Functional characterization of selected LEA proteins from Arabidopsis thaliana in yeast and in vitro

Main conclusion

Expression of eight LEA genes enhanced desiccation tolerance in yeast, including two LEA_2 genes encoding atypical, stably folded proteins. The recombinant proteins showed enzyme, but not membrane protection during drying. To screen for possible functions of late embryogenesis abundant (LEA) proteins in cellular stress tolerance, 15 candidate genes from six Arabidopsis thaliana LEA protein families were expressed in Saccharomyces cerevisiae as a genetically amenable eukaryotic model organism. Desiccation stress experiments showed that eight of the 15 LEA proteins significantly enhanced yeast survival. While none of the proteins belonging to the LEA_1, LEA_5 or AtM families provided protection to yeast cells, two of three LEA_2 proteins, all three LEA_4 proteins and three of four dehydrins were effective. However, no significantly enhanced tolerance toward freezing, salt, osmotic or oxidative stress was observed. While most LEA proteins are highly hydrophilic and intrinsically disordered, LEA_2 proteins are “atypical”, since they are more hydrophobic and possess a stable folded structure in solution. Because nothing was known about the functional properties of LEA_2 proteins, we expressed the three Arabidopsis proteins LEA1, LEA26 and LEA27 in Escherichia coli. The bacteria expressed all three proteins in inclusion bodies from which they could be purified and refolded. Correct folding was ascertained by Fourier transform Infrared (FTIR) spectroscopy. None of the proteins was able to stabilize liposomes during freezing or drying, but they were all able to protect the enzyme lactate dehydrogenase (LDH) from inactivation during freezing. Significantly, only LEA1 and LEA27, which also protected yeast cells during drying, were able to stabilize LDH during desiccation and subsequent rehydration.     

Expression analysis of dehydrin multigene family across tolerant and susceptible barley (Hordeum vulgare L.) genotypes in response to terminal drought stress

Dehydrins are one of the characteristic families of plant proteins that usually accumulate in response to drought. In the present study, gene expressions of dehydrin multigene family (13 genes) were examined in flag leaves of tolerant (Yousef) and susceptible (Moroco) barley varieties under terminal drought to characterize the involvement of dehydrins in the adaptive processes. The stomatal conductance, RWC, and Chl a, b contents had more reduction in Moroco than the Yousef which has more elevated osmotic adjustment. Drought stress increased significantly MDA and electrolyte leakage levels, but greater in Moroco, indicating a poor protection of cell and cytoplasmic membrane in this variety. Yousef variety had no reduction in grain yield under drought condition. Five genes (Dhn1, Dhn3, Dhn5, Dhn7 and Dhn9) were exclusively induced in Yousef under drought stress. In the stress condition, relative gene expression of Dhn3, Dhn9 had the direct correlations (P < 0.05) with Chl a, b contents, osmotic adjustment, stomatal conductance, plant biomass and grain yield, and the negative correlations (P < 0.05) with MDA and electrolyte leakage levels. The results supported the impending functional roles of dehydrin K_n and particularly Y_nSK_n types in dehydration tolerance of barley during the reproductive stage.     

Genetic variations in the KIR gene family may contribute to susceptibility to ankylosing spondylitis: a meta-analysis

The present meta-analysis of relevant case–control studies was conducted to investigate the possible relationships between genetic variations in the killer cell immunoglobulin-like receptor (KIR) gene clusters of the human KIR gene family and susceptibility to ankylosing spondylitis (AS). The following electronic databases were searched for relevant articles without language restrictions: the Web of Science, the Cochrane Library Database, PubMed, EMBASE, CINAHL, the Chinese Biomedical Database (CBM) and Chinese National Knowledge Infrastructure (CNKI) databases, covering all papers published until 2013. STATA statistical software was adopted in this meta-analysis as well. We also calculated the crude odds ratios (OR) and its 95 % confidence intervals (95 % CI). Seven case–control studies with 1,004 patients diagnosed with AS and 2,138 healthy cases were implicated in our meta-analysis, and 15 genes in the KIR gene family were also evaluated. The results of our meta-analysis show statistical significance between the genetic variations in the KIR2DL1, KIR2DS4, KIR2DS5 and KIR3DS1 genes and an increased susceptibility to AS (KIR2DL1: OR 7.82, 95 % CI 3.87–15.81, P< 0.001; KIR2DS4: OR 1.91, 95 % CI 1.16–3.13, P = 0.010; KIR2DS5: OR1.51, 95 % CI 1.14–2.01, P = 0.004; KIR3DS1: OR 1.58, 95 % CI 1.34–1.86, P< 0.001; respectively). However, we failed to found positive correlations between other genes and susceptibility to AS (all P >0.05). The current meta-analysis provides reliable evidence that genetic variations in the KIR gene family may contribute to susceptibility to AS, especially for the KIR2DL1, KIR2DS4, KIR2DS5 and KIR3DS1 genes.     

