Monitoring of Gene Expression Profiles and Isolation of Candidate Genes Involved in Pollination and Fertilization in Rice (Oryza Sativa L.) with a 10K cDNA Microarray

To monitor gene expression profiles during pollination and fertilization in rice at a genome scale, we generated 73,424 high-quality expressed sequence tags (ESTs) derived from the green/etiolated shoot and pistil (0-5 h after pollination, 5hP) of rice, which were subsequently used to construct a cDNA microarray containing ca. 10 000 unique rice genes. This microarray was used to analyze gene expression in pistil unpollinated (UP), 5hP and 5DAP(5 days after pollination), anther, shoot, root, 10-day-old embryo (10EM) and 10-day-old endosperm (10EN). Clustering analysis revealed that the anther has a gene-expression profile more similar to root than to pistil and most pistil-preferentially expressed genes respond to pollination and/or fertilization. There are 253 ESTs exhibiting differential expression (e +/- 2-fold changes) during pollination and fertilization, and about 70% of them can be assigned a putative function. We also recovered 20 genes similar to pollination-related and/or fertility-related genes previously identified as well as genes that were not implicated previously. Microarray and real-time PCR analyses showed that the array sensitivity was estimated at 1-5 copies of mRNA per cell, and the differentially expressed genes showed a high correlation between the two methods. Our results indicated that this cDNA microarray constructed here is reliable and can be used for monitoring gene expression profiles in rice. In addition, the genes that differentially expressed during pollination represent candidate genes for dissecting molecular mechanism of this important biological process in rice.     

ZNRF3 acts as a tumour suppressor by the Wnt signalling pathway in human gastric adenocarcinoma

E3 ubiquitin ligases regulate a variety of biological processes through the ubiquitin–proteasome system, together with ubiquitin activating enzyme E1 and ubiquitin-conjugating enzyme E2. Previous studies have demonstrated that zinc and ring finger 3 (ZNRF3), which belongs to the E3 ubiquitin ligases family is involved in the Wnt signalling pathway, which plays an important role in causing cancer. However, the expression and function of ZNRF3 in human gastric adenocarcinoma still remains unclear. Immunohistochemical and western blot analysis showed a significant down-regulation of ZNRF3 protein in gastric adenocarcinoma tissues compared with adjacent normal gastric tissues. In addition, there was a correlation between the down-regulation of ZNRF3 and poor tissue differentiation in gastric adenocarcinoma. To investigate the potential function of ZNRF3 in cell proliferation and apoptosis, a gastric cell line SGC7901 was employed. The over-expression of wild-type ZNRF3, which was accomplished by the transient transfection of recombinant pEGFP-ZNRF3 (or empty plasmids as control) into the cell line SGC7901, was confirmed by western blot analysis. Flow-cytometry-based and Cell Counting Kit-8 assays showed that over-expression of wt ZNRF3 induced apoptosis and suppressed proliferation. ZNRF3-overexpressing gastric cells displayed partly attenuated protein levels of beta-catenin and TCF-4 compared with those transfected with the empty plasmid. Our study demonstrates a novel gastric adenocarcinoma suppressor and reveals that ZNRF3 inhibits gastric cancer cell growth and promotes the cell apoptosis by affecting the Wnt/beta-catenin/TCF signalling pathway.     

Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck)

Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of “Washington” navel orange and its red-fleshed mutant “Cara Cara”. Results showed that phytoene was exclusively accumulated in peel and pulp of “Cara Cara”. Although phytoene was observed accumulating with fruit ripening of “Cara Cara”, the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in “Cara Cara” and “Washington” genomes. During “Cara Cara” and “Washington” fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of “Cara Cara” was higher than that in “Washington”, as evidenced by phytoene accumulation in the peel.     

