Suppression and restoration of primordial germ cell marker gene expression in channel catfish,Ictalurus punctatus,using knockdown constructs regulated by copper transport protein gene promoters: Potential for reversible transgenic sterilization

Complementary DNA overexpression and short hairpin RNA interference approaches were evaluated for decreasing expression of primordial germ cell (PGC) marker genes and thereby sterilizing channel catfish, Ictalurus punctatus, by delivering knockdown constructs driven by a constitutive promoter from yeast and a copper transport protein gene into fish embryos by electroporation. Two PGC marker genes, nanos and dead end, were the target knockdown genes, and their expressions, along with that of an off-target gene, vasa, were evaluated temporally using real-time polymerase chain reaction. Copper sulfate was evaluated as a repressor compound. Some of the constructs knocked down PGC marker gene expression, and some of the constructs were partially repressed by application of 0.1-ppm copper sulfate. When the rate of sexual maturity was compared for three-year-old broodfish that had been exposed to the sterilizing constructs during embryologic development and controls that had not been exposed, several treatments had reduced sexual maturity for the exposed fish. Of two promoter systems evaluated, the one which had been designed to be less sensitive to copper generally was more effective at achieving sterilization and more responsive to repression. Knockdown constructs based on 3′ nanos short hairpin RNA interference appeared to result in the best repression and restoration of normal sexual maturity. We conclude that these copper-based systems exhibited good potential for repressible transgenic sterilization. Optimization of this system could allow environmentally safe application of transgenic technology and might be applicable to other applications for aquatic organisms.     

Co-transformation of canola by chimeric chitinase and <Emphasis Type="Italic">tlp</Emphasis> genes towards improving resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T₂ generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29 mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant.     

Overexpression of <Emphasis Type="Italic">MuHSP70</Emphasis> gene from <Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> confers multiple abiotic stress tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A 70-KD heat shock protein (HSP70) is one of the most conserved chaperones. It is involved in de novo protein folding and prevents the aggregation of unfolded proteins under lethal environmental factors. The purpose of this study is to characterise a MuHSP70 from horsegram (Macrotyloma uniflorum) and elucidating its role in stress tolerance of plants. A MuHSP70 was cloned and characterised from a natural drought stress tolerant HPK4 variety of horsegram (M. uniflorum). For functional characterization, MuHSP70 was overexpressed in transgenic Arabidopsis. Overexpression of MuHSP70 was found to provide tolerance to the transgenic Arabidopsis against various stresses such as heat, cold, drought, salinity and oxidative stress. MuHSP70 transgenics were observed to maintain the shoot biomass, root length, relative water content, and chlorophyll content during exposure to multi-stresses relative to non-transgenic control. Transgenic lines have further shown the reduced levels of MDA, H₂O₂, and proteolytic activity. Together, these findings suggest that overexpression of MuHSP70 plays an important role in improving abiotic stress tolerance and could be a crucial candidate gene for exploration in crop improvement program.     

Enhancement of syringin contents in soybean seeds with seed-specific expression of a chimeric <Emphasis Type="Italic">UGT72E3</Emphasis>/<Emphasis Type="Italic">E2</Emphasis> gene

Syringin, sinapyl alcohol 4-O-glucoside, is well known as a plant-derived bioactive monolignol glucoside. In Arabidopsis, recombinant chimeric protein UGT72E3/2 has been previously reported to lead to significantly higher syringin production than the parental enzymes UGT72E2 and UGT72E3. To enhance syringin content in Korean soybean (Glycine max L. ‘Kwangan’), we cloned the UGT72E3/2 gene under the control of the β-conglycinin or CaMV-35S promoter to generate β-UGT72E3/2 and 35S-UGT72E3/2 constructs, respectively, and then transformed them into soybean to obtain transgenic plants using the modified half-seed method. Real-time semi-quantitative PCR (RT-PCR) analysis showed that the UGT72E3/2 gene was expressed in the leaves of the β-UGT72E3/2 and 35S-UGT72E3/2 transgenic lines. HPLC analysis of the seeds and mature tissues of the T2 generation plants revealed that the β-UGT72E3/2 transgenic seeds accumulated 0.15 µmol/g DW of total syringin and 0.29 µmol/g DW of total coniferin, whereas coniferin and syringin were not detected in non-transgenic seeds. Moreover, coniferin and syringin also accumulated at high levels in non-seed tissues, particularly the leaves of β-UGT72E3/2 transgenic lines. In contrast, 35S-UGT72E3/2 lines showed no differences in the contents of coniferin and syringin between transgenic and non-transgenic soybean plants. Thus, the seed-specific β-conglycinin promoter might be an effective tool to apply to the nutritional enhancement of soybean crops through increased syringin production.     

Production of heterozygous alpha 1,3-galactosyltransferase (<Emphasis Type="Italic">GGTA1</Emphasis>) knock-out transgenic miniature pigs expressing human <Emphasis Type="Italic">CD39</Emphasis>

Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig’s cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig’s cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.     

Overexpression of <Emphasis Type="Italic">AlTMP2</Emphasis> gene from the halophyte grass <Emphasis Type="Italic">Aeluropus littoralis</Emphasis> in transgenic tobacco enhances tolerance to different abiotic stresses by improving membrane stability and deregulating some stress-related genes

Herein, we report isolation of the AlTMP2 gene from the halophytic C4 grass Aeluropus littoralis. The subcellular localization suggested that AlTMP2 is a plasma membrane protein. In A. littoralis exposed to salt and osmotic stresses, the AlTMP2 gene was induced early and at a high rate, but was upregulated relatively later in response to abscisic acid and cold treatments. Expression of AlTMP2 in tobacco conferred improved tolerance against salinity, osmotic, H₂O₂, heat, and freezing stresses at the germination and seedling stages. Under control conditions, no growth or yield penalty were mentioned in transgenic plants due to the constitutive expression of AlTMP2. Interestingly, under greenhouse conditions, the seed yield of transgenic plants was significantly higher than that of non-transgenic (NT) plants grown under salt or drought stress. Furthermore, AlTMP2 plants had less electrolyte leakage, higher membrane stability, and lower Na⁺ and higher K⁺ accumulation than NT plants. Finally, six stress-related genes were shown to be deregulated in AlTMP2 plants relative to NT plants under both control and stress conditions. Collectively, these results indicate that AlTMP2 confers abiotic stress tolerance by improving ion homeostasis and membrane integrity, and by deregulating certain stress-related genes.     

Overexpression and oral immunogenicity of a dengue antigen transiently expressed in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

To understand the genetic and expression stability of transgenic insect-resistant poplar 741, this study compared the experimental plantations of transgenic insect-resistant poplar 741 lines (pb1, pb6, pb11, pb17, and pb29) with non-transgenic poplar 741, P. tomentosa Carr.f.yixianensis (poplar 84 K) and transgenic hybrid progeny lines cultured from immature embryos. The insect resistance and growth stability of transgenic poplar 741 were investigated by detecting exogenous genes by polymerase chain reaction (PCR), measuring the diameter at breast height (DBH) and volume growth, and performing insect-resistance tests against Clostera anachoreta and Hyphantria cunea. The inheritance and expression of the exogenous gene was also examined in transgenic hybrid progeny lines. The results revealed that the exogenous gene was stable, remaining stable in 8–10-year-old transgenic poplar 741 trees. No significant difference was found between the height of 10-year-old transgenic poplar 741 and non-transgenic poplar 741 in the experimental plantations in Baoding, China. The DBH and volume growth of pb17 was significantly greater than that of pb29 and pb11. The 8-year-old transgenic poplar 741 pb29 grown in Zhuozhou showed no significant difference from poplar 741 in terms of height growth, DBH, and volume. From 1999 to 2013, pb29-fed larvae (C. anachoreta larvae and H. cunea) exhibited stable mortality rates >79%. Likewise, pb11-fed larvae showed stable mortality rates (C. anachoreta larvae had mortality rates >75%, and H. cunea larvae exhibited rates >80%). pb17 conferred low insect-resistant stability, showing mortality rates that varied from 28.2 to 99.27% in C. anachoreta and H. cunea larvae. Among the hybrid progeny lines acquired by hybridization of pb1, pb29, and pb11 with 84 K poplar, the ratios of PCR-positive to PCR-negative lines for the BtCry1Ac gene were 1.31, 1.15, and 0.86, respectively. X ² tests showed that the ratio was consistent with the Mendelian law of 1:1 segregation controlled by an allele pair. The hybrid progeny of pb6?×?84 K had a segregation ratio of 3:1. The nptII gene followed the same segregation rule as Cry1Ac. The transgenic hybrid progeny that contained Cry1Ac gene exhibited the same insect resistance as the parent plants.     

