Core Proteins of Rat Liver Heterogeneous Nuclear Ribonucleoprotein (hnRNP) Particles

To study the nature of the four core proteins A, B, C, and D, of rat liver heterogeneous nuclear ribonucleoprotein (hnRNP) particles, the proteins were isolated and fractionated by preparative sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. The structure of the four purified proteins were compared by partial peptide mapping on SDS-polyacrylamide gels, using the Cleveland Method. The enzymes used for the partial peptide mapping were a-Chymotrypsin and S. aureus V8 Protease. Products of the proteolytic treatment were detected on the SDS-polyacrylamide gels either by silver-staining or by autoradiography. In the latter case core proteins iodinated in their tyrosine residues were used and therefore only the tyrosine-containing fragments were observed. Experiments were further conducted to determine whether the four proteins represent glycosylated variants of one protein. As an aid for the structural study of the proteins, polyclonal antibodies were raised to total core proteins and separately to core protein A and B. Immunoblots of the core proteins with the antibody preparation were used to establish the relationship between the four core proteins. Several approaches were made to determine the cause of the observed charge heterogeneity of each of the core proteins when they are fractionated on a pH-gradient. 1) Artefactual formation of the charge isomers was considered. 2) The charge isomers of core proteins A and B were subjectd to partial peptide mapping by the Cleveland Method. One dimensional SDS-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis were used to map the peptides produced after V8 protease treatment. 3) The core proteins were subjected to both in vitro and in vivo phosphorylation. For the in vitro studies, qualitative and quantitative phosphorylation patterns were analysed. The in vivo phosphorylation was performed with core proteins of a Buffalo Rat Liver Cell Line, and 4) Preliminary attempts were made to determine whether the core proteins are poly ADP-ribosylated. Results from the comparison of the partial peptide maps of isolated core proteins A, B, C and D indicated that the four proteins are a family of closely related species. A majority of the peptide products from each protein were identical, and there were relatively few unique bands. The immunological experiments confirmