Reduction of methylviologen-mediated oxidative stress tolerance in antisense transgenic tobacco seedlings through restricted expression of StAPX

Ascorbate peroxidases are directly involved in reactive oxygen species (ROS) scavenging by reducing hydrogen peroxide to water. The tomato thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco. RNA gel blot analysis confirmed that StAPX in tomato leaves was induced by methylviologen-mediated oxidative stress. The sense transgenic seedlings exhibited higher tAPX activity than that of the wild type (WT) plants under oxidative stress conditions, while the antisense seedlings exhibited lower tAPX activity. Lower APX activities of antisense transgenic seedlings caused higher malondialdehyde contents and relative electrical conductivity. The sense transgenic seedlings with higher tAPX activity maintained higher chlorophyll content and showed the importance of tAPX in maintaining the optimal chloroplast development under methylviologen stress conditions, whereas the antisense lines maintained lower chlorophyll content than WT seedlings. Results indicated that the over-expression of StAPX enhanced tolerance to methylviologen-mediated oxidative stress in sense transgenic tobacco early seedlings, whereas the suppression of StAPX in antisense transgenic seedlings showed high sensitivity to oxidative stress.     

Transgenic Brassica chinensis plants expressing a bacterial codA gene exhibit enhanced tolerance to extreme temperature and high salinity

Transgenic Brassica compestris L.spp.chinensis plants expressing a choline oxidase(codA) gene from Arthrobacter globiformis were obtained through Agrobacterium tumefaciens-mediated transformation.In the transgenic plants,codA gene expression and its product transportation to chloroplasts were detected by the enzyme-linked immunosorbent assay(ELISA) examination,immunogold localization,and 1 H-nuclear magnetic resonance( 1 H-NMR) . Stress tolerance was evaluated in the T3 plants under extreme temperature and salinity conditions.The plants of transgenic line 1(L1) showed significantly higher net photosynthetic rate(Pn) and Pn recovery rate under high(45°C,4 h) and low temperature(1°C,48 h) treatments,and higher photosynthetic rate under high salinity conditions(100,200,and 300 mmol/L NaCl,respectively) than the wild-type plants.The enhanced tolerance to high temperature and high salinity stresses in transgenic plants is associated with the accumulation of betaine,which is not found in the wild-type plants.Our results indicate that the introduction of codA gene from Arthrobacter globiformis into Brassica compestris L.spp.chinensis could be a potential strategy for improving the plant tolerance to multiple stresses.     

Identification of a heat shock cognate protein 70 gene in Chinese soft-shell turtle (Pelodiscus sinensis) and its expression profiles under thermal stress

The heat shock cognate protein 70 (Hsc70) is a member of a 70-kDa heat shock protein (HSP70) family that functions as molecular chaperones. In this study, a novel Hsc70 gene from Chinese soft-shelled turtle (Pelodiscus sinensis) (tHsc70) was identified. The tHsc70 full-length complementary DNA (cDNA) is 2 272 bp long with a 1 941-bp open reading frame (ORF) encoding 646 amino acids. Three characteristic signature regions of the HSP70 family, two major domains of an adenosine triphosphate (ATP)/guanosine triphosphate (GTP) binding domain (ABD), and a substrate-binding domain (SBD) were present in the predicted tHsc70 amino acid sequence. The tHsc70 gene was expressed in Escherichia coli BL21 and the expression product reacted with the anti-Hsc70 mouse monoclonal antibody by Western blotting. Homology analysis revealed that tHsc70 shared identity from 53.9% to 87.7% at the nucleotide level, and 49.1% to 99.5% at the amino acid level with the known Hsc70s. Phylogenetic analysis showed that tHsc70 was clustered together with the Hsc70 gene of another reptile species (Alligator mississippiensis). The tHsc70 was expressed in the liver, lung, heart, and skeletal muscle. The expression patterns of tHsc70 messenger RNA (mRNA) differed among different tissues under different durations of heat stress at 40 °C. Adaptation at 25 °C for 1 h after heat stress was also different among tissues and length of heat stress. Irrespective of different profiles of expression under heat stress, tHsc70 may play roles in protecting turtles from thermal stress.     

