MsSAMS,a cold stress-responsive gene,provides resistance to environmental stress in T2-generation transgenic plants

The SAMS (S-adenosylmethionine synthetase) gene is known to play an important role in the mechanism of cold resistance, as overexpression of this gene results in phenotypic changes in T₁-generation transgenic plants. Accordingly, this study was conducted to test the expression of the MsSAMS gene in T₂-generation transgenic plants and to investigate the resistance of these plants and the function of the transgene in response to various environmental stresses. For the morphological analysis of T₂-generation transgenic plants overexpressing the MsSAMS gene, observations using scanning electron microscopy (SEM) were performed. T₂-generation transgenic plants were obtained by planting a total of 5 lines, and their characteristics were tested by comparisons with those of the control. SEM revealed that the thickest leaves were produced by the T6 transgenic line—161.24?±?8.05 µm. The number of stomata ranged from 20.00?±?2.65 to 34.00?±?1.00 in the T₂-generation transgenic plants, but the control had more stomata. Resistance to various factors, such as low temperature, drought, and oxidative stress, in the T₂-generation transgenic plants was also confirmed. Under cold-stress conditions, the T6 transgenic line presented the lowest value (22.73%) of ion leakage, and under drought-stress conditions, compared with the control, the transgenic lines presented lower ion leakage after being treated with various concentrations of mannitol. Even under oxidative-stress conditions, the T₂-generation transgenic plants presented ion leakage levels that were 32.91?±?4.24 to 48.33?±?3.54% lower than those of the control after treatment with various concentrations of methyl viologen. Regarding SAMS enzyme activity, as the duration of cold treatment increased, the activity in the transgenic plants tended to decrease and then increase. During 48 h of cold treatment, the control showed a decrease in SAM content, while the T₂-generation transgenic plants presented an increase in SAM content, from 13.58?±?1.04 to 22.75?±?1.95 mg protein/g FW. The results suggest that the MsSAMS gene may be important to the mechanisms of resistance to oxidative and drought stresses in addition to its previously known association with cold resistance. Based on these results, it was suggested that the MsSAMS gene, whose expression is induced by cold stress, can serve as a marker of various responses to environmental stresses, because resistance to cold damage and various environmental stresses are stably inherited in the T₂ generation.

     

Physiological and photosynthetic characteristics of indica Hang2 expressing the sugarcane PEPC gene

Phosphoenolpyruvate carboxylase (PEPC) is known to play a key role in the initial fixation of CO₂ in C4 photosynthesis. The PEPC gene from sugarcane (a C4 plant) was introduced into indica rice (Hang2), a process mediated by Agrobacterium tumefaciens. Integration patterns and copy numbers of the gene was confirmed by DNA blot analysis. RT-PCR and western blotting results showed that the PEPC gene was expressed at both the mRNA and protein levels in the transgenic lines. Real-time PCR results indicated that expression of the sugarcane PEPC gene occurred mostly in green tissues and changed under high temperature and drought stress. All transgenic lines showed higher PEPC enzyme activities compared to the untransformed controls, with the highest activity (11.1 times higher than the controls) being observed in the transgenic line, T34. The transgenic lines also exhibited higher photosynthetic rates. The highest photosynthetic rate was observed in the transgenic line, T54 (22.3 μmol m^?2 s^?1; 24.6 % higher than that in non-transgenic plants) under high-temperature conditions. Furthermore, the filled grain and total grain numbers for transgenic lines were higher than those for non-transgenic plants, but the grain filling (%) and 1,000-grain weights of all transgenic lines remained unchanged. We concluded that over-expression of the PEPC gene from sugarcane in indica rice (Hang2) resulted in higher PEPC enzyme activities and higher photosynthesis rates under high-temperature conditions.     

拟南芥AtCIPK23基因对烟草抗旱能力的影响

为了探索拟南芥AtCIPK23基因对烟草耐旱能力的影响,对3个转AtCIPK23基因阳性纯合株系KA13、KA14和KA44与野生型烟草K326（对照）进行了自然干旱处理,测定离体叶片的失水速率、叶绿素含量、相对电导率、脯氨酸和可溶性糖含量,并分析了转基因及野生型材料对活性氧的清除能力,对活性氧清除基因NtSOD、NtCAT和NtAPX及干旱胁迫相关基因NtDREB、NtLEA5和NtCDPK2的表达量进行检测。结果表明：（1）转基因烟草离体叶片的失水速率明显低于K326;自然干旱7 d后,野生型K326出现了明显的干旱胁迫症状;干旱7 d进行复水后,转基因株系的复水存活率明显高于K326。（2）转基因株系中的叶绿素、脯氨酸及可溶性糖含量比K326显著提高,电导率则明显降低。（3）野生型烟草K326中H₂O₂的积累量明显高于3个转基因株系,转基因株系中ROS清除机制的3个关键基因NtSOD、NtCAT和NtAPX被诱导上调表达。（4）抗旱相关基因NtDREB、NtLEA5和NtCDPK2仅在转基因烟草中受干旱诱导。研究认为,AtCIPK23基因可能具有提高植物抗旱能力的功能。     

