that the nucleotide sequence of the polymerase coding region differed by more than 20% indicated that PVA is a member of a new species the genus Caulimovirus, family Caulimoviridae.

First Report of Alternaria alternata Causing Leaf Spot on Kidney Bean in China.

Kidney bean (Phaseolus vulgaris), also being known as common bean, dry bean or french bean, is one of the most precious and highly nutritious legume crop cultivated and consumed worldwide(Blair et al.,2012; Choudhary et al.,2018) , which is an important edible foods or one of the most economically important vegetable crops in China. It is widely grown in the Heilongjiang Province in China. In July of 2020, leaf spot symptoms were found on the old or new leaves of Kidney bean plants (Phaseolus vulgaris L.) in our experimental fields located in Zhaozhou County(N45°42 '20.16 ", E125°15' 58.63" ), Daqing City, Heilongjiang Province, China. This field had disease incidences of approximately 20%. The leaf spot is conducive to the onset at high temperature and humidity environment, and this disease spreads very quickly after rainy days, therefore it is potentially a large risk for the development of Kidney bean industry. In its early occurrence phase, the infected leaves showed yellowish halo on the leaves, in which the middle mesophyll lost green. Thereafter, the yellow halo turned brown, and the middle leaf tissue of the halo appeared brown, ultimately the whole leaves had many brown spots (Supplementary Figure S1). To isolate the pathogen, diseased tissue (5×5 mm) was excised from the margins of individual lesions from the leaves of diseased plants with typical symptoms, and was disinfected with 75% ethanol for 10s followed by 2% NaClO for 3 min and then washed five to eight times with sterile water. Afterwards, the samples were transferred to potato dextrose agar (PDA) plates and incubated. After 5 to 7 days of incubation at 25°C (Wei et al.,2018), the mycelia were dark green with white margins in obverse and dark in reverse. Conidiophores were light brown with 2 to 4 septa and obclavate, 17.5to 44.0 × 6.5 to 14.5μm, with a short beak, and with 1 to 5 transverse septa and 0 to 2 longitudinal septa, light brown to olive-brown (Supplementary Figure S2). Based on morphological features and sporulation pattern, the pathogen was similar to characteristics described Alternaria alternate (Zhou et al,2014), being identified as A. alternata. To confirm pathogenicity, the isolates were cultured on PCA for 7days to prepare conidial suspensions, then being produced a final concentration of 1×108 spores/ml. Five potted Kidney bean plants were sprayed with conidial suspensions, and five control potted plants were sprayed with sterile distilled water, in which these potted Kidney bean plants were treated after wiping each leaf surface with 75% ethanol and washing each leaf with sterilized distilled water five times. These plants were incubated in an artificial growth chamber at 26 to 28°C with a 12 h light/dark photoperiod, with 85% relative humidity. After 3 days, yellowish halo lesions appeared on the inoculated plants, and pale lesions with distinct dark brownish red borders on Kidney bean leaves were observed after eight days, but no lesions were observed on the control leaves. Pathogenicity tests were repeated three times. The internal transcribed spacer (ITS) region of rDNA was amplified and sequenced with primers ITS1/ITS4. BLAST analysis of the sequences showed 100% sequence identity with a pathogenic A. alternata (Fr.) Keissl (Supplementary Figure S3), and the nucleotide sequence of the ITS region was submitted to GenBank under accession MZ951052. In China, there are no detailed records about the causal agent of this disease on Kidney bean in a paper in Chinese. To our knowledge, this is the first confirmed report of leaf spot causing by A. alternata on Kidney bean in China.

Excessive replacement changes drive evolution of global sheep prion protein (PRNP) sequences.

Sheep prion protein (PRNP) is the major host genetic factor responsible for susceptibility to scrapie. We aimed to understand the evolutionary history of sheep PRNP, and primarily focused on breeds from Turkey and Ethiopia, representing genome-wise ancient sheep populations. Population molecular genetic analyses are extended to European, South Asian, and East Asian populations, and for the first time to scrapie associated haplotypes. 1178 PRNP coding region nucleotide sequences were analyzed. High levels of nucleotide diversity driven by extensive low-frequency replacement changes are observed in all populations. Interspecific analyses were conducted using mouflon and domestic goat as outgroup species. Despite an abundance of silent and replacement changes, lack of silent or replacement fixations was observed. All scrapie-associated haplotype analyses from all populations also showed extensive low-frequency replacement changes. Neutrality tests did not indicate positive (directional), balancing or strong negative selection or population contraction for any of the haplotypes in any population. A simple negative selection history driven by prion disease susceptibility is not supported by the population and haplotype based analyses. Molecular function, biological process enrichment, and protein-protein interaction analyses suggested functioning of PRNP protein in multiple pathways, and possible other functional constraint selections. In conclusion, a complex selection history favoring excessive replacement changes together with weak purifying selection possibly driven by frequency-dependent selection is driving PRNP sequence evolution. Our results is not unique only to the Turkish and Ethiopian samples, but can be generalized to global sheep populations.