High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties

The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase (ALAS), was expressed in Escherichia coli Rosetta (DE3) and the enzymatic properties of the purified recombinant ALAS (RC-ALAS) were studied. Compared with ALASs encoded by hemA genes from Agrobacterium radiobacter (AR-ALAS) and Rhodobacter sphaeroides (RS-ALAS), the specific activity of RC-ALAS reached 198.2 U/mg, which was about 31.2% and 69.5% higher than those of AR-ALAS (151.1 U/mg) and RS-ALAS (116.9 U/mg), respectively. The optimum pH values and temperatures of the three above mentioned enzymes were all pH 7.5 and 37 °C, respectively. Moreover, RC-ALAS was more sensitive to pH, while the other two were sensitive to temperature. The effects of metals, ethylene diamine tetraacetic acid (EDTA), and sodium dodecyl sulfate (SDS) on the three ALASs were also investigated. The results indicate that they had the same effects on the activities of the three ALASs. SDS and metal ions such as Co²⁺, Zn²⁺, and Cu²⁺ strongly inhibited the activities of the ALASs, while Mn²⁺ exerted slight inhibition, and K⁺, Ca²⁺, Ba²⁺, Mg²⁺, or EDTA had no significant effect. The specificity constant of succinyl coenzyme A [(k _cat/K _m)_S-CoA] of RC-ALAS was 1.4989, which was higher than those of AR-ALAS (0.7456) and RS-ALAS (1.1699), showing its high catalytic efficiency. The fed-batch fermentation was conducted using the recombinant strain containing the R. capsulatus hemA gene, and the yield of 5-aminolevulinic acid (ALA) achieved was 8.8 g/L (67 mmol/L) under the appropriate conditions.     

Family-based association study of ZNF533, DOCK4 and IMMP2L gene polymorphisms linked to autism in a northeastern Chinese Han population简

Objective

A study in a Caucasian population has identified two single-nucleotide polymorphisms (SNPs) in ZNF533, one in DOCK4, and two in IMMP2L, which were all significantly associated with autism. They are located in AUTS1 and AUTS5, which have been identified as autism susceptibility loci in several genome-wide screens. The present study aimed to investigate whether ZNF533, DOCK4, and IMMP2L genes are also associated with autism in a northeastern Chinese Han population.

Methods

We performed a similar association study using families with three individuals (one autistic child and two unaffected parents). A family-based transmission disequilibrium test (TDT) was used to analyze the results.

Results

There were significant associations between autism and the two SNPs of ZNF533 gene (rs11885327: χ ²=4.5200, P=0.0335; rs1964081: χ ²=4.2610, P=0.0390) and the SNP of DOCK4 gene (rs2217262: χ ²=5.3430, P=0.0208).

Conclusions

Our data suggest that ZNF533 and DOCK4 genes are linked to a predisposition to autism in the northeastern Chinese Han population.     

Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene

Insect resistance and glyphosate tolerance have been two of the most important traits in the genetic improvement of various crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, Cry1Ac and Cry1Ig, and a modified glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (G10) were combined into a single transferred DNA (T-DNA) fragment and introduced into rice by Agrobacterium-mediated transformation. A transgenic line with single-copy T-DNA insertion named GAI-14 was found to be highly resistant to striped stem borer and rice leaf roller, and tolerant to glyphosate. Analysis of T-DNA border sequence suggested that the transgenes were inserted at the chromosome 3 and appeared to have not interrupted any known or putative genes. A field trial observed no significant difference in the basic agronomic traits between GAI-14 and the recipient rice.     

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites

To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase (CHS) gene fragments of Demodex folliculorum, Demodex brevis, and Demodex canis (three isolates from each species) from Xi’an China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was successful only in three D. canis isolates and one D. brevis isolate out of the nine Demodex isolates. The obtained fragments were sequenced to be 339 bp for D. canis and 338 bp for D. brevis. The CHS gene sequence similarities between the three Xi’an D. canis isolates and one Japanese D. canis isolate ranged from 99.7% to 100.0%, and those between four D. canis isolates and one D. brevis isolate were 99.1%–99.4%. Phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods shared the same clusters, according with the traditional classification. Two open reading frames (ORFs) were identified in each CHS gene sequenced, and their corresponding amino acid sequences were located at the catalytic domain. The relatively conserved sequences could be deduced to be a CHS class A gene, which is associated with chitin synthesis in the integument of Demodex mites.     

