Tetracycline-dependent expression of the human erythropoietin gene in transgenic chickens

A critical problem in the production of transgenic animals is the uncontrolled constitutive expression of the foreign gene, which occasionally results in serious physiological disorders in the transgenic animal. In this study, we report successful production of transgenic chickens that express the human erythropoietin (hEPO) gene under the control of a tetracycline-inducible promoter. A recombinant Moloney murine leukemia virus (MoMLV)-based retrovirus vector encapsidated with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of unincubated chicken embryos (stage X). Out of 198 injected eggs, 15 chicks hatched after 21 days of incubation and 14 hatched chicks expressed the vector-encoded hEPO gene when fed doxycycline, a tetracycline derivative, without any significant physiological dysfunctions. The expression of hEPO reverted to the pre-induction state by removing doxycycline from the diet. The biological activity of the hEPO produced in the transgenic chickens was comparable to commercially available CHO cell-derived hEPO. Successful germline transmission of the transgene was also confirmed in G1 transgenic chicks produced from crossing G0 transgenic roosters with non-transgenic hens. Tetracycline-inducible expression of the hEPO gene was also confirmed in the blood and eggs of the transgenic chickens.     

Enhanced salinity stress tolerance in transgenic chilli pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) plants overexpressing the wheat antiporter (<Emphasis Type="Italic">TaNHX2</Emphasis>) gene

Transgenic chilli pepper (Capsicum annuum L.) plants tolerant to salinity stress were produced by introducing the wheat Na⁺/H⁺ antiporter gene (TaNHX2) via Agrobacterium-mediated transformation. Cotyledonary explants were infected with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBin438 that contains a wheat antiporter (TaNHX2) gene driven by the double CaMV 35S promoter and NPT II gene as a selectable marker. PCR and semiquantitative RT-PCR analysis confirmed that the TaNHX2 gene had been integrated and expressed in the T₁ generation of transgenic pepper plants as compared to the non-transformed plants. Southern blot analysis further verified the integration and presence of TaNHX2 gene in the genome of chilli pepper plants. Biochemical assays of these transgenic plants revealed enhanced levels of proline, chlorophyll, superoxide dismutase, ascorbate peroxidase, relative water content, and reduced levels of hydrogen peroxide (H₂O₂), malondialdehyde compared to wild-type plants under salt stress conditions. The present investigation clearly showed that overexpression of the TaNHX2 gene enhanced salt stress tolerance in transgenic chilli pepper plants.     

The effects of polymorphisms in <Emphasis Type="Italic">growth hormone</Emphasis> and <Emphasis Type="Italic">growth hormone receptor</Emphasis> genes on production and reproduction traits in Aberdeen-Angus cattle (<Emphasis Type="Italic">Bos taurus</Emphasis> L., 1758)

The study was aimed to analyze the relation between individual genotypes and allelic variants of SNPs g.2141C>G of growth hormone gene, g.914T>A and g.257A>G of growth hormone receptor gene with growth and reproduction traits and to evaluate the populationgenetic structure in Aberdeen-Angus cattle (Bos taurus L., 1758) sample of Eastern Ukraine according SNPs studied. Allele C of SNP g.2141C>G has a positive correlation with birth weight, body stature, bigger rump, udder and total exterior evaluation score, shorter calving interval and better calve birth weight and negative correlation with calve average daily gain. Allele T of SNP g.914T>A has positive correlation with the muscle and udder size; live weight in each age, average daily gain, weight and average daily gain of calves born conform to the principle AA>TT≥TA. SNP g.257A>G showed a positive correlation for G allele with muscle size. The population is in equilibrium for SNPs g.2141C>G and g.257A>G, and in disequilibrium for SNP g.914T>A. The analysis showed no linkage disequilibrium between SNPs g.914T>A and g.257A>G. Inbreeding coefficient F_ST in Aberdeen-Angus group studied was 16.1%.     