RNA virus accumulation is inhibited by ribonuclease activity of 3D8 scFv in transgenic Nicotiana tabacum

A mannose selection system was adapted for Agrobacterium-mediated transformation of plum (Prunus domestica L.) hypocotyl explants and the recovery of transgenic plants. Adventitious regeneration from non-transformed hypocotyl sections was inhibited when 3 mg/l mannose, combined with 10 mg/l sucrose, was added to the medium. Mature seed hypocotyl slices from the cultivar ‘Claudia Verde’ were infected with A. tumefaciens AGL1, carrying the pNOVgus vector, and placed onto different selective media with mannose. A low mannose selection (1.5 g/l, regeneration below the inhibitory concentration) applied for 16 weeks led to the regeneration of escapes. However, when mannose at 1.5 g/l or at 3 g/l (the regeneration-inhibiting concentration) was applied for 6 weeks from the beginning of the experiments and, after that, was increased to 5 g/l, several independent transgenic lines were obtained. The transformation events were monitored by detection of the GUS enzymatic activity at different stages of the process. Nevertheless, stable integration of transgenes into the genome of the plum plants was confirmed by PCR and Southern blot analysis. The transformed shoots were rooted on a medium supplemented with 10 g/l sucrose and 4 g/l mannose. The transformation procedure described here, using the pmi/mannose system for selection of transgenic plum plants, represents an alternative for the production of transgenic plum plants under conditions that are safe regarding human health and the environment, and would permit the insertion of more transgene/s in a pre-existing transgenic line.     

Prevalence, distribution and antibiotic resistance pattern among enterococci species in two traditional fermented dairy foods

The prevalence, distributions and antibiotic resistance pattern among enterococci species were determined. A total of 30 samples of Nigerian traditional fermented dairy food were positive to presence of enterococci, with viable counts of 4.17 log CFU/g in nunu, lower than 4.55 log CFU/g observed in wara samples. Twenty-five representative strains were characterized by a combination of phenotypic and genomic typing based on 16S rRNA gene and multi-locus sequencing analysis (MLSA) of RNA polymerase A (rpoA) and phenylanaline synthase (pheS) genes sequencing; these strains were identified as Enterococcus faecium (84 %) and Enterococcus faecalis (16 %). All the 95 enterococci isolated from wara and nunu samples were alpha haemolytic with multi-drug resistance to 10 antimicrobials regardless of class. Four strains were sensitive to chloramphenicol (30 μg) while 33.7 % of the total isolates were resistant to vancomycin from 5 μg. This information will enhance understanding of Enterococcus drug resistance and distribution in traditional fermented foods to support safety and guarantee quality of traditional foods in West Africa.     

Effects of single and combined genotypes of MC4R and POU1F1 genes on two production traits in Langshan chicken