Origin and phylogenetic analysis of Pasundan cattle based on D-loop of Mitochondrial genome

Pasundan cattle is one of the native Indonesian species and molecular data is needed to further generate historical and ancestral information about cattle breeds. Therefore, to understand the maternal inheritance and phylogeny of Pasundan cattle, the nucleotide sequence of the D-loop region was analyzed. The DNA samples were collected from the Research Center for Biotechnology, BRIN, Cibinong, Indonesia. The phylogenetic tree reconstruction was analyzed using the Neighbor-joining method based on Kimura 2 Parameters. Based on the phylogenetic analysis, the Pasundan cattle showed two clusters, namely Bos indicus and Bos javanicus CERZA. Furthermore, the result showed that they originated from two maternal lineages and native cattle in Indonesia, including Madura cattle.

Genetic Variation Analysis between Wild and Cultured Pangasianodon hypopthalmus Using COI and Cytochrome b Among Asian Countries

One of the top species in the aquaculture sector, known as striped catfish or Pangasianodon hypophthalmus, is an important and valuable freshwater fish in many countries. Due to the high demand for this species, their number has declined to "threatened" levels. Hence, the purpose of this study is to analyse the genetic variation of wild and cultured striped catfish collected from five producers in Asian countries; Thailand, Vietnam, Indonesia, India, and Philippines, by using mitochondrial DNA partial region data sequence; CO1 and cytochrome b gene. Population analyses using 395 base pairs length for CO1 and 275 base pairs length of cytochrome b partial region nucleotide sequence have shown no significance difference between wild and cultured striped catfish. Vietnam species had shown a wide range of genetic distance of the intrapopulation compared with other countries in the range of 0.000-0.040 for CO1 gene and 0.003-0.008 for cytochrome b gene. The Neighbour-joining method has also been used to construct phylogenetic trees using CO1 gene; the tree formed few subclades with mixed populations, and the tree using cytochrome b showed only Vietnam species divided into a few sub-populations. For the other four countries, Thailand, Indonesia, India, and Philippines were in the same group. Hence, this study's findings may provide a reference for inter and intra-relationships of P. hypophthalmus that may help in the aquaculture activity of this striped catfish.

Characterization and validation of an alternative reference bacterium Korean Pharmacopoeia Staphylococcus aureus strain.

The National Culture Collection of Pathogens (NCCP) is a microbial resource bank in Korea that collects pathogen resources causing infectious disease in human and distributes them for research and education. The NCCP bank attempts to discover strains with various characteristics and specific purposes to provide diverse resources to researchers. Staphylococcus aureus American Type Culture Collection (ATCC) 6538P is used as a reference strain in the microbial assay for antibiotics in the Korean and in the United States Pharmacopoeias. We aimed to analyze domestically isolated microbial resources from the NCCP to replace the S. aureus reference strain. Staphylococcus aureus strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the VITEK-2 system and characterized by multilocus sequence typing, 16S rRNA sequencing, and antibiotic susceptibility testing. Several candidate strains had similar characteristics as the reference strain. Among them, the nucleotide sequence of the 16S rRNA region of NCCP 16830 was 100% identical to that of the reference strain; it was sensitive to six types of antibiotics and showed results most similar to the reference strain. A validity evaluation was conducted using the cylinder-plate method. NCCP 16830 presented valid results and had the same performance as ATCC 6538P; therefore, it was selected as an alternative candidate strain.

First records of three Lepiota species (Agaricales, Basidiomycota) from Ukraine, with notes on a poorly known species, Lepiota subalba

New records of four species of the genus Lepiota (Agaricales, Basidiomycota) are reported from Ukraine. Three species, L. fuscovinacea, L. griseovirens, and L. roseolivida, are recorded in Ukraine for the first time, whereas a poorly known species, L. subalba, earlier known in Ukraine from a few records, is confirmed using molecular identification methods. All species reports are supplemented with original descriptions and drawings based on newly collected material, as well as data on general distribution, habitat, references to new collections and public databases. Original nucleotide sequence of the ITS region of ribosomal DNA obtained from our voucher specimen of L. subalba is provided.

