Agrobacterium-mediated transformation of the CrDAT gene and selection of transgenic periwinkle lines with a high vincristine accumulation

Catharanthus roseus contains vincristine and vinblastine, which are outstanding drugs for cancer. In the biosynthetic pathways of terpenoid indole alkaloids (TIAs) in C. roseus, deacetylvindoline 4-O-acetyltransferase (DAT) is a key enzyme that catalyses the last reaction of vindoline biosynthesis to form vinblastine and vincristine. In this study, the CrDAT transgene was transferred into the periwinkle by Agrobacterium-mediated transformation and generated transgenic periwinkle lines with an increase in vincristine accumulation. The C. roseus DAT gene was introduced into C. roseus plants and it was confirmed that CrDAT was successfully transferred into the genome of periwinkle plants and efficiently translated to synthesise recombinant DAT protein. Four transgenic periwinkle lines in T1 generation, T1-1, T1-3, T1-6, and T1-7, expressed recombinant DAT protein with the total protein content in the range of 2.86 μg.mg^?1 to 5.12 μg.mg^?1. Moreover, the vincristine contents of four transgenic lines increased by 1.63?2.48-fold compared to non-transgenic plants, ranging from 6.91 µg.g^?1 (fresh weight) to 10.53 µg.g^?1 (fresh weight). The T1-1 line had the highest vincristine content. Hence, the overexpression of the recombinant DAT protein can improve the vincristine accumulation of transgenic C. roseus plants.

Abbreviation: CrDAT - Catharanthus roseus Deacetylvindoline-4-O-Acetyl Transferase; D4H - Deacetoxyvindoline 4-hydroxylase; ELISA - Enzyme-Linked Immunosorbent Assay Monoterpene indole alkaloid; T0, T1 - Generations of transgenic plants; TIAs - Terpenoid indole alkaloids; WT- The wild-type tobacco plants (non transgenic plant); 35S - Cauliflower mosaic virus 35S promoter     

Neuroprotective Metabolites from Vietnamese Marine Derived Fungi of Aspergillus and Penicillium Genera

Low molecular weight secondary metabolites of marine fungi Aspergillus flocculosus, Aspergillus terreus and Penicillium sp. from Van Phong and Nha Trang Bays (Vietnam) were studied and a number of polyketides, bis-indole quinones and terpenoids were isolated. The structures of the isolated compounds were determined by 1D and 2D NMR and HR-ESI-MS techniques. Stereochemistry of some compounds was established based on ECD data. A chemical structure of asterriquinone F (6) was thoroughly described for the first time. Anthraquinone (13) was firstly obtained from a natural source. Neuroprotective influences of the isolated compounds against 6-OHDA, paraquat and rotenone toxicity were investigated. 4-Hydroxyscytalone (1), 4-hydroxy-6-dehydroxyscytalone (2) and demethylcitreoviranol (3) have shown significant increasing of paraquat- and rotenone-treated Neuro-2a cell viability and anti-ROS activity.     

Milk vetch dwarf virus infection in the Solanaceae and Caricaceae families in Southeast Asia

Milk vetch dwarf virus (MDV) is an important member of the genus Nanovirus and is transmitted by the aphid Aphis craccivora. MDV has multiple single-stranded DNA genome components, each approximately 1 kb, and two or three alpha-satellite molecules. It mainly infects plants of the legume family Fabaceae. Recently, papaya (Carica papaya) collected in Yesan, South Korea, displaying symptoms of leaf yellowing and dwarfism, was identified as a new host for MDV. To examine the geographical distribution of MDV, papaya samples with symptoms were harvested in South Korea, Vietnam, and Taiwan in August 2018, along with tomato and pepper samples grown in adjacent fields in Vietnam. The results revealed the presence of MDV not only in papaya but also in pepper and tomato. This MDV infection in members of the Solanaceae family was confirmed by Southern blot hybridization performed using a PCR product of segment S as a probe. Based on sequence analysis of three MDV components (M, S, and C3), we verified the presence of three different isolates of MDV in these three countries and homology between sequences of isolates from papaya and from members of the Solanaceae from Vietnam. Taken together, our results clearly demonstrate MDV infection in Vietnam and Taiwan for the first time and confirm that MDV can infect economically important pepper and tomato.     