Expression of an endochitinase gene from <Emphasis Type="Italic">Trichoderma virens</Emphasis> confers enhanced tolerance to <Emphasis Type="Italic">Alternaria</Emphasis> blight in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) czern and coss lines

An endochitinase gene ‘ech42’ from the biocontrol fungus ‘Trichoderma virens’ was introduced to Brassica juncea (L). Czern and Coss via Agrobaterium tumefaciens mediated genetic transformation method. Integration and expression of the ‘ech42’ gene in transgenic lines were confirmed by PCR, RT-PCR and Southern hybridization. Transgenic lines (T₁) showed expected 3:1 Mendelian segregation ratio when segregation analysis for inheritance of transgene ‘hpt’ was carried out. Fluorimetric analysis of transgenic lines (T₀ and T₁) showed 7 fold higher endochitinase activity than the non-transformed plant. Fluorimetric zymogram showed presence of endochitinase (42 kDa) in crude protein extract of transgenic lines. In detached leaf bioassay with fungi Alternaria brassicae and Alternaria brassicicola, transgenic lines (T₀ and T₁) showed delayed onset of lesions as well as 30–73 % reduction in infected leaf area compared to non-transformed plant.     

Salt tolerance conferred by expression of a global regulator IrrE from <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in oilseed rape

As salinity is a major threat to sustainable agriculture worldwide, cultivation of salt-tolerant crops becomes increasingly important. IrrE acts as a global regulator and a general switch for stress resistance in Deinococcus radiodurans. In this study, to determine whether the irrE gene can improve the salt tolerance of Brassica napus, we introduced the irrE gene into B. napus by the Agrobacterium tumefaciens-mediated transformation method. Forty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) analyses confirmed that the irrE gene had integrated into the plant genome. Northern as well as Western blot analyses revealed that the transgene was expressed at various levels in transgenic plants. Analysis for the T₁ progenies derived from four independent transformants showed that irrE had enhanced the salt tolerance of T₁ in the presence of 350 mM NaCl. Furthermore, under salt stress, transgenic plants accumulated more compatible solutes (proline) and a lower level of malondialdehyde (MDA), and they had higher activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). However, agronomic traits were not affected by irrE gene overexpression in the transgenic B. napus plants. This study indicates that the irrE gene can improve the salt tolerance of B. napus and represents a promising candidate for the development of crops with enhanced salt tolerance by genetic engineering.     

Expression of a rice <Emphasis Type="Italic">GLP</Emphasis> in <Emphasis Type="Italic">Medicago truncatula</Emphasis> exerting pleiotropic effects on resistance against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> through enhancing FeSOD-like activity

To evaluate the effectiveness of a germin-like protein (GLP) in legumes against the serious soil-borne pathogen Fusarium oxysporum f. sp. lentis, an Oryza sativa root-expressed GLP (OsRGLP1) was expressed in the model legume Medicago truncatula using the recombinant vector pCOsRGLP1. The transgene was highly expressed in M. truncatula transformed lines as assessed by RT-qPCR. Consistent with the active status of the transgene there was an elevated accumulation of H₂O₂ in transformed progeny. Enzymatic characterization of T₁ transgenic progeny showed increased superoxide dismutase (SOD) activity. The additional SOD activity in transgenic lines was insensitive to potassium cyanide and sensitive to H₂O₂ indicating its resemblance to FeSOD. The effectiveness of the OsRGLP1 gene was tested by monitoring the root disease after infection of wild-type and transgenic lines. Wild-type plants were greatly affected by the pathogen infection showing a percent disease index value of 50 compared to 10–18 for the transgenic lines. The tolerance of the transgenic lines leads to recovery in fresh weight and pod production to an almost normal level. Analysis of defense-related genes downstream of hydrogen peroxide (H₂O₂) in transgenic plants showed induction of salicylic acid and jasmonate signaling pathways and increased expression of some pathogenesis-related-1 (PR-1) genes and a plant defensin gene. Overall, the findings suggest that OsRGLP1 provides protection against the fungal pathogen F. oxysporum that may involve the direct influence of H₂O₂ on signaling pathways leading to the activation of defense-related genes.     