Nitric oxide induced by polyamines involves antioxidant systems against chilling stress in tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.) seedling

Polyamines (PAs) and nitric oxide (NO) are vital signals in modulating plant response to abiotic stress. However, to our knowledge, studies on the relationship between NO and PAs in response to cold stress in tomato are limited. Accordingly, in this study, we investigated the effects of putrescine (Put) and spermidine (Spd) on NO generation and the function of Spd-induced NO in the tolerance of tomato seedling under chilling stress. Spd increased NO release via the nitric oxide synthase (NOS)-like and nitrate reductase (NR) enzymatic pathways in the seedlings, whereas Put had no such effect. Moreover, H₂O₂ might act as an upstream signal to stimulate NO production. Both exogenous NO donor (sodium nitroprusside (SNP)) and Spd enhanced chilling tolerance in tomato, thereby protecting the photosynthetic system from damage. Compared to chilling treatment alone, Spd enhanced the gene expressions of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and their enzyme activities in tomato leaves. However, a scavenger or inhibitor of NO abolished Spd-induced chilling tolerance and blocked the increased expression and activity due to Spd of these antioxidant enzymes in tomato leaves under chilling stress. The results showed that NO induced by Spd plays a crucial role in tomato’s response to chilling stress.     

Functional characterization of an invertase inhibitor gene involved in sucrose metabolism in tomato fruit

In this study, we produced tomato plants overexpressing an invertase inhibitor gene (Sly-INH) from tomato, using a simple and efficient transient transformation system. Compared with control plants, the expression of Sly-INH was highly upregulated in Sly-INH overexpressing plants, as indicated by real-time polymerase chain reaction (PCR). Physiological analysis revealed that Sly-INH inhibited the activity of cell wall invertase (CWIN), which increased sugar accumulation in tomato fruit. Furthermore, Sly-INH mediated sucrose metabolism by regulating CWIN activity. Our results suggest that invertase activity is potentially regulated by the Sly-INH inhibitor at the post-translational level, and they demonstrate that the transient transformation system is an effective method for determining the functions of genes in tomato.     

An Arabidopsis mutant atcsr-2 exhibits high cadmium stress sensitivity involved in the restriction of H2S emission

The gene AtCSR encodes peptidyl-prolyl cis/trans isomerases (PPIases) that accelerate energetically unfavorable cis/trans isomerization of the peptide bond preceding proline production.In our studies,we found that AtCSR was associated with cadmium (Cd)-sensitive response in Arabidopsis.Our results show that AtCSR expression was triggered by Cd-stress in wild type Arabidopsis.The expression of some genes responsible for Cd2+ transportation into vacuoles was induced,and the expression of the iron-regulated transporter 1 (IRT1) related to Cd2+ absorption from the environment was not induced in wild type with Cd2+ treatment.The expression of Cd-transportation related genes was not in response to Cd-stress,whereas IRT expression increased dramatically in atcsr-2 with Cd2+ treatment.The expression of glutathione 1 (GSH1) was consistent with GSH being much lower in atcsr-2 in comparison with the wild type with Cd2+ treatment.Additionally,malondialdehyde (MDA),hydrogen peroxide,and Cd2+ contents,and activities of some antioxidative enzymes,differed between the wild type and atcsr-2.Hydrogen sulfide (H2S) has been confirmed as the third gas-transmitter over recent years.The findings revealed that the expression pattern of H2 S-releasing related genes and that of Cd-induced chelation and transportation genes matched well in the wild type and atcsr-2,and H2S could regulate the expression of the Cd-induced genes and alleviate Cd-triggered toxicity.Finally,one possible suggestion was given:down-regulation of atcsr-2,depending on H2S gas-transmitter not only weakened Cd2+ chelation,but also reduced Cd2+ transportation into vacuoles,as well as enhancing the Cd2+ assimilation,thus rendering atcsr-2 mutant sensitive to Cd-stress.     