Effect of sulfide on the immune response and susceptibility to Vibrio alginolyticus in the kuruma shrimp Marsupenaeus japonicus

Kuruma shrimp Marsupenaeus japonicus held in 34 per thousand seawater were injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus (2.7x10(6)cfu shrimp(-1)), and then placed in water containing concentrations of sulfide at 0 (control), 51, 106, 528 and 1050microgl(-1), respectively. After 12-144h, mortality of V. alginolyticus-injected shrimp exposed to 528 and 1102microgl(-1) sulfide was significantly higher than that of shrimp exposed to 51microgl(-1) sulfide and the control solution. In another experiment, M. japonicus which had been exposed to 0, 56, 112, 525 and 1076microgl(-1) sulfide for 6, 12, 24 and 48h were examined for immune parameters, and phagocytic activity and clearance efficiency of V. alginolyticus. Sulfide concentrations at 525microgl(-1) or greater for 12h resulted in decreased total haemocyte count (THC) and phenoloxidase activity, phagocytic activity and bacterial clearance efficiency, whereas a sulfide concentration at 1076microgl(-1) for 24h caused a significant increase in respiratory burst and superoxide dismutase activity of M. japonicus. It is concluded that concentrations of sulfide at 528microgl(-1) or greater increased the susceptibility of M. japonicus against V. alginolyticus infection by a depression in immune ability. The increased production of superoxide anion by M. japonicus exposed to 525microgl(-1) sulfide or greater was considered to be cytotoxic to the host.     

Molecular dynamics simulations on hydrogen adsorption in finite single walled carbon nanotube bundles

Molecular dynamics simulations of the adsorption of hydrogen molecules in finite single-walled carbon nanotube bundles are presented using a curvature dependent force field. The heat of formation and the effective adsorption capacity are expressed as a function of H₂ distance from adsorbent. The heat of adsorption decreases rapidly with the distance and increasing H₂ loading results in weakening adsorption strength. The effects of nanotube packing and bundle thickness on hydrogen adsorption strength were investigated and the results show that the heat of adsorption can be improved slightly if hydrogen molecules are placed in thicker and inhomogeneously packed nanotube bundles. Only very small diameter nanotube bundles were found to hold promise for significant hydrogen storage for onboard applications.     

Effects of GCK,GCKR, G6PC2 and MTNR1B Variants on Glucose Metabolism and Insulin Secretion

Background

Single nucleotide polymorphisms (SNPs) from GCK, GCKR, G6PC2 and MTNR1B were found to modulate the fasting glucose levels. The current study aimed to replicate this association in the Chinese population and further analyze their effects on biphasic insulin secretion.

Methods/Principal Findings

SNPs from GCK, GCKR, G6PC2 and MTNR1B were genotyped in the Shanghai Chinese, including 3,410 type 2 diabetes patients and 3,412 controls. The controls were extensively phenotyped for the traits related to glucose metabolism and insulin secretion. We replicated the association between GCK rs1799884, G6PC2 rs16856187 and MTNR1B rs10830963 and fasting glucose in our samples (p = 0.0003∼2.0×10⁻⁸). GCK rs1799884 and G6PC2 rs16856187 showed association to HOMA-β, insulinogenic index and both first- and second-phases insulin secretion (p = 0.0030∼0.0396). MTNR1B rs10830963 was associated to HOMA-β, insulinogenic index and first-phase insulin secretion (p = 0.0102∼0.0426), but not second-phase insulin secretion (p = 0.9933). Combined effect analyses showed individuals carrying more risk allele for high fasting glucose tended to have a higher glucose levels at both fasting and 2 h during OGTTs (p = 1.7×10⁻¹³ and 0.0009, respectively), as well as lower HOMA-β, insulinogenic index and both first- and second-phases insulin secretion (p = 0.0321∼1.1×10⁻⁷).

Conclusions/Significance

We showed that SNPs from GCK, G6PC2 and MTNR1B modulated the fasting glucose levels in the normoglycaemic population while SNPs from G6PC2 and GCKR was associated with type 2 diabetes. Moreover, we found GCK and G6PC2 genetic variants were associated to both first- and second-phases insulin secretion while MTNR1B genetic variant was associated with first-phase insulin secretion, but not second-phase insulin secretion.     