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.     

Cloning <Emphasis Type="Italic">PIP</Emphasis> genes in drought-tolerant vetiver grass and responses of transgenic <Emphasis Type="Italic">VzPIP2;1</Emphasis> soybean plants to water stress

Vetiver grass [Vetiveria zizanioides (L.) Nash] displays comprehensive abiotic stress tolerance closely related to fine maintenance of plant water relation mediated by plasma membrane intrinsic proteins (PIPs). Two open reading frame sequences of PIPs (867 and 873 bp) were cloned from vetiver grass and named as VzPIP1;1 and VzPIP2;1, respectively. Expression of green fluorescent protein revealed only subcellular localization of VzPIP2;1 in the plasma membrane. Agrobacterium tumefaciens mediated transgenic (VzPIP2;1) soybean plants had a higher water content in above-ground parts under sufficient water supply through enhancing transpiration as compared to the non-transgenic plants but displayed a more severe drought injury because of a lower photosynthesis and a higher transpiration rate. However, A. rhizogenes mediated transgenic soybean plants kept a higher water content in above-ground parts by improving root water transport and kept a more effective photosynthesis under normal and drought conditions.     

Transformation of bahiagrass (<Emphasis Type="Italic">Paspalum notatum</Emphasis> Flugge)

Bahiagrass (Paspalum notatum Flugge), a forage species widely used in the southeastern United States, and from Central Mexico to Argentina, was targeted for improvement through genetic engineering. Embryogenic callus, initiated from germinating seedlings, was bombarded with a vector containing the bar selectable marker/reporter gene that confers resistance to phosphinothricin (glufosinate) herbicide (trade names Liberty, Ignite and Finale). Thirty-two transgenic plants were recovered. These plants were identified by the polymerase chain reaction (PCR) and verified by Southern analysis. Transgenic plants with bar, as well as non-transgenic plants without bar, regenerated from bombarded callus and selected with glufosinate, developed strong and stable resistance to glufosinate during selection. This unusual resistance in non-transgenic plants has persisted for over a year and is passed on to new tillers. The development of resistance in non-transgenic cells reduced the herbicide selection efficiency and made it necessary to identify transgenic plants by PCR where the 32 transgenic plants were recovered from 674 glufosinate-resistant plants, giving a very low selection efficiency.     

Molecular analysis reveals hidden diversity in <Emphasis Type="Italic">Zungaro</Emphasis> (Siluriformes: Pimelodidade): a genus of giant South American catfish

The genus Zungaro contains some of the largest catfish in South America. Two valid species are currently recognized: Zungaro jahu, inhabiting the Paraná and Paraguay basins, and Zungaro zungaro, occurring in the Amazonas and Orinoco basins. Analysing Zungaro specimens from the Amazonas, Orinoco, Paraguay and Paraná basins, based on the sequencing of COI and D-loop, we found at least three MOTUs, indicating the existence of hidden diversity within this fish group. Considering the ecological and economic values of this fish, our results are surely welcomed for its conservation, disclosing new findings on its diversity and pointing out the necessity for a detailed taxonomic revision.     

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.     

UV-B stress-induced ABA production in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> mutants defective in ethylene signal transduction pathway

In this study, we examined the influence of UV-B radiation (280–320 nm) on ABA accumulation in 14-day-old Arabidopsis thaliana (L.) Heynh plants of wild type (WT), ethylene receptor mutant (etr1-1), and mutant with a constitutively active ethylene signal transduction pathway (ctr1-1). ABA content in nonirradiated WT plants was twice higher than in each mutant. UV-B irradiation caused dose-dependent ABA accumulation in WT plants. In the etr1-1 mutant, the amount of accumulated ABA was significantly less. In the ctr1-1 mutant, ABA content didn’t increase after UV-B irradiation. These data suggest that start of stress-induced ABA formation requires the adjustable ethylene signal pathway. In the ctr1-1 mutant, a constitutively active (nonadjustable) ethylene signal pathway blocks stress-induced ABA accumulation.     

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.