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     

Growth performance,carcass quality characteristics and colonic microbiota profiles in finishing pigs fed diets with different inclusion levels of rice distillers’ by‐product

The aim of this study was to evaluate the effects of diets containing rice distillers’ by‐product (RDP) on growth performance, carcass characteristics, meat quality, and gut microbiota of fattening pigs. Twenty‐four crossbred finishing pigs (Duroc × Landrace × Yorkshire), 56.9 ± 3.1 kg initial body weight, were randomly allocated to three groups. For 56 days, pigs were fed one of three diets including RDP0 (control), RDP15 (15% RDP in DM), and RDP30 (30% RDP in DM). With RDP level in diet, average daily gain and backfat thickness linearly increased (p < 0.05), and drip loss tended to increase (p ≤ 0.08). In addition, 16S ribosomal RNA gene amplicon profiling showed that RDP was associated with modulation of colonic microbiota composition, especially at family and genus levels. Relative abundance of Porphyromonadaceae and Erysipelotrichaceae families in colonic digesta increased with inclusion of RDP, while that of Enterobacteriaceae decreased. The proportion of genera unclassified Erysipelotrichaceae, and Butyrivibrio increased as inclusion of RDP. These results indicate that up to 30% inclusion in diet of finishing pigs, RDP can modulate colonic microbiota composition, and induces an improvement of animal growth and fat deposition.     

Comparative analysis of evaluation parameters in E. acervulina,E. maxima and E. tenella-infected broilers

Three parameters, body weight gain (BWG), intestinal lesion score (LS) and fecal oocyst shedding, were compared in broilers infected with major parasitic species; Eimeria acervulina, E. maxima, and E. tenella. First, two- and three-week-old chickens with Eimeria infection showed LS of approximately 3, but two-week-old chickens were more correlated with BWG. Second, significant differences in BWG were observed between male and female broilers challenged with Eimeria. Finally, E. maxima-infected broilers among three Eimeria species showed a higher relationship between BWG and LS, suggesting three considerations such as genders, age and Eimeria species for Eimeria experiments.     

Seasonal effects on the aerobic bacterial and fungal conjunctival flora of normal thoroughbred brood mares in Florida

Objective To evaluate seasonal effects on the presence or absence of fungal and aerobic bacterial flora of the conjunctival fornix of normal Florida Thoroughbred horses. Sample population Both eyes of 100 horses. Procedure Horses with normal anterior segment ophthalmic examinations from three farms in north central Florida were included. Each animal had the ventral conjunctival fornix of each eye swabbed with sterile culturettes. Samples were taken in October, January, April, and July (1999–2000). Aerobic and fungal cultures were plated. Bacterial cultures were reviewed at 24 and 48 h. Fungal cultures were reviewed weekly for 4 weeks. Logistic regression analysis with season as a factor and age of the horse as a covariate was performed. Statistical significance was set at P < 0.01. Results Horses ranged from 3 to 24 years of age, with a median age of 9 years. Twenty‐four genera of bacteria and 35 genera of fungi were recovered. Corynebacterium sp., Staphylococcus sp., Bacillus sp. and Moraxella sp. were the bacteria most frequently isolated. Mold species, dematiaceous mold species, Chrysosporium sp., Cladosporium sp., and Aspergillus sp. were the most frequently recovered fungi. Season did not have a significant effect on the presence of microorganisms isolated for individual horses adjusted for age. Younger horses had an increased incidence of gram‐negative rods and fungal isolates. The number of bacteria and fungi isolated are not uniform across seasons. Conclusion There were no significant differences between the number or type of organisms cultured during the sampling seasons in normal Florida horses. A large range of normal bacterial and fungal flora were isolated from these horses. The number of bacteria and fungi isolated are not uniform across seasons. The likelihood of detecting an organism depends on the horses’ age.     

Adipose tissue gene expression in obese dogs after weight loss

Body weight (BW) mainly depends on a balance between fat storage (lipogenesis) and fat mobilization (lipolysis) in adipocytes. BW changes play a role in insulin resistance (IR), the inability of insulin target tissue to respond to physiological levels of insulin. This results in inhibition of lipogenesis and stimulation of lipolysis. Weight gain leads to IR whereas, weight loss improves insulin sensitivity (IS). The aim of this study was to evaluate the effect of weight loss and recovery of IS on the expression of genes involved in lipogenesis and lipolysis in weight losing dogs. Gene expression was studied in both subcutaneous and visceral adipose tissue. Obese dogs received a hypoenergetic low fat high protein diet (0.6 x NRC recommendation). Before and after weight loss, IS was assessed using the euglycaemic hyperinsulinaemic clamp. Gene expression of IRS-2, SREBP, intracellular insulin effectors, ACC, FAS, FABP, ADRP, PEPCK, lipogenesis key proteins, perilipin and HSL, lipolysis key proteins were quantified using real-time RT-PCR in subcutaneous and visceral fat. BW decreased from 15.2 +/- 0.5 to 11.4 +/- 0.4 kg (p < 0.05) over 78 +/- 8 days. When obese, dogs were insulin resistant. After weight loss, IS was improved. In the subcutaneous adipose tissue, the expression of only the IRS-2 was increased. In the visceral adipose tissue, the expression of the genes involved in the lipogenesis was decreased whereas one of the genes implied in the lipolysis did not change. The expression profile of genes involved in lipid metabolism, as measured after weight loss, is indicative for a lower lipogenesis after weight loss than in obese dogs. Our results also confirm dramatic differences in the lipid metabolism of visceral and subcutaneous fat. They should be completed by comparing gene expression during weight losing and normal weight steady state.     