The objective of this study was to analyze the effects of single and combined genotypes of MC4R and POU1F1 genes in Chinese well-known indigenous chicken (Langshan chicken) population. Genetic variants within MC4R gene and POU1F1 gene were screened through PCR-SSCP and DNA sequencing methods. A C/T mutation at nt 944 in MC4R gene (NC_006089.2:g. 944C>T) and a G/A mutation at nt 3109 in POU1F1 gene (NC_006088.2:g. 3109 G>A) were identified. Associations between the mutations of the two genes with two production traits were analyzed. The results showed that, at MC4R locus, individuals with BB and AB genotypes had highly significantly higher body weight at 16 weeks (p < 0.01) than did those with the AA genotype. And, individuals within AA and AB genotypes had significantly higher egg numbers at 300 days (p < 0.05). At POU1F1 locus, individuals with CD genotype had higher body weight at 16 weeks and egg numbers at 300 days (p < 0.05). Furthermore, combined genotypes from these two loci were found to be associated with egg numbers at 300 days (p < 0.05). The individuals within combined genotype AB/CD had higher egg production. Therefore, variations identified within the MC4R and POU1F1genes are suitable for future use in identifying chickens with the genetic potential of higher body weight and reproductive traits, at least in the population of Langshan chickens.     

Effects of phosphogypsum on the growth of potato plants overexpressing the StDREB1 transcription factor

We developed an efficient system for agrobacterial transformation of plum (Prunus domestica L.) leaf explants using the PMI/mannose and GFP selection system. The cultivar ‘Startovaya’ was transformed using Agrobacterium tumefaciens strain CBE21 carrying the vector pNOV35SGFP. Leaf explants were placed onto a nutrient medium containing various concentrations and combinations of mannose and sucrose to develop an efficient selection system. Nine independent transgenic lines of plum plants were obtained on a regeneration medium containing 20 g/L sucrose and 15 g/L mannose. The highest transformation frequency (1.40?%) was produced using a delayed selection strategy. Starting from the 1st days after transformation and ending by regeneration of shoots from the transgenic callus, selection of transgenic cells was monitored by GFP fluorescence that allowed avoiding formation of escapes. Integration of the manA and gfp transgenes was confirmed by PCR and Southern blotting. The described transformation protocol using a positive PMI/mannose system is an alternative selection system for production of transgenic plum plants without genes of antibiotic and herbicide resistance, and the use of leaf explants enables retention of cultivar traits of plum plants.     

Expression of dehydrin gene from Arctic Cerastium arcticum increases abiotic stress tolerance and enhances the fermentation capacity of a genetically engineered Saccharomyces cerevisiae laboratory strain

We investigated Arctic plants to determine if they have a specific mechanism enabling them to adapt to extreme environments because they are subject to such conditions throughout their life cycles. Among the cell defense systems of the Arctic mouse-ear chickweed Cerastium arcticum, we identified a stress-responsive dehydrin gene CaDHN that belongs to the SK₅ subclass and contains conserved regions with one S segment at the N-terminus and five K segments from the N-terminus to the C-terminus. To investigate the molecular properties of CaDHN, the yeast Saccharomyces was transformed with CaDHN. CaDHN-expressing transgenic yeast (TG) cells recovered more rapidly from challenge with exogenous stimuli, including oxidants (hydrogen peroxide, menadione, and tert-butyl hydroperoxide), high salinity, freezing and thawing, and metal (Zn²⁺), than wild-type (WT) cells. TG cells were sensitive to copper, cobalt, and sodium dodecyl sulfate. In addition, the cell survival of TG cells was higher than that of WT cells when cells at the mid-log and stationary stages were exposed to increased ethanol concentrations. There was a significant difference in cultures that have an ethanol content >16 %. During glucose-based batch fermentation at generally used (30 °C) and low (18 °C) temperatures, TG cells produced a higher alcohol concentration through improved cell survival. Specifically, the final alcohol concentrations were 13.3 and 13.2 % in TG cells during fermentation at 30 and 18 °C, respectively, whereas they were 10.2 and 9.4 %, respectively, in WT cells under the same fermentation conditions. An in vitro assay revealed that purified CaDHN acted as a reactive oxygen species scavenger by neutralizing H₂O₂ and a chaperone by preventing high temperature-mediated catalase inactivation. Taken together, our results show that CaDHN expression in transgenic yeast confers tolerance to various abiotic stresses by improving redox homeostasis and enhances fermentation capacity, especially at low temperatures (18 °C).     