Inclusion levels and modes of whole grain incorporation into wheat-based rations differentially influence the performance of broiler chickens

1. The objective was to compare three whole grain (WG) inclusion levels (7.5, 15 and 30%) offered to broiler chickens by three modes of WG incorporation: (i) pre-pellet WG inclusion, (ii) post-pellet WG inclusion as a blend of WG and pelleted concentrate and (iii) post-pellet WG inclusion where WG and pelleted concentrate were provided in separate feed trays against a ground-grain, wheat-based control diet.

2. Ten dietary treatments were offered to 6 replicate cages (6 birds per cage) of male Ross 308 chickens from 7 to 28 d post-hatch. The effects of treatment on relative gizzard weights, gizzard contents, pancreatic weights and pH of gizzard digesta were monitored. Parameters of growth performance, nutrient utilisation (apparent metabolisable energy [AME], metabolisable to gross energy [ME:GE] ratios, nitrogen [N] retention and N-corrected AME [AMEn]), apparent starch and protein (N) digestibility coefficients and disappearance rates in for small intestinal segments and concentrations of free amino acids in plasma taken from the anterior mesenteric vein were determined.

3. Whole grain feeding (WGF) did not influence weight gain, but 30% post-pellet blended and 15 and 30% post-pellet separated treatments significantly depressed (P < 0.05) feed intakes while the 30% post-pellet separated treatment improved (P < 0.01) feed conversion ratios (FCR). WGF regimes significantly increased relative gizzard weights.

4. Overall, WGF generated profound responses in AME, ME:GE ratios, N retention and AMEn that were highly correlated with relative gizzard weights. In general, WGF improved starch and protein (N) digestibilities and again there were some correlations with these outcomes and relative gizzard weights.

5. Post-pellet WG inclusions where WG and pelleted concentrate were offered separately provided chickens with the opportunity to choice feed. Birds showed a preference for the relatively high-protein pelleted concentrate and at 30% WG, this resulted in an improvement in FCR of 7.69% (1.260 versus 1.365; P < 0.001) relative to the ground-grain control diet.     

Effects of Dietary Supplementation of Barodon on Growth Performance,Innate Immunity and Disease Resistance of Juvenile Olive Flounder,Paralichthys olivaceus,Against Streptococcus iniae

This study was conducted to evaluate the effects of dietary supplementation of Barodon, an anionic alkali mineral complex, on growth, feed utilization, humoral innate immunity and disease resistance of olive flounder. A basal experimental diet was used as a control and supplemented with 0.1, 0.2, 0.3, 0.4, or 0.5% Barodon. Triplicate groups of fish (26.4 ± 0.2 g) were fed one of the diets to apparent satiation twice daily for 10 wk. The growth performance was enhanced (P < 0.05) linearly and quadratically in fish fed diets containing Barodon compared with that in fish fed the control. Feed utilization was significantly improved by Barodon supplementation. Serum lysozyme and antiprotease activities were increased quadratically in Barodon fed groups. Also, significantly higher superoxide dismutase activity was found in Barodon‐fed fish. Dietary supplementation of 0.1–0.3% Barodon resulted in significant enhancement of fish disease resistance against Streptococcus iniae. The findings in this study indicate that dietary supplementation of Barodon can enhance growth, feed utilization, innate immunity, and disease resistance of olive flounder and that the optimum level seems to be 0.1% in diets.     

Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR

BackgroundThe microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.ObjectivesThe present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees.MethodsA procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.ResultsUR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.ConclusionsUR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.     

A morphological analysis of the skull size and shape of Kerivoulinae (Chiroptera: Vespertilionidae) from Vietnam

Southeast Asia is a region of high biodiversity, containing species of plants and animals that are yet to be discovered. In this region, bats of the subfamily Kerivoulinae are widespread and diverse with six species recorded in Vietnam. However, the taxonomy of the Kerivoulinae in Asia is complicated. In our study, we used diagnostic characters and multivariate analysis to determine morphological differences between the genera Kerivoula and Phoniscus. We showed that the two genera are distinguishable by the size of second upper incisors, the shape of skull, nasal sinus, canines, second upper and lower premolars. In addition, the two genera can be osteometrically separated by measurements of the braincase height, interorbital width and shape of anterior palatal emargination. Our data clearly revealed the morphological variations in the skull shape of Kerivoula hardwickii in Vietnam. This suggests a possible separation into three morphotypes, representing cryptic species supported by statistical differences with wide variation in skull shape, size and teeth. These results demonstrated Kerivoula hardwickii can be separated three subspecies, and the result will serve as the basis for the future assessment and classification of this group in Southeast Asia.