Towards sustainable farming systems: Effectiveness and deficiency of the French procedure of sustainable agriculture

There is a widespread consensus about the importance of sustainable agriculture. In France, the Farming Orientation Law of July 1999 has set down a precise procedure for the implementation of sustainable agriculture: the contrat territorial d’exploitation – or territorial farm contract (TFC). This paper aims to analyse the TFC effectiveness. An analysis has been made of the TFCs signed in the Midi-Pyrenees Region in south-western France, using statistical analysis and qualitative surveys of a sample of farms. The results show that the most valuable effects have been mainly economic. Effects regarding social and environmental aspects were very limited. But from the point of view of an integrated approach, indirect effects of TFCs can be observed on social and environmental aspects. These results are explained, on the one hand, by farmers’ motivations guided by economic objectives, and on the other hand, by the dominance of professional farming organisations in the implementation of TFC’s procedure. Given these results, two ways of improving the elaboration procedure for TFCs are proposed. Firstly, technical improvements can be made to the diagnostic assessment of farms, and to the choice of actions addressed by the contract. Secondly, organisational improvements can be applied to TFCs elaboration and implementation process, in order to adopt a participatory approach which would involve all rural stakeholders.     

Water Deficit Reduced Fertility of Young Microspores Resulting in a Decline of Viable Mature Pollen and Grain Set in Rice

Pollen formation in rice ( Oryza sativa L.) is highly vulnerable to environmental stresses such as heat, chilling and drought. In rice plants exposed to drought during male reproductive development, the most obvious damage often observed is a decline in the number of engorged pollen and grain set. This has been well characterized in rice under chilling and to a lesser extent under drought stress. Moreover, detailed literature on the immediate effects of drought on developing young microspores in rice is still limited. Here, we report findings from experiments on rice plants exposed to water deficit for three consecutive days during early stages of anther development. When the osmotic potential of the growing medium was equal to or less than −0.5 MPa, as induced by polyethylene glycol, the leaf water potential was significantly lowered and grain set was reduced. A strong correlation between grain set and viable young microspores (P < 0.001, r² = 0.8223) indicates that water deficit immediately reduced fertility of rice plants at the time of exposure. This result suggests a new underlying mechanism of water deficit-induced pollen abortion in rice.     

Mapping QTL controlling soybean seed sucrose and oligosaccharides in a single family of soybean nested association mapping (SoyNAM) population

The meal value of Soybean for monogastric animals is determined partly by sucrose, raffinose and stachyose. Of these, sucrose is desirable, while raffinose and stachyose are indigestible, causing flatulence and abdominal discomfort. The objective of this study was to identify quantitative trait loci (QTL) controlling seed sucrose, raffinose, and stachyose in a set of 140 SoyNAM (Nested Association Mapping) recombinant inbred lines (RILs), developed from the cross between lines IA3023 and LD02‐4485. A total of 3,038 SNP markers from the Illumina SoyNAM BeadChip SNP were used to map the QTLs for sucrose and the RFOs, raffinose, and stachyose. Significant genotypic differences (p < .001) among RILs were observed for sucrose, raffinose and stachyose contents across years. A 3038 Illumina SoyNAM BeadChip SNPs identified three QTLs for sucrose, one on chromosome 1, explaining 10% variance and two on chromosome 3 each explaining 22%. Raffinose QTL was detected on chromosome 6, explaining 6% variance. The mapped QTLs were novel and spanned regions harbouring candidate genes with roles in plant growth including seed development.     

Crystallographic structure of the octameric histone core of the nucleosome at a resolution of 3.3 A

The structure of the (H2A-H2B-H3-H4)2 histone octamer has been determined by means of x-ray crystallographic techniques at a resolution of 3.3 angstroms. The octamer is a prolate ellipsoid 110 angstroms long and 65 to 70 angstroms in diameter, and its general shape is that of a rugby ball. The size and shape are radically different from those determined in earlier studies. The most striking feature of the histone octamer is its tripartite organization, that is, a central (H3-H4)2 tetramer flanked by two H2A-H2B dimers. The DNA helix, placed around the octamer in a path suggested by the features on the surface of the protein, appears like a spring holding the H2A-H2B dimers at either end of the (H3-H4)2 tetramer.     

Genetic control of acquired high temperature tolerance in winter wheat

Summary The development of high temperature-tolerant wheat (Triticum aestivum L.) germplasm is necessary to improve plant productivity under high-temperature stress environments. The quantification of high temperature tolerance and the characterization of its genetic control are necessary for germplasm enhancement efforts. This study was conducted to determine the genetic control of acquired high temperature tolerance in common bread wheat cultivars. Reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by heat-stressed seedling leaves was used as a quantitative measure to characterize acquired high temperature tolerance. Eleven-day-old seedlings of 20 F₁ progeny produced through a complete 5×5 (Payne, Siouxland, Sturdy, TAM W-101, and TAM 108) diallel mating design were acclimated at 37° C for 24 hours, followed by a 2-hour incubation at 50° C. Under these test conditions, acquired high temperature tolerance ranged from a high of 75.7% for the genotype TAM W-101 × TAM 108, to a low of 37.3% for the genotype Payne × Siouxland. Partitioning of genotypic variance revealed that only the general combining ability component effect was statistically highly significant, accounting for 67% of the total genotypic variation. These results suggest that enhancing the level of high temperature tolerance in wheat germplasm is feasible utilizing existing levels of genetic variability and exploiting additive genetic effects associated with high temperature tolerance.Contribution of the Texas Tech College of Agric. Sci. Journal no T-4-386. This work was supported by USDA specific agreement No. 58-7MNI-6-114 from the Plant Stress and Water Conservation Laboratory, USDA-ARS, Lubbock, Texas, USA