Lactoferrin gene polymorphism of exons 8 and 13 in Murrah buffalo

Lactoferrin is one of the important candidate genes for mastitis resistance in dairy animals. The gene is located on chromosome BBU21 and consists of 17 exons spanning over 32.95 kb of genomic DNA. The present study was undertaken with the objectives to identify allelic variants in exon 8 and 13 of lactoferrin gene and to analyze their association with incidence of clinical mastitis in Murrah buffalo. A total of 200 lactating Murrah buffaloes, grouped as mastitis affected and non-affected, were included in the study. Genomic DNA was isolated from the whole blood sample of each animal. Two primer sets were used to amplify exon 8 and 13 of lactoferrin gene, which yielded respective amplicons of 216 bp and 211 bp. The PCR-RFLP analysis of lactoferrin gene revealed monomorphic pattern in exon 8 and polymorphic pattern in exon 13. Hpy 188I -RFLP for exon 13 exhibited polymorphism with three genotypes: AA, AB and BB with respective frequencies of 0.20, 0.58 and 0.22 whereas, frequencies for A and B alleles were estimated as 0.49 and 0.51. Comparison of nucleotide sequence of exonic region of lactoferrin gene in Murrah buffalo with that of Bos taurus cattle revealed a total of 5 mutations out of which 2 were transition and 3 were transversion. The SNPs in exon 8 were found to be non synonymous and revealed two amino acid changes in exon 8 of Murrah buffalo as compared to Bos taurus cattle. Chi-square (χ2) analysis indicated non significant association between genetic variants of exon 13 and incidence of clinical mastitis.

Molecular identification and technological properties of yeasts isolated from spontaneously fermented cassava waste pulp

The aim of this work was to report on molecular identification and technological properties of the yeast flora isolated from spontaneously fermented cassava waste pulp. This was done with a view of obtaining yeast strains that could be used as a starter culture for the fermentation of cassava waste pulp. Molecular identification was based on the nucleotide sequence of the ITS region of the genomic DNA of the yeast isolates while the technological properties evaluated include linamarase (U/mL), gelatinase, and haemolytic activity; growth at pH 2.5 and tolerance to 2 % bile salt. All the representative five isolated yeasts were identified as Geotrichum silvicola KLP04 – KLP08. The isolates exhibited linamarase activity ranging between 3.3 and 4.2 with strain KLP04 having the highest value and strain KLP05 the least. None of the isolates demonstrated gelatinase and haemolytic activity except strain KLP08 which was partially haemolytic. All the examined yeasts exhibited good growth at pH 2.5, with strain KLP08 having the highest viable counts of 4.1 log10cfu/ml and strain KLP04 the least value of 3.5 log10cfu/ml after 72 h of growth. All the identified yeasts showed strain-specific tolerance to 2% bile salt with strain KLP04 having the highest viable count of 4.3 log10cfu/ml and strain KLP08 the least value of 2.2 log10cfu/ml at the end of 72 h of incubation. Based on all the examined technological properties, Candida silvicola KLP04 strain had the highest potential to be considered for starter culture for the fermentation of cassava waste pulp.

The perspectives for DNA barcoding of Rhaponticum carthamoides (Willd.) Iljin using rbcL gene sequence

The methods of biological species identification using nucleotide sequences of short genome regions (DNA barcoding) are actively developed. The universal DNA barcode for plants remains to be discovered, and one of the leading candidates is the plastid gene of the large subunit of ribulose-bisphosphate carboxylase gene (rbcL). In our study, we estimated the part of rbcL gene as a possible marker for molecular identification of Rhaponticum carthamoides (Willd.) Iljin. Due to its officinal properties, the species is susceptible to uncontrolled and illegal harvesting from natural populations. Today, the species needs to be protected and therefore is included into the Red Data Books of the Russian Federation and certain regions. The study was carried out using plants from the natural populations sampled from the Khamar-Daban Ridge (South Siberia) and considering now as Rh. carthamoides var. chamarense (Peschkova) O S Zhirova. It was shown that rbcL gene can be used to identify Rh. carthamoides at least from the populations of the Khamar-Daban Ridge using a fragment of the maximum length or its 3’ region. Apparently, the 5’ region of the gene (rbcLa) most often used as DNA barcode for plants may be of lesser importance for Rh. carthamoides. The rbcL gene sequences can be also used for the development of approaches for Rh. carthamoides identification in the medicinal preparations and products containing dried tissues to prevent their falsification and illegal harvesting of this species. The combination of rbcL gene with additional markers seems to be highly desirable to create effective DNA barcodes for Rhaponticum species.