Search for associations between <Emphasis Type="Italic">G/A</Emphasis> polymorphism of the <Emphasis Type="Italic">EPAS1</Emphasis> gene and the maximal oxygen consumption in Russian athletes

This study investigates associations between G/A polymorphism of the epithelial PAS domain protein 1 (EPAS1) gene (rs1867785) and the maximum rate of oxygen consumption (VO_2max) in male Russian athletes. The study engaged 241 male athletes from different sports; the control group of nonathletes included 92 subjects. Increased frequencies of the AA and AG genotypes of the EPAS1 gene (χ² = 14.16, p = 0.03) were found in the cohort of male athletes. The frequencies of these alleles in the subgroups with moderate (EPAS1*A 38.1% and EPAS1*G 61.9%) and high (EPAS1*A 41.8% and EPAS1*G 58.2%) VO_2max values significantly differed from those in the control group (χ² = 7.53, p = 0.006 and χ² = 6.58, p = 0.01, respectively). The higher aerobic capacities are probably associated with the presence of at least one minor A allele of the EPAS1 gene in the genome.     

Over-expressing a UDP-glucosyltransferase gene (<Emphasis Type="Italic">Ta-UGT</Emphasis><Subscript><Emphasis Type="Italic">3</Emphasis></Subscript>) enhances <Emphasis Type="Italic">Fusarium</Emphasis> Head Blight resistance of wheat

Wheat Fusarium Head Blight (FHB), mainly caused by Fusarium graminearum (F.g), is a destructive fungal disease worldwide. FHB can not only cause considerable reduction in yield, but more seriously, can contaminate grain by trichothecene toxins released by the fungus. Here, we report new insights into the function and underlying mechanisms of a UDP-glycosyltransferase gene, Ta-UGT ₃ , that is involved in FHB resistance in wheat. In our previous study, Ta-UGT ₃ was found to enhance host tolerance against deoxynivalenol (DON) in Arabidopsis. In this study, four transgenic lines over-expressing Ta-UGT ₃ in a FHB highly susceptible wheat variety, Alondra’s, were obtained and characterized. 3 years of assays using single floret inoculation with F.g indicated that all four transgenic lines exhibited significantly enhanced type II resistance to FHB and less DON accumulation in the grains compared to the untransformed control. Histological observation using GFP labelled F.g was in agreement with the above test results since over-expression of Ta-UGT ₃ dramatically inhibited expansion of F.g. To explore the putative mechanism of resistance mediated by Ta-UGT ₃ , microarray analysis, qRT-PCR and hormone measurements were performed. Microarray analysis showed that DON up-regulated genes, such as TaNPR1, in the susceptible control, and down-regulated genes in F.g inoculated transgenic lines, while qRT-PCR showed that some defence related genes were up-regulated in F.g inoculated transgenic lines. Ta-UGT ₃ over-expression also changed the contents of the endogenous hormones SA and JA in the spikes. These data suggest that Ta-UGT ₃ positively regulates the defence responses to F.g, perhaps by regulating defence-related and DON-induced downstream genes.     

Structural and functional analysis of new plant promoter pro-SmAMP1 from <Emphasis Type="Italic">Stellaria media</Emphasis>

The nucleotide sequence of a fragment of the promoter region of pro-SmAMP1 gene, having a length of 1257 bp and encoding antifungal peptides, was determined in chickweed (Stellaria media (L.) Vill.). Computer analysis of the nucleotide sequence revealed a number of cis-elements that are typical strong plant promoters. Five 5′-deletion variants were created taking into account the distribution of cis-elements:–1235,–771,–714,–603, and–481 bp of pro-SmAMP1 gene promoter, which were fused to the coding region of the uidA reporter gene in pCambia1381Z plant expression vector. The efficacy of pro-SmAMP1 promoter deletion variants was determined by transient expression in plants of Nicotiana benthamiana and using sequential generations of transgenic Nicotiana tabacum plants. It was found that the levels of GUS reporter protein activity in the extracts from transgenic and agroinfiltrated plants using all deletion variants of pro-SmAMP1 gene promoter were 3–5 times higher than those of 35S CaMV viral promoter. The highest activity of GUS protein was observed in the leaves of transgenic tobacco plants and closely correlated with the mRNA level of encoding gene. The levels of GUS activity did not differ significantly among 11 independent homozygous lines of T₂ generation of N. tabacum plants with different deletion variants of pro-SmAMP1 promoter. The results give reason to assume that all deletion variants of pro-SmAMP1 promoter provide stable and high level of expression of controlled genes. The shortest deletion variant–481 bp of pro-SmAMP1 promoter should be viewed as a potentially strong plant promoter for the genetic engineering of plants.     

