Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

Efficient direct plant regeneration from immature leaf roll explants of sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) using polyamines and assessment of genetic fidelity by SCoT markers

A rapid and efficient method for in vitro direct plant regeneration from immature leaf roll explants of Saccharum officinarum L. (sugarcane) cv. Co 86032 was developed by the application of exogenous polyamines (PA). The effect of explant source from apical meristems and pre-culture of explants in the dark on shoot regeneration was studied. Adventitious shoot regeneration occurred on the proximal regions of immature leaf roll explants when pre-incubated in the dark for 2 wk and the regeneration response was decreased from the middle to distal end. A higher number of direct shoots (130 primary shoots explant^?1) and multiple shoots (657 secondary shoots explant^?1), were obtained with a combination of spermidine (103.27 μM), spermine (49.42 μM), and putrescine (31.04 μM) along with plant growth regulators. Shoot induction was increased up to twofold and multiplication was increased up to threefold in the medium supplemented with PA. Profuse rooting was observed in putrescine (93.12 μM), spermidine (68.84 μM), and spermine (24.71 μM), with mean number of 57 roots. A twofold increase in the number of roots was observed in medium supplemented with PA with respect to control cultures, which facilitated the successful transplantation and acclimatization process of in vitro propagated sugarcane plants. Histology and scanning electron microscopy analyses supported adventitious direct shoot regeneration from immature leaf roll explants. The genetic stability of in vitro regenerated plants was confirmed using start codon targeted polymorphism marker system.     

Interaction of boron and aluminum on the physiological characteristics of rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) seedlings

It has been reported that aluminum (Al) toxicity is a major limiting factor for plant growth and production on acidic soils. Boron (B) is indispensable micronutrient for normal growth of higher plants, and its addition could alleviate Al toxicity. The rape seedlings were grown under three B (0.25, 25 and 500 μM) and two Al concentrations [0 (?Al) and 100 μM (+Al) as AlCl₃·6H₂O]. The results indicated that Al stress severely hampered root elongation and root activity at 0.25 μM B while the normal (25 μM) and excess (500 μM) B improved the biomass of rape seedlings under Al exposure. Additionally, normal and excess B treatment reduced accumulation of Al in the roots and leaves under Al toxicity, which was also confirmed by hematoxylin with light staining. This indicates that both normal and excess B could alleviate Al toxicity. Furthermore, it also decreased the contents of malondialdehyde and soluble protein under Al toxicity. Likewise, superoxide dismutase activity (SOD) improved by 97.82 and 131.96% in the roots, and 168 and 119.88% in the leaves at 25 and 500 µM B, respectively, while the peroxidase and catalase activities dropped as a result of Al stress. The study results demonstrated that appropriate B application is necessary to avoid the harmful consequences of Al toxicity in rape seedlings.     

Condition and feeding behaviour of subadult burbot (<Emphasis Type="Italic">Lota lota</Emphasis>) in riverine and lacustrine environments

The condition and feeding behaviour of burbot, a widespread potamodromous species in riverine and lacustrine environments, were compared in order to evaluate the importance of both in three artificial systems. Subadult burbot were sampled in three temperate reservoirs in spring, and one of them also in summer and autumn. Standardised abundance and sizes of burbot were comparable between the reservoirs, but the conditions were significantly different. The Clark’s condition coefficient and index of fullness were independent of burbot size and individuals. Feeding behaviour in terms of abundance and composition of consumed food was environment-dependent. The most common prey category was aquatic insect larvae, dominating in the riverine environment. Permanent water invertebrates, microcrustaceans, beetles and crayfish were less common food and eaten more often in the lacustrine environment. Prey-fish were usually the most common species of suitable size. The food analyses demonstrated opportunistic feeding behaviour with selection of prey associated with benthic habitat and suitable size. Burbot is therefore flexible not only in environments utilisation, but also feeding strategy.     

Genomewide association study for economic traits in the large yellow croaker with different numbers of extreme phenotypes

A traditional genomewide association study (GWAS) detects genotype–phenotype associations by the vast number of genotyped individuals. This method requires large-scale samples and considerable sequencing costs. Extreme phenotypic sampling proposes make GWAS more cost-efficient and are applied more widely. With extreme phenotypic sampling, we performed a GWAS for n-3 highly unsaturated fatty acids (HUFA) and eviscerated weight (EW) traits in the large yellow croaker population. Of the 32,249 and 29,748 detected SNPs for the two traits, three candidate regions were found in each trait. Three candidate regions associated with HUFA were known near genes on chromosomes 4 and 11, and three candidate regions were on chromosome 6, and 15 for the EW trait. By combing through our GWAS results and the biological functional analysis of the genes, we suggest that the FABP, DGAT, ATP8B1, FAF2 and CERS2 genes,  as well as the IGF2, BORA, CYP1A1, GRTP1 and HOX genes are promising candidate genes for n-3 HUFA and EW, respectively, in the large yellow croaker. Moreover, compared with the different numbers of the extreme phenotypic sampling, we conclude that 60% of the extreme phenotypic subsample can obtain a similar result as GWAS with whole phenotypes. Thus, extreme phenotypic sampling could save 40% of the cost for genotyping and DNA extraction without loss of the candidate regions and functional genes. Our study may provide a basis for further genomic breeding and a reference for others who want to perform GWAS with extreme phenotypes.     