Prevalence of sensitization to weed pollens of Humulus scandens, Artemisia vulgaris, and Ambrosia artemisiifolia in northern China

Objective:Weed pollens are common sources of allergens worldwide.The prevalence of weed pollen sensitization is not yet fully known in China.The purpose of this study was to investigate the prevalence of sensitization to weed allergens from Artemisia,Ambrosia,and Humulus in northern China.Methods:A total of 1144 subjects(aged from 5 to 68 years) visiting our clinic from June to October 2011 underwent intradermal testing using a panel of 25 allergen sources.Subjects with positive skin responses to any pollen were further tested for their serum concentrations of IgE antibodies against Artemisia vulgaris,Ambrosia artemisiifolia,and Humulus scandens,and against the purified allergens,Art v 1 and Amb a 1.Results:Of 1144 subjects,170 had positive intradermal reactions to pollen and 144 donated serum for IgE testing.The prevalence of positive intradermal responses to pollens of Artemisia sieversiana,Artemisia annua,A.artemisiifolia,and H.scandens was 11.0%,10.2%,3.7%,and 6.6%,respectively.Among the intradermal positive subjects,the prevalence of specific IgE antigens to A.vulgaris was 58.3%,to A.artemisiifolia 14.7%,and to H.scandens 41.0%.The prevalence of specific IgE antigens to the allergen Art v 1 was 46.9%,and to Amb a 1 was 11.2%.The correlation between the presence of IgE antibodies specific to A.vulgaris and to the Art v 1 antigen was very high.Subjects with A.artemisiifolia specific IgE also had A.vulgaris specific IgE,but with relatively high levels of A.vulgaris IgE antibodies.There were no correlations between the presence of IgE antibodies to H.scandens and A.vulgaris or to H.scandens and A.artemisiifolia.Conclusions:The intradermal prevalence of weed pollen sensitization among allergic subjects in northern China is about 13.5%.Correlations of specific IgE antibodies suggest that pollen allergens from Artemisia and Humulus are independent sources for primary sensitization.     

Partial AZFc duplications not deletions are associated with male infertility in the Yi population of Yunnan Province, China

Objective: There are many reports on associations between spermatogenesis and partial azoospermia factor c (AZFc) deletions as well as duplications; however, results are conflicting, possibly due to differences in methodology and ethnic background. The purpose of this study is to investigate the association of AZFc polymorphisms and male infertility in the Yi ethnic population, residents within Yunnan Province, China. Methods: A total of 224 infertile patients and 153 fertile subjects were selected in the Yi ethnic population. The study was performed by sequence-tagged site plus/minus (STS+/−) analysis followed by gene dosage and gene copy definition analysis. Y haplotypes of 215 cases and 115 controls were defined by 12 binary markers using single nucleotide polymorphism on Y chromosome (Y-SNP) multiplex assays based on single base primer extension technology. Results: The distribution of Y haplotypes was not significantly different between the case and control groups. The frequencies of both gr/gr (7.6% vs. 8.5%) and b2/b3 (6.3% vs. 8.5%) deletions do not show significant differences. Similarly, single nucleotide variant (SNV) analysis shows no significant difference of gene copy definition between the cases and controls. However, the frequency of partial duplications in the infertile group (4.0%) is significantly higher than that in the control group (0.7%). Further, we found a case with sY1206 deletion which had two CDY1 copies but removed half of DAZ genes. Conclusions: Our results show that male infertility is associated with partial AZFc duplications, but neither gr/gr nor b2/b3 deletions, suggesting that partial AZFc duplications rather than deletions are risk factors for male infertility in Chinese-Yi population.     