<Emphasis Type="Italic">In Planta</Emphasis> transformation for conferring salt tolerance to a tissue-culture unresponsive indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivar

Many farmer-popular indica rice (Oryza sativa L.) cultivars are recalcitrant to Agrobacterium-mediated transformation through tissue culture and regeneration. In planta transformation using Agrobacterium could therefore be a useful alternative for indica rice. A simple and reproducible in planta protocol with higher transformation efficiencies than earlier reports was established for a recalcitrant indica rice genotype. Agrobacterium tumefaciens containing the salt tolerance-enhancing Pea DNA Helicase45 (PDH45) gene, with the reporter and selectable marker genes, gus-INT (β-glucuronidase with intron) and hygromycin phosphotransferase (hpt), respectively, were used. Overnight-soaked mature embryos were infected and allowed to germinate, flower, and set T₁ seeds. T₀ plants were considered positive for the transgene if the spikelets of one or more of their panicles were positive for gus. Thereafter, selection at T₁ was done by germination in hygromycin and transgenic status re-confirmation by subjecting plantlet DNA/RNA to gene-specific PCR, Southern and semi-quantitative RT-PCR. Additionally, physiological screening under saline stress was done at the T₂ generation. Transformation efficiency was found to be 30–32% at the T₀ generation. Two lines of the in planta transformed seedlings of the recalcitrant rice genotype were shown to be saline tolerant having lower electrolyte leakage, lower Na⁺/K⁺, minimal leaf damage, and higher chlorophyll content under stress, compared to the WT at the T₂ generation.     

Mapping of <Emphasis Type="BoldItalic">Id</Emphasis> locus for dermal shank melanin in a Chinese indigenous chicken breed

The dermal shank pigmentation, one of the defining traits of chicken breeds, is caused by an abnormal deposition of melanin in the dermis of the shank. The abnormal deposition is controlled by the sex-linked inhibitor of dermal melanin (Id). In this study, we aim to locate the gene responsible for the dermal shank pigmentation in chickens by an association analysis and a differential expression analysis. Based on our results, 72 single-nucleotide polymorphisms (SNPs) located in Z chromosome (chrZ): 71–73 Mb (galGal3) were selected to further explore their relationships with the dermal shank pigmentation in pure lines of 96 Gushi hens and 96 Gushi hens with a yellow shank skin colour. The results of the association analysis showed that the SNPs located in chrZ: 72.58–72.99 Mb (galGal3) (chrZ: 79.02–79.44 Mb (galGal4)) are significantly associated with the dermal shank pigmentation. Based on the results of our previous studies and the present association analysis, the zinc-finger protein 608 (ZNF608), GRAM domain containing 3 (GRAMD3), aldehyde dehydrogenase 7 family member A1 (ALDH7A1), fem-1 homologue C (FEM1C), beta-1,4-galactosyltransferase 1 (B4GALT1) and versican (VCAN) genes were selected for the differential expression analysis. The gene expression profiles showed that the expression of GRAMD3 gene in the dermis tissues of the shank was significantly (\(P= 0.010738 < 0.05\)) higher in 350-day-old Gushi chickens characterized by the dermal shank pigmentation than in one-day-old Gushi chickens. The dermal shank pigmentation was not present in the one-day-old Gushi chickens. Additionally, the results of the association analysis and the expression analysis showed that GRAMD3 could be the most likely candidate gene for the Id locus. However, we did not detect a mutation, i.e. significantly associated with this trait within GRAMD3. Therefore, we concluded that the variations located in the flanking region of GRAMD3 led to the abnormal expression of GRAMD3, which requires further study.     