Genomic analysis identifies candidate pathogenic variants in 9 of 18 patients with unexplained West syndrome

West syndrome, which is narrowly defined as infantile spasms that occur in clusters and hypsarrhythmia on EEG, is the most common early-onset epileptic encephalopathy (EOEE). Patients with West syndrome may have clear etiologies, including perinatal events, infections, gross chromosomal abnormalities, or cases followed by other EOEEs. However, the genetic etiology of most cases of West syndrome remains unexplained. DNA from 18 patients with unexplained West syndrome was subjected to microarray-based comparative genomic hybridization (array CGH), followed by trio-based whole-exome sequencing in 14 unsolved families. We identified candidate pathogenic variants in 50 % of the patients (n = 9/18). The array CGH revealed candidate pathogenic copy number variations in four cases (22 %, 4/18), including an Xq28 duplication, a 16p11.2 deletion, a 16p13.1 deletion and a 19p13.2 deletion disrupting CACNA1A. Whole-exome sequencing identified candidate mutations in known epilepsy genes in five cases (36 %, 5/14). Three candidate de novo mutations were identified in three cases, with two mutations occurring in two new candidate genes (NR2F1 and CACNA2D1) (21 %, 3/14). Hemizygous candidate mutations in ALG13 and BRWD3 were identified in the other two cases (14 %, 2/14). Evaluating a panel of 67 known EOEE genes failed to identify significant mutations. Despite the heterogeneity of unexplained West syndrome, the combination of array CGH and whole-exome sequencing is an effective means of evaluating the genetic background in unexplained West syndrome. We provide additional evidence for NR2F1 as a causative gene and for CACNA2D1 and BRWD3 as candidate genes for West syndrome.     

The genetic contribution of CIDEA polymorphisms, haplotypes and loci interaction to obesity in a Han Chinese population

To investigate the association of tag-SNPs and haplotype structures of the CIDEA gene with obesity in a Han Chinese population. Five single nucleotide polymorphisms (SNPs) (rs1154588/V115F, rs4796955/SNP1, rs8092502/SNP2, rs12962340/SNP3 and rs7230480/SNP4) in the CIDEA gene were genotyped in a case–control study. Genotyping was performed using the sequenom matrix-assisted laser desorption/ionization time-of-flight mass spectrometry iPLEX platform. There were significant differences between the obese and control groups in genotype distributions of V115F (P < 0.001), SNP1 (P = 0.006) and SNP2 (P = 0.005). Carriers of V115F-TT, SNP1-GG and SNP2-CC genotypes had a 2.84-fold (95 % CI 1.73–4.66), 2.19-fold (95 % CI 1.09–4.38) and 4.37-fold (95 % CI 1.21–15.08) increased risk for obesity, respectively. Haplotype analysis showed that GTTC (SNP1/SNP2/V115F/SNP4) had 1.41-fold (95 % CI 1.02–1.95) increased risk for obesity; whereas, haplotype TTGC had 0.48-fold (95 % CI 0.24–0.96) decreased risk for obesity. Using the multifactor dimensionality reduction method, the best model including SNP1, SNP2, V115F and SNP4 polymorphisms was identified with a maximum testing accuracy to 59.32 % and a perfect cross-validation consistency of 10/10 (P = 0.011). Logistic analysis indicated that there was a significant interaction between SNP1 and V115F associated with obesity. Subjects having both genotypes of SNP1/GG and V115F/TT were more susceptible to obesity in the Han Chinese population (OR 2.66, 95 %: 1.22–5.80). Genotypes of V115F/TT, SNP1/GG and SNP2/CC and haplotype GTTC of CIDEA gene were identified as risk factors for obesity in the Han Chinese population. The interaction between SNP1 and V115F could play a joint role in the development of obesity.     