Enhanced drought tolerance in transgenic Leymus chinensis plants with constitutively expressed wheat TaLEA3

Leymus chinensis is an important grassland perennial grass. However, its drought tolerance requires to be improved. LEA (late embryogenesis abundant) genes are believed to confer resistance to drought and water deficiency. Using Agrobacterium-mediated transformation, a wheat LEA gene, TaLEA ₃ , was integrated into L. chinensis. The transgenic lines showed enhanced growth ability under drought stress during which transgenic lines had increased the relative water content, leaf water potential, relative average growth rate, but decreased the malondialdehyde content compared with the non-transgenic plant. Thus, transgenic breeding is an efficient approach to enhance drought tolerance in L. chinensis.     

Salt stress alleviation in transgenic Vigna mungo L. Hepper (blackgram) by overexpression of the glyoxalase I gene using a novel Cestrum yellow leaf curling virus (CmYLCV) promoter

A reproducible and efficient transformation system utilizing the nodal regions of embryonal axis of blackgram (Vigna mungo L. Hepper) has been established via Agrobacterium tumefaciens. This is a report of genetic transformation of Vigna mungo for value addition of an agronomic trait, wherein the gene of interest, the glyoxalase I driven by a novel constitutive Cestrum yellow leaf curling viral promoter has been transferred for alleviating salt stress. The overexpression of this gene under the constitutive CaMV 35S promoter had earlier been shown to impart salt, heavy metal and drought stress tolerance in the model plant, tobacco. Molecular analyses of four independent transgenic lines performed by PCR, Southern and western blot revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 2.25% and the time required for the generation of transgenic plants was 10–11 weeks. Exposure of T1 transgenic plants as well as untransformed control plants to salt stress (100 mM NaCl) revealed that the transgenic plants survived under salt stress and set seed whereas the untransformed control plants failed to survive. The higher level of Glyoxalase I activity in transgenic lines was directly correlated with their ability to withstand salt stress. To the best of our knowledge this is the only report of engineering abiotic stress tolerance in blackgram. Prasanna Bhomkar, Chandrama P. Upadhyay are contributed equally. An erratum to this article can be found at     

Effects of drought on water content and photosynthetic parameters in potato plants expressing the trehalose-6-phosphate synthase gene of Saccharomyces cerevisiae

Two transgenic potato lines, T1 and T2, expressing the trehalose-6-phosphate synthase (TPS1) gene of yeast were isolated. In our experimental approach, we applied two novelties, namely the fusion of the drought-inducible promoter StDS2 to TPS1 and a marker-free transformation method. In contrast to the expected drought-induced expression, only a very low constitutive TPS1 expression was detected in the transgenic lines, probably due to chromosomal position effects. The observed expression pattern, however, was sufficient to alter the drought response of plants. Detached leaves of T1 and T2 showed an 8 h delay in wilting compared to the non-transformed control. Potted plants of T1 and T2 kept water 6 days longer than control plants and maintained high stomatal conductance and a satisfactory rate of net photosynthesis. During drought treatment, CO₂ assimilation rate measured at saturating CO₂ level was maintained at maximum level for 6–9 days in transgenic plants while it decreased rapidly after 3 days in the wild type plants. Under optimal growth conditions, lower CO₂ fixation was detected in the transgenic than in the control plants. Stomatal densities of T1 and T2 leaves were reduced by 30–40%. This may have contributed to the lower CO₂ fixation rate and altered drought response. Ibolya Stiller, and Sándor Dulai contributed equally to this work.     

Expression of Arabidopsis Bax Inhibitor‐1 in transgenic sugarcane confers drought tolerance

The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor‐1 from Arabidopsis thaliana (AtBI‐1), can confer increased tolerance of sugarcane plants to long‐term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C₄ grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long‐term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.     