Identification of Fungal Pathogens to Control Postharvest Passion Fruit (Passiflora edulis) Decays and Multi-Omics Comparative Pathway Analysis Reveals Purple Is More Resistant to Pathogens than a Yellow Cultivar.

Production of passion fruit (Passiflora edulis) is restricted by postharvest decay, which limits the storage period. We isolated, identified, and characterized fungal pathogens causing decay in two passion fruit cultivars during two fruit seasons in China. Morphological characteristics and nucleotide sequences of ITS-rDNA regions identified eighteen isolates, which were pathogenic on yellow and purple fruit. Fusarium kyushuense, Fusarium concentricum, Colletotrichum truncatum, and Alternaria alternata were the most aggressive species. Visible inspections and comparative analysis of the disease incidences demonstrated that wounded and non-wounded yellow fruit were more susceptible to the pathogens than the purple fruit. Purple cultivar showed higher expression levels of defense-related genes through expression and metabolic profiling, as well as significantly higher levels of their biosynthesis pathways. We also found fungi with potential beneficial features for the quality of fruits. Our transcriptomic and metabolomics data provide a basis to identify potential targets to improve the pathogen resistance of the susceptible yellow cultivar. The identified fungi and affected features of the fruit of both cultivars provide important information for the control of pathogens in passion fruit industry and postharvest storage.

Detection and molecular characteristics of Pyelosomum cochlear (Digenea: Pronocephalidae) in the urinary bladder of the green sea turtle (Chelonia mydas) in the Northwest Pacific Ocean.

The genus Pyelosomum consists of parasitic flukes occurring primarily in marine turtles; Pyelosomum cochlear Looss 1899 is the only species of this genus that parasitizes the urinary bladder. In this study, we detected flukes in the urinary bladders of 20 of 88 green sea turtles (Chelonia mydas) harvested in the Ogasawara Islands, in the Northwest Pacific Ocean. We identified the flukes as P. cochlear based on detailed morphological observations and comparisons of morphometric measurements of the species reported previously. Nucleotide sequences of nuclear ribosomal 18S and 28S regions and the mitochondrial cytochrome c oxidase subunit 1 (COI) region were determined for the flukes. The 18S and 28S phylogenetic trees revealed that the species of the superfamily Pronocephaloidea, including P. cochlear, constituted a single clade, but the species of the family Pronocephalidae did not constitute a single taxon. These findings suggest that Pronocephalidae is a paraphyletic group. The COI sequences of P. cochlear exhibited high genetic diversity, suggesting that they would be useful markers to understand the genetic structure of the parasite and its evolutionary relationship with the host turtle populations. This is the first study to provide the nucleotide sequences of Pyelosomum species; these data will be available for further molecular studies of this genus and its related taxa.

Identification and Validation of Four Novel Promoters for Gene Engineering with Broad Suitability across Species.

The transcriptional capacities of target genes are strongly influenced by promoters, whereas few studies have focused on the development of robust, high-performance and cross-species promoters for wide application in different bacteria. In this work, four novel promoters (Pk.rtufB, Pk.r1, Pk.r2, and Pk.r3) were predicted from Ketogulonicigenium robustum and their inconsistency in the -10 and -35 region nucleotide sequences indicated they were different promoters. Their activities were evaluated by using green fluorescent protein (gfp) as a reporter in different species of bacteria, including K. vulgare SPU B805, Pseudomonas putida KT2440, Paracoccus denitrificans PD1222, Bacillus licheniformis and Raoultella ornithinolytica, due to their importance in metabolic engineering. Our results showed that the four promoters had different activities, with Pk.r1 showing the strongest activity in almost all of the experimental bacteria. By comparison with the commonly used promoters of E. coli (tufB, lac, lacUV5), K. vulgare (Psdh, Psndh) and P. putida KT2440 (JE111411), the four promoters showed significant differences due to only 12.62% nucleotide similarities, and relatively higher ability in regulating target gene expression. Further validation experiments confirmed their ability in initiating the target minCD cassette because of the shape changes under the promoter regulation. The overexpression of sorbose dehydrogenase and cytochrome c551 by Pk.r1 and Pk.r2 resulted in a 22.75% enhancement of 2-KGA yield, indicating their potential for practical application in metabolic engineering. This study demonstrates an example of applying bioinformatics to find new biological components for gene operation and provides four novel promoters with broad suitability, which enriches the usable range of promoters to realize accurate regulation in different genetic backgrounds.