Polymorphisms of killer immunoglobulin-like receptors (KIRs) and HLA ligands in northeastern Thais

Killer cell immunoglobulin-like receptors (KIRs) are cell surface receptors on natural killer (NK) cells and subsets of T cells. The functions of NK cells are partly regulated by interactions between KIRs and HLA ligands on target cells. In this study, the presence or absence of 17 KIR genes and their known HLA ligands have been investigated in 235 unrelated individuals living in northeastern Thailand (NET). Subtypes of KIR2DS4 including full length (KIR2DS4F) and deleted forms (KIR2DS4D) have also been determined. Framework genes (KIR2DL4, 3DL2, 3DL3, and 3DP1) were found in all individuals and KIR genes belonging to the A haplotype (KIR2DL1, 2DL3, 3DL1, and 2DS4) were present in more than 90 % of NET. KIR2DS4D (61.7 %) was more common than KIR2DS4F (52.8 %). A total of 33 different KIR genotypes were observed. Of these, three new genotypes were identified. The most common genotype (AA) was observed in 35.7 % of NET, and HLA-C alleles bearing the C1 epitope (HLA-C1) had the highest frequency (97 %). All individuals had at least one inhibitory KIR and its corresponding HLA ligand; 40.9 % of NET had three pairs of receptor–ligand combinations, and 18.3 % had all three receptor–ligand combinations of KIR2DL3+C1, 3DL1+Bw4, and 3DL2+A11. Surprisingly, the patterns of KIR gene frequencies in NET are more similar to those of Caucasians than Japanese, Korean, and Chinese. This is the first report on complete analysis of KIR and known HLA ligands in Thais. These data provide basic knowledge on KIR for further studies on disease associations and transplantation in northeastern Thais.     

Genetic variants in the JAK1 gene confer higher risk of Behcet’s disease with ocular involvement in Han Chinese

Recent surveys have identified SLC22A4, SLC22A5, RUNX1, JAK1 as susceptibility genes for various immune-related diseases. An association study was performed in 738 Behcet’s patients with ocular involvement and 1,873 controls using the iPLEX system method. The first-stage study for 30 SNPs showed that SNPs rs2780815, rs310241, rs3790532 in JAK1 were associated with Behcet’s disease in Han Chinese (Pc_{(Bonferroni correction)} = 0.022–7.7 × 10^?3). The G allele and AA genotype of SNP rs2834643 in RUNX1 (Pc = 0.041–1.75 × 10^?3), but none of the other SNPs, were associated with Behcet’s disease. Haplotype analysis for the SLC22A4, SLC22A5 genes showed an increased tendency for AGTCTGCCGC frequency in patients compared with controls; however, the significance was lost after Bonferroni correction (P = 0.004, Pc > 0.05). Subsequently, we further replicated the significantly associated SNPs using another independent cohort. Replication and combining studies showed that three SNPs rs2780815, rs310241, rs3790532 in JAK1, but not SNP rs2834643 in RUNX1, were consistently associated with Behcet’s disease (replication: Pc = 0.012–9.60 × 10^?4; combining: Pc = 0.030–1.90 × 10^?4). SNPs rs2780815, rs310241, rs3790532 were estimated to confer a population attributable risk of 35.0, 28.0, 27.0 %, respectively. We found a strong association between HLA-B51 with Behcet’s disease in Chinese Han population (P = 1.35 × 10^?73; OR = 5.15; 95 % CI 4.28–6.19). GMDR analysis showed that no gene–gene interaction was detectable between JAK1 and HLA-B51. Logistic analysis indicated that the JAK1 gene was an independent risk factor for Behcet’s disease (P > 0.05). Real-time PCR analysis showed that no difference on the expression of JAK1 in PBMCs or LPS-stimulated PBMCs between individuals with the different rs1762780815 genotypes studied (P > 0.05). In conclusion, this study suggests that JAK1, but not SLC22A4, SLC22A5 and RUNX1, contributes to the genetic susceptibility to Behcet’s disease with ocular involvement.     

Y chromosome diversity and paternal origin of Chinese cattle

To determine the Y chromosome genetic diversity and paternal origin of Chinese cattle, 369 bulls from 17 Chinese native cattle breeds and 30 bulls from Holstein and four bulls from Burma were analyzed using a recently discovered USP9Y marker that could distinguish between taurine and indicine cattle more efficiently. In total, the taurine Y1, Y2 haplogroup and indicine Y3 haplogroup were detected in 7 (1.9 %), 193 (52.3 %) and 169 (45.8 %) individuals of 17 Chinese native breeds, respectively, although these frequencies varied amongst the Chinese native cattle breeds examined. Y2 dominates in northern China (91.4 %), while Y3 dominates in southern China (81.2 %). Central China is an admixture zone with Y2 predominating overall (72.0 %). Our results demonstrate that Chinese cattle have two paternal origins, one from B. taurus (Y2) and the other from B. indicus (Y3). The Y1 haplogroup may originate from the imported beef cattle breeds in western countries. The geographical distributions of the Y2 and Y3 haplogroup frequencies reveal a pattern of male indicine introgression from south to north China, and male taurine introgression from north to south China.     