DEP and AFO Regulate Reproductive Habit in Rice

Sexual reproduction is essential for the life cycle of most angiosperms. However, pseudovivipary is an important reproductive strategy in some grasses. In this mode of reproduction, asexual propagules are produced in place of sexual reproductive structures. However, the molecular mechanism of pseudovivipary still remains a mystery. In this work, we found three naturally occurring mutants in rice, namely, phoenix (pho), degenerative palea (dep), and abnormal floral organs (afo). Genetic analysis of them indicated that the stable pseudovivipary mutant pho was a double mutant containing both a Mendelian mutation in DEP and a non-Mendelian mutation in AFO. Further map-based cloning and microarray analysis revealed that dep mutant was caused by a genetic alteration in OsMADS15 while afo was caused by an epigenetic mutation in OsMADS1. Thus, OsMADS1 and OsMADS15 are both required to ensure sexual reproduction in rice and mutations of them lead to the switch of reproductive habit from sexual to asexual in rice. For the first time, our results reveal two regulators for sexual and asexual reproduction modes in flowering plants. In addition, our findings also make it possible to manipulate the reproductive strategy of plants, at least in rice.     

Diversified chromosomal distribution of tandemly repeated sequences revealed evolutionary trends in Secale (Poaceae)

Genomic in situ hybridization (GISH) with Secale cereale cv. ‘Jingzhou rye’ DNA as a probe to chromosomes of hexaploid triticale line Fenzhi-1 revealed that not only were all chromosomes of rye strongly hybridized along the entire chromosome length, but there were also stronger signals in terminal or subtelomeric regions. This pattern of hybridization signals is referred to as GISH banding. After GISH banding, sequential fluorescene in situ hybridizaion (FISH) with tandem repeated sequence pSc200 and pSc250 as probes showed that the chromosomal distribution of pSc200 is highly coincident with the GISH banding pattern, suggesting that GISH banding revealed chromosomal distribution of pSc200 in rye. In addition, FISH using pSc200 and pSc250 as probes to chromosomes of 11 species of the genus Secale and two artificial amphiploids (Triticum aestivum-S. strictum subsp. africanum amphiploid and Aegilops tauschii-S. silvestre amphiploid) showed that (1) the chromosomal distribution of pSc200 and pSc250 differed greatly in Secale species, and the trend towards an increase in pSc200 and pSc250 binding sites from wild species to cultivated rye suggested that pSc200 and pSc250 sequences gradually accumulated during Secale evolution; (2) the chromosomal distribution of pSc200 and pSc250 presented polymorphism on homologous chromosomes, suggesting that the same species has two heterogeneous homologous chromosomes; (3) the intensity and number of hybridization signals varied differently on chromosomes between pSc200 and pSc250, suggesting that each repetitive family evolved independently.     

Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain

We have observed extensive interindividual differences in DNA methylation of 8590 CpG sites of 6229 genes in 153 human adult cerebellum samples, enriched in CpG island “shores” and at further distances from CpG islands. To search for genetic factors that regulate this variation, we performed a genome-wide association study (GWAS) mapping of methylation quantitative trait loci (mQTLs) for the 8590 testable CpG sites. cis association refers to correlation of methylation with SNPs within 1 Mb of a CpG site. 736 CpG sites showed phenotype-wide significant cis association with 2878 SNPs (after permutation correction for all tested markers and methylation phenotypes). In trans analysis of methylation, which tests for distant regulation effects, associations of 12 CpG sites and 38 SNPs remained significant after phenotype-wide correction. To examine the functional effects of mQTLs, we analyzed 85 genes that were with genetically regulated methylation we observed and for which we had quality gene expression data. Ten genes showed SNP-methylation-expression three-way associations—the same SNP simultaneously showed significant association with both DNA methylation and gene expression, while DNA methylation was significantly correlated with gene expression. Thus, we demonstrated that DNA methylation is frequently a heritable continuous quantitatively variable trait in human brain. Unlike allele-specific methylation, genetic polymorphisms mark both cis- and trans-regulatory genetic sites at measurable distances from their CpG sites. Some of the genetically regulated DNA methylation is directly connected with genetically regulated gene expression variation.