A Raman-spectroscopy-based approach for detection and discrimination of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis> phages at low titer in raw milk

In this study, a method combining Raman spectroscopy with chemometric analysis was developed for detection of phage presence in raw milk and discrimination of Streptococcus thermophilus and Lactobacillus bulgaricus phages which are among the main phages causing problems in dairy industry. For this purpose, S. thermophilus and L. bulgaricus phages were added into raw milk separately, and then some pretreatments such as fat separation, removal of casein, and filtration were applied to the raw milk samples. Raman spectra of the samples were collected and then analyzed using principal component analysis in order to discriminate these phages in raw milk. In the next step, dilutions of S. thermophilus phages in pretreated raw milk were prepared, and Raman spectra were collected. These spectra were analyzed by using partial least squares method to quantify phages in low titer. Consequently, it has been demonstrated that S. thermophilus and L. bulgaricus phages, which have titers sufficient to fail the fermentation (~?10⁷ pfu/mL) and have lower titers (10²–10³ pfu/mL), could be discriminated from antibiotic and each other. Additionally, low concentrations of S. thermophilus phages (10² pfu/mL) could be detected through Raman spectroscopy with a short analysis time (60 min) and high coefficient of determination (R²) values for both calibration (0.985) and validation (0.906) with a root mean square error of calibration of 70.54 and root mean square error of prediction of 165.47. However, a lower success was achieved with L. bulgaricus phages and the obtained coefficient of determination values were not sufficiently high (0.649).     

Comparative sequence and structure analysis of eIF1A and eIF1AD

Background

Eukaryotic translation initiation factor 1A (eIF1A) is universally conserved in all organisms. It has multiple functions in translation initiation, including assembly of the ribosomal pre-initiation complexes, mRNA binding, scanning, and ribosomal subunit joining. eIF1A binds directly to the small ribosomal subunit, as well as to several other translation initiation factors. The structure of an eIF1A homolog, the eIF1A domain-containing protein (eIF1AD) was recently determined but its biological functions are unknown. Since eIF1AD has a known structure, as well as a homolog, whose structure and functions have been extensively studied, it is a very attractive target for sequence and structure analysis.

Results

Structure/sequence analysis of eIF1AD found significant conservation in the surfaces corresponding to the ribosome-binding surfaces of its paralog eIF1A, including a nearly invariant surface-exposed tryptophan residue, which plays an important role in the interaction of eIF1A with the ribosome. These results indicate that eIF1AD may bind to the ribosome, similar to its paralog eIF1A, and could have roles in ribosome biogenenesis or regulation of translation. We identified conserved surfaces and sequence motifs in the folded domain as well as the C-terminal tail of eIF1AD, which are likely protein-protein interaction sites. The roles of these regions for eIF1AD function remain to be determined. We have also identified a set of trypanosomatid-specific surface determinants in eIF1A that could be a promising target for development of treatments against these parasites.

Conclusions

The results described here identify regions in eIF1A and eIF1AD that are likely to play major functional roles and are promising therapeutic targets. Our findings and hypotheses will promote new research and help elucidate the functions of eIF1AD.

     

CITRIC: cold-inducible translational readthrough in the chloroplast of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> using a novel temperature-sensitive transfer RNA

Background

The chloroplast of eukaryotic microalgae such as Chlamydomonas reinhardtii is a potential platform for metabolic engineering and the production of recombinant proteins. In industrial biotechnology, inducible expression is often used so that the translation or function of the heterologous protein does not interfere with biomass accumulation during the growth stage. However, the existing systems used in bacterial or fungal platforms do not transfer well to the microalgal chloroplast. We sought to develop a simple inducible expression system for the microalgal chloroplast, exploiting an unused stop codon (TGA) in the plastid genome. We have previously shown that this codon can be translated as tryptophan when we introduce into the chloroplast genome a trnW_UCA gene encoding a plastidial transfer RNA with a modified anticodon sequence, UCA.

Results

A mutated version of our trnW_UCA gene was developed that encodes a temperature-sensitive variant of the tRNA. This allows transgenes that have been modified to contain one or more internal TGA codons to be translated differentially according to the culture temperature, with a gradient of recombinant protein accumulation from 35 °C (low/off) to 15 °C (high). We have named this the CITRIC system, an acronym for cold-inducible translational readthrough in chloroplasts. The exact induction behaviour can be tailored by altering the number of TGA codons within the transgene.

Conclusions

CITRIC adds to the suite of genetic engineering tools available for the microalgal chloroplast, allowing a greater degree of control over the timing of heterologous protein expression. It could also be used as a heat-repressible system for studying the function of essential native genes in the chloroplast. The genetic components of CITRIC are entirely plastid-based, so no engineering of the nuclear genome is required.