Identification of a mosquitocidal toxin from Bacillus thuringiensis using mass spectrometry

The Bacillus thuringiensis strain S2160-1 has previously been identified as being highly toxic to mosquito larvae and a viable alternative to strains currently used commercially to control these insects. A PCR approach had identified the presence of four putative insecticidal toxin genes (cry30Ea, cry30 Ga, cry50Ba and cry54Ba) in this strain, but did not identify the genes that encoding three of the main crystal toxin proteins of size 140 and 130 and 30 kDa. In this study we used mass spectrometry to identify the 130 kDa toxin as a rare Cry4 toxin (Cry4Cb3). The gene encoding this toxin was cloned and expressed and the toxin shown to have mosquitocidal activity against Culex quinquefasciatus.     

Gene loss/retention and evolutionary pattern of ascorbic acid biosynthesis and recycling genes in <Emphasis Type="Italic">Brassica rapa</Emphasis> following whole genome triplication

Ascorbic acid (AsA) is an inevitable antioxidant found abundantly in higher plants. Despite the importance of AsA in plants, how AsA biosynthesis (ABGs; d-mannose/l-galactose pathway) and AsA recycling genes (ARGs) evolved through polyploidization has not been addressed to date. Here, we evaluated the impacts of whole genome triplication (WGT) on ABGs and ARGs in Chinese cabbage (Brassica rapa ssp. pekinensis), which diverged from Arabidopsis thaliana before the WGT event. Twenty-three ABGs coded in 13 loci representing nine different enzyme classes and 29 ARGs coded in 19 loci representing five different enzyme classes were identified in the B. rapa genome by whole-genome screening through comparative genomic analyses. Five of these loci maintained three gene copies, 10 loci maintained two gene copies and the majority of the loci (n = 17) maintained single gene copies. Segmental (62 %) and tandem duplication (6 %), and fragment (21 %) and large-scale recombination (10 %) events accelerated the diversification of ABGs and ARGs. Thirteen of the 52 (25 %) identified genes experienced intron losses and two (4 %) experienced intron gains implying that intron losses outnumbered intron gains. The expansion and the retention of ABGs and ARGs were similar to the whole genome gene expansion and retention (P > 0.05). These findings provide new insights into the structural characteristics and evolutionary trends of ABGs and ARGs. In addition, our data could become a useful resource to further the functional characterization of these genes.     

Study on genetic variations of PPARα gene and its effects on thermal tolerance in Chinese Holstein

Peroxisome proliferator-activated receptor alpha (PPARα) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. In this research, polymerase chain reaction (PCR) technique was used to amplify 766 and 589 bp fragments of intron 3 and 7 of PPARα gene in Chinese Holstein (n = 771). Sequencing results showed that three novel single nucleotide polymorphisms (SNPs) were identified at position 44087 (G/A), 65550 (G/A), and 65676(G/A) in the PPARα gene. PCR–restriction fragment length polymorphism technology was used to genotype the three SNPs. Association analysis showed that cows with H1H8 (P < 0.05), H2H8 (P < 0.01), H5H7 (P < 0.05), H5H8 (P < 0.05), and H8H8 (P < 0.05) haplotype combinations had lower potassium content in erythrocytes than those with H2H6 haplotype combination. Cows with H1H8, and H8H8 haplotype combinations had lower decrease rate of milk yield than those with H2H6 and H6H8 haplotype combinations (P < 0.05). Cows with H2H8 and H8H8 haplotype combinations had lower rectal temperature than those with H5H8 and H7H7 haplotype combinations (P < 0.05). In conclusion, H8H8 haplotype combination may be advantageous for heat resistance traits in Chinese Holstein cattle.