Generation of GTKO <Emphasis Type="Italic">Diannan</Emphasis> Miniature Pig Expressing Human Complementary Regulator Proteins hCD55 and hCD59 via T2A Peptide-Based Bicistronic Vectors and SCNT

Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assays using human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.     

Expression Profiles of <Emphasis Type="Italic">IGF</Emphasis>-<Emphasis Type="Italic">1R</Emphasis> Gene and Polymorphisms of its Regulatory Regions in Different Pig Breeds

Insulin like growth factor 1 receptor (IGF-1R) is a candidate gene for growth and carcass traits in regulating animal growth, metabolism and endocrine. It is widely expressed in liver, muscle, bone tissues where the IGF-1R functions as a factor that promotes cell growth. In this study, the protein expression level of IGF-1R gene in liver and muscle tissues of three periods (birth, weaning and adult) of three pig breeds (BamaXiang pigs (BM), Tibetan pigs (TM) and Junmu No.1 pigs (JM)) were tested by western blot. SNPs within the regulatory region of pig IGF-1R gene were detected using direct sequencing and then the genotypes were identified through AS-PCR approach. Results showed expression profiles of IGF-1R gene between liver and muscle tissues were different and significant differences were also found among pig breeds. In the same time, four SNPs were detected in the regulatory region of IGF-1R gene, among which the genotype frequency of three (g.?1468G > C, g.?1192 C > T and g.330,424 C > T) were significantly different among the pig breeds. BM tended to heterozygous (GC/CT) of the anterior two loci, while TM and JM preferred the other two homozygotes respectively. For the g.330,424 C > T, all pig breeds were tended to be the heterozygous. In conclusion, the SNPs with different genotype distribution among the three pig breeds may explain the gene expression difference between the different pig breeds.     

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.     

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.     

Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

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.     

Overexpression of <Emphasis Type="Italic">ThGSTZ1</Emphasis> from <Emphasis Type="Italic">Tamarix hispida</Emphasis> improves tolerance to exogenous ABA and methyl viologen

Key message

Molecular analysis of a zeta subfamily GST gene from T. hispida involved in ABA and methyl viologen tolerance in transgenic Arabidopsis and Tamarix.

Abstract

Glutathione S-transferase (GST) genes are important for the improvement of plant abiotic stress tolerance, and our previous study demonstrated that the ThGSTZ1 gene from Tamarix hispida improves plant salt and drought tolerance. To further understand the role of ThGSTZ1 in the response of plants to abscisic acid (ABA) and oxidative stress, three ThGSTZ1-overexpressing transgenic Arabidopsis thaliana lines were analyzed in the current study. The results showed that the transgenic lines exhibited higher biomass accumulation, higher activities of GST and other protective enzymes, and less reactive oxygen species (ROS) and cell damage than wild-type (WT) plants under ABA and methyl viologen (MV) stress. In addition, the analysis of a transgenic T. hispida line transiently expressing ThGSTZ1 confirmed these results. The activities of GST, glutathione peroxidase, and superoxide dismutase were markedly higher in the ThGSTZ1-overexpressing lines compared with the control lines under both ABA and MV treatments, and the transgenic lines also exhibited a lower degree of electrolyte leakage (EL) and a decreased H₂O₂ content. All these results suggested that ThGSTZ1 can also improve plant ABA and oxidation tolerance by regulating ROS metabolism and that ThGSTZ1 represents an excellent candidate gene for molecular breeding to increase plant stress tolerance.

     

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.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

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.     

Improving tobacco freezing tolerance by co-transfer of stress-inducible <Emphasis Type="Italic">CbCBF</Emphasis> and <Emphasis Type="Italic">CbICE53</Emphasis> genes

Cold stress is one of the major limitations to crop productivity worldwide. We investigated the effects of multiple gene expression from cold tolerant Capsella bursa-pastoris in transgenic tobacco (Nicotiana tabaccum) plants. We combined CblCE53 and CbCBF into a reconstruct vector by isocaudomers. Plant overexpression of CbICE53 under the stress inducible CbCOR15b promoter and CbCBF under a constitutive promoter showed increased tolerance to both chilling and freezing temperatures in comparison to wild-type plants, according to the electrolyte leakage and relative water content. The expressions of endogenous cold-responsive genes in transgenic tobacco (NtDREB1, NtDREB3, NtERD10a and NtERD10b) were obviously upregulated under normal and low temperature conditions. These results suggest that the CbICE53 + CbCBF transgenic plants showed a much greater cold tolerance as well as no dwarfism and delayed flowering. Thus they can be considered as a potential candidate for transgenic engineering for cold tolerant tobacco.