Genetic variation in alkaloid accumulation in leaves of Nicotiana

Alkaloids are plant secondary metabolites that are widely distributed in Nicotiana species and contribute greatly to the quality of tobacco leaves. Some alkaloids, such as nornicotine and myosmine, have adverse effects on human health. To reduce the content of harmful alkaloids in tobacco leaves through conventional breeding, a genetic study of the alkaloid variation among different genotypes is required. In this study, alkaloid profiles in leaves of five Nicotiana tabacum cultivars and Nicotiana tomentosiformis were investigated. Six alkaloids were identified from all six genotypes via gas chromatograph-mass spectrometry (GC-MS). Significant differences in alkaloid content were observed both among different leaf positions and among cultivars. The contents of nornicotine and myosmine were positively and significantly correlated (R ²=0.881), and were also separated from those of other alkaloids by clustering. Thus, the genotype plays a major role in alkaloid accumulation, indicating a high potential for manipulation of alkaloid content through traditional breeding.     

Genomic organization and sequence dynamics of the AvrPiz-t locus in Magnaporthe oryzae

Plants utilize multiple layers of defense mechanisms to fight against the invasion of diverse pathogens. The R gene mediates resistance, in most cases, dependent on the co-existence of its cognate pathogen-derived avirulence (Avr) gene. The rice blast R gene Piz-t corresponds in gene-for-gene fashion to the Magnaporthe oryzae Avr gene AvrPiz-t. In this study, we determined and compared the genomic sequences surrounding the AvrPiz-t gene in both avirulent and virulent isolates, designating as AvrPiz-t-ZB15 and avrPiz-t-70-15 regions, respectively. The sequence of the AvrPiz-t-ZB15 region is 120 966 bp whereas avrPiz-t-70-15 is 146 292 bp in length. The extreme sequence similarity and good synteny in gene order and content along with the absence of two predicted genes in the avrPiz-t-70-15 region were observed in the predicted protein-coding regions in the AvrPiz-t locus. Nevertheless, frequent presence/absence and highly dynamic organization of transposable elements (TEs) were identified, representing the major variation of the AvrPiz-t locus between different isolates. Moreover, TEs constitute 27.3% and 43.2% of the genomic contents of the AvrPiz-t-ZB15 and avrPiz-t-70-15 regions, respectively, indicating that TEs contribute largely to the organization and evolution of AvrPiz-t locus. The findings of this study suggest that M. oryzae could benefit in an evolutionary sense from the presence of active TEs in genes conferring avirulence and provide an ability to rapidly change and thus to overcome host R genes.     

FLT3 and NPM1 mutations in Chinese patients with acute myeloid leukemia and normal cytogenetics

Mutations of fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) exon 12 genes are the most common abnormalities in adult acute myeloid leukemia (AML) with normal cytogenetics. To assess the prognostic impact of the two gene mutations in Chinese AML patients, we used multiplex polymerase chain reaction (PCR) and capillary electrophoresis to screen 76 AML patients with normal cytogenetics for mutations in FLT3 internal tandem duplication (FLT3/ITD) and exon 12 of the NPM1 gene. FLT3/ITD mutation was detected in 15 (19.7%) of 76 subjects, and NPM1 mutation in 20 (26.3%) subjects. Seven (9.2%) cases were positive for both FLT3/ITD and NPM1 mutations. Significantly more FLT3/ITD aberration was detected in subjects with French-American-British (FAB) M1 (42.8%). NPM1 mutation was frequently detected in subjects with M5 (47.1%) and infrequently in subjects with M2 (11.1%). FLT3 and NPM1 mutations were significantly associated with a higher white blood cell count in peripheral blood and a lower CD34 antigen expression, but not age, sex, or platelet count. Statistical analysis revealed that the FLT3/ITDpositive group had a lower complete remission (CR) rate (53.3% vs. 83.6%). Survival analysis showed that the FLT3/ITD-positive/NPM1 mutation-negative group had worse overall survival (OS) and relapse-free survival (RFS). The FLT3/ITD-positive/NPM1 mutation-positive group showed a trend towards favorable survival compared with the FLT3/ITD-positive/NPM1 mutation-negative group (P=0.069). Our results indicate that the FLT3/ITD mutation might be a prognostic factor for an unfavorable outcome in Chinese AML subjects with normal cytogenetics, while NPM1 mutation may be a favorable prognostic factor for OS and RFS in the presence of FLT3/ITD.