Analysis of selected promoter polymorphisms and haplotypes of the CYBA gene encoding the p22phox,subunit of NADPH oxidases,in patients with coronary artery disease

Abstract

The p22phox is a critical component of vascular NADPH oxidases and is encoded by the CYBA gene. It was shown that functionally relevant polymorphisms of the CYBA gene ?930A?>?G, ?852C?>?G, ?675A?>?T, ?536C?>?T, 214C?>?T (previously described as 242C?>?T), *24A?>?G (previously described as 640A?>?G), and *49A?>?G modulate generation of reactive oxygen species (ROS). To analyse whether the CYBA gene polymorphisms ?852C?>?G, ?675A?>?T, and ?536C?>?T were associated with coronary artery disease (CAD), and to designate haplotype blocks. Four hundred and ninety subjects: 245 patients with CAD and 245 age and sex-matched controls. The polymorphisms were genotyped using the PCR-RFLP method and the TagMan^® Pre-designed SNP Genotyping Assay. The analysed polymorphisms do not form haplotype blocks. Case–control study revealed that the ?930?G/-675T and ?930G/*49G diplotypes were a CAD risk factor. The 675T/*49G diplotype can modulate CAD risk in women. The protective effect reducing CAD risk in women was related to the ?930A/?675T and ?930A/*49A diplotypes. Carrier state of the ?852C allele (?852C?>?G) was associated with multivessel stenosis while the CC genotype of the ?536C?>?T polymorphism was more frequent in patients with peripheral artery disease. Hypercholesterolemic, cigarette smokers had an increased risk of CAD, especially C???852 allele (?852C?>?G) carriers (SIM?=?3.54; odds ratios (OR)?=?10.01, p?<?0.000). The CYBA gene polymorphisms modulate the risk of CAD but do not form a haplotype blocks.     

Enhanced tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein,sHSP17.7

Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42°C) for 24 h resulted in a significant increase in tolerance to drought stress. To try to determine the mechanisms of acquisition of tolerance to drought stress by heat shock, the rice small heat-shock protein gene, sHSP17.7, the product of which was shown to act as molecular chaperones in vitro and in vivo in our previous study, was overexpressed in the rice cultivar “Hoshinoyume”. Western and Northern blot analyses showed higher expression levels of sHSP17.7 protein in three transgenic lines than in one transgenic line. Drought tolerance was assessed in these transgenic lines and wild-type plants by withholding water for 6 days for evaluation of the ability of plants to continue growth after water-stress treatments. Although no significant difference was found in water potential of seedlings between transgenic lines and wild-type plants at the end of drought treatments, only transgenic seedlings with higher expression levels of sHSP17.7 protein could regrow after rewatering. Similar results were observed in survival rates after treatments with 30% polyethylene glycol (PEG) 3640 for 3 days. These results suggest that overproduction of sHSP17.7 could increase drought tolerance in transgenic rice seedlings.     

Polymorphism identification in GDF9 gene and its association analysis with reproduction traits in Jinghai Yellow chicken

Abstract

GDF9 (growth differentiation factor 9) belongs to the transforming growth factor-β (TGF-β) superfamily and plays an irreplaceable role in female fertility. To reveal its genetic effects on productivity performance in chickens, 373 Jinghai Yellow chickens were chosen randomly to detect SNPs in GDF9 by PCR-SSCP and DNA sequencing methods. Eventually, four SNPs (g.2053G?>?A, g.2275T?>?C, g.2338C?>?T, g.2420T?>?C) in total had been detected. Amongst them, g.2420T?>?C was first found significantly associated with reproduction trait in chickens and heterozygous type C₂T₂ had higher average egg weight at 300?days of age (AEWD300) than T₂T₂ (p?<?0.01). Least squares analysis showed that age at first laying (AFE) of H1 and H1H1 chickens were significantly earlier than that of H7 and H7H7 ones, respectively (p?<?0.05). H1H5 hens showed higher AEWD300 than H4H7 ones (p?<?0.05). For total egg number at 300?days of age (END300), mean of H5H5 was significantly higher than that of H4H4 (p?<?0.05). Hence, the study suggested that hybrid vigor at g.2420T?>?C could be utilized in practice. H1H1, H1H5 and H5H5 could be the dominant diplotypes for chicken breeding. The study may contribute to the breeding progress of productive chickens and supply reference for oviparous animal production practice.     

Generation of marker free salt tolerant transgenic plants of Arabidopsis thaliana using the gly I gene and cre gene under inducible promoters

Despite the advances in transgenesis, transformation technologies still rely on the introduction of a selectable marker gene to identify cells and tissues that have integrated the gene of interest in their genome. The continuous presence of the marker genes in the transgenics is often controversial as it can potentially have multiple undesirable impacts. The present study employed the self-excising Cre-loxP system to generate marker-free Arabidopsis thaliana expressing the agronomically important glyoxalase I (glyI) gene from Brassica juncea to confer salt stress tolerance. A binary vector was constructed wherein the salt-inducible rd29A promoter was used to drive the expression of the glyI gene and the transformants of A. thaliana were recovered using kanamycin resistance as the selectable marker. The neomycin phosphotransferase II (nptII) gene was flanked by the loxP sites followed by the introduction of a heat-inducible Cre-recombinase in between the loxP sites. The kanamycin-resistant transgenic lines of A. thaliana using this vector showed an ability to withstand stress imposed by 150 mM NaCl. The exposure of the T2 transgenic lines to a mild heat shock (37°C) resulted in the recovery of salt-tolerant, kanamycin-sensitive T3 progeny. Molecular analyses of the T3 transgenic lines following the heat shock treatment confirmed the excision of the nptII gene and the completion of their life cycle in the presence of 150 mM NaCl-induced stress.     

Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane

A reproducible method for transformation of sugarcane using various strains of Agrobacterium tumefaciens (A. tumefaciens) (AGL0, AGL1, EHA105 and LBA4404) has been developed. The selection system and co-cultivation medium were the most important factors determining the success of transformation and transgenic plant regeneration. Plant regeneration at a frequency of 0.8–4.8% occurred only when callus was transformed with A. tumefaciens carrying a newly constructed superbinary plasmid containing neomycin phosphotransferase (nptII) and β-glucuronidase (gusA) genes, both driven by the maize ubiquitin (ubi-1) promoter. Regeneration was successful in plants carrying the nptII gene but not the hygromycin phosphotransferase (hph) gene. NptII gene selection was imposed at a concentration of 150 mg/l paromomycin sulphate and applied either immediately or 4 days after the co-cultivation period. Co-cultivation on Murashige and Skoog (MS)-based medium for a period of 4 days produced the highest number of transgenic plants. Over 200 independent transgenic lines were created using this protocol. Regenerated plants appeared phenotypically normal and contained both gusA and nptII genes. Southern blot analysis revealed 1–3 transgene insertion events that were randomly integrated in the majority of the plants produced.     

Analysis of transgene integration and expression following biolistic transfer of different quantities of minimal expression cassette into sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. hybrids)

Sugarcane (Saccharum spp. hybrids) is an interspecific hybrid with a highly polyploid and frequently aneuploid genome. This C4 grass accounts for nearly 70% of the global sugar production and more recently has become an important biofuel feedstock. Biolistic gene transfer of plasmid DNA is the most frequently used approach for genetic transformation of sugarcane. Minimal expression cassettes lacking vector backbone sequences (MC) have been reported to support simple transgene integration in other species. In this study, we introduced a MC of nptII into embryogenic callus derived from immature leaf whorl cross-sections by biolistic gene transfer. The precipitation equivalents of 12.5, 25 or 50 ng of the nptII MC were delivered per shot to the target tissue with 1.0 μm gold particles. A total of 203 independent putative transgenic plants were regenerated following 80 bombardments and selection on geneticin or paromomycin containing media and 176 transgenic lines were confirmed with PCR. Twenty independent transgenic lines were selected for Southern blot analysis and expression analysis by NPTII ELISA from each of the three treatments. Genomic DNA from transgenic sugarcane plants displayed two to 13 nptII hybridization signals on Southern blots. There was a trend toward reduced transgene integration complexity and reduced transgene expression levels when lower (12.5 ng) MC was used per shot. These results demonstrate that backbone free MCs can be efficiently integrated and expressed in sugarcane.     

Transgenic Agrostis mongolica Roshev. with enhanced tolerance to drought and heat stresses obtained from Agrobacterium-mediated transformation

Efficient procedures for regeneration and Agrobacterium-mediated transformation were established for Agrostis mongolica Roshev. and generated transgenic plants tolerant to drought and heat stresses using a regulatory gene from Arabidopsis, ABF3, which controls the ABA-dependent adaptive responses. The identification and selection of regenerable and reproducible callus type was a key factor for successful transformation. The transformation efficiency was 49.2% and gfp expression was detected in hygromycin-resistant calli and stem of putative transgenic plants. The result of Southern blot analysis showed that the ABF3 transgene was stably integrated into the genome of transgenic plants. Of the five transgenic lines analyzed, single transgene integration was observed in two lines and two copy integration was observed in three transgenic lines. Northern blot analysis confirmed that ubi::ABF3 was expressed in all transgenic lines. Transgenic plants exhibited neither growth inhibition nor visible vegetative phenotypic alternations. However, both transgenic and wild-type plants were highly sterile and did not flower during 3 years of growth period in the open field under subtropical Jeju Island climate. The stomata of the transgenic plants opened less than did stomata of the wild-type plants, and water content of the transgenic leaves remained about 3–4 fold higher than observed for wild-type leaves under drought stress. The transgenic plants showed about 2 fold higher survival rates under drought stress and about 3 fold higher survival rates under heat stress when compared to wild-type plants. Thus, overexpression of the Arabidopsis ABF3 gene results in enhancement of both drought and heat stress tolerance in Agrostis mongolica Roshev.