Development of leaffolder resistant transgenic rice expressing <Emphasis Type="Italic">cry2AX1</Emphasis> gene driven by green tissue-specific <Emphasis Type="Italic">rbcS</Emphasis> promoter

The insecticidal cry genes of Bacillus thuringiensis (Bt) have been successfully used for development of insect resistant transgenic rice plants. In this study, a novel cry2AX1 gene consisting a sequence of cry2Aa and cry2Ac gene driven by rice rbcS promoter was introduced into a rice cultivar, ASD16. Among 27 putative rice transformants, 20 plants were found to be positive for cry2AX1 gene. The expression of Cry2AX1 protein in transgenic rice plants ranged from 5.95 to 122.40 ng/g of fresh leaf tissue. Stable integration of the transgene was confirmed in putative transformants of rice by Southern blot hybridization analysis. Insect bioassay on T₀ transgenic rice plants against rice leaffolder (Cnaphalocrosis medinalis) recorded larval mortality up to 83.33 %. Stable inheritance and expression of cry2AX1 gene in T₁ progenies was demonstrated using Southern and ELISA. The detached leaf bit bioassay with selected T₁ plants showed 83.33–90.00 % mortality against C. medinalis. The whole plant bioassay for T₁ plants with rice leaffolder showed significant level of resistance even at a lower level of Cry2AX1 expression varying from 131 to 158 ng/g fresh leaf tissue during tillering stage.     

Effect of <Emphasis Type="Italic">BBX-B8</Emphasis> overexpression on development,body weight,silk protein synthesis and egg diapause of <Emphasis Type="Italic">Bombyx mori</Emphasis>

Bombyxin (BBX) is an insulin-like peptide exists in the silkworm Bombyx mori. Our previous studies on the effects of inhibiting BBX-B8 expression found that BBX-B8 is important for the development of organ, reproduction and trehalose metabolism in the silkworms. In this paper, we investigated the expression profile of the BBX-B8 gene and effect of BBX-B8 overexpression on the development, body weight, silk protein synthesis and egg diapause of B. mori to further understand BBX-B8 functions. BBX-B8 gene expression could be detected in the brains, midguts, anterior silkglands, ovaries, testes, fat bodies, hemolymph, malpighian tubules and embryos by RT-PCR, however it was mainly expressed in the brain. Western blots showed that the change in BBX-B8 expression was not obvious in the brain of 1- to 4-day-old larvae of fifth instar silkworms, but expression increased substantially at 5- to 6-day-old larvae of fifth instar silkworms. Transgenic silkworms overexpressing BBX-B8 were obtained by introducing non-transposon transgenic vector pIZT-B8 containing a BBX-B8 gene driven by Orgyia pseudotsugata nucleopolyhedrovirus IE2 promoter into the genome. Development duration of the transgenic silkworms was delayed by 2.5–3.5 days. Cocoon shell weight of transgenic silkworms was reduced by 4.79 % in females and 7.44 % in males, pupal weight of transgenic silkworms was reduced 6.75 % in females and 13.83 % in males compared to non-transgenic silkworms, and 5.56–14.29 % of transgenic moths laid nondiapausing eggs. All results indicated that BBX-B8 plays an important role in the development, silk protein synthesis and egg diapause of silkworm.     

Over-Expression of <Emphasis Type="Italic">PmHSP17.9</Emphasis> in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Confers Thermotolerance

Small heat shock proteins (sHSPs) have been shown to be involved in stress tolerance. However, their functions in Prunus mume under heat treatment are poorly characterized. To improve our understanding of sHSPs, we cloned a sHSP gene, PmHSP17.9, from P. mume. Sequence alignment and phylogenetic analysis indicated that PmHSP17.9 was a member of plant cytosolic class III sHSPs. Besides heat stress, PmHSP17.9 was also upregulated by salt, dehydration, oxidative stresses and ABA treatment. Leaves of transgenic Arabidopsis thaliana that ectopically express PmHSP17.9 accumulated less O₂ ^? and H₂O₂ compared with wild type (WT) after 42 °C treatment for 6 h. Over-expression of PmHSP17.9 in transgenic Arabidopsis enhanced seedling thermotolerance by decreased relative electrolyte leakage and MDA content under heat stress treatment when compared to WT plants. In addition, the induced expression of HSP101, HSFA2, and delta 1-pyrroline-5-carboxylate synthase (P5CS) under heat stress was more pronounced in transgenic plants than in WT plants. These results support the positive role of PmHSP17.9 in response to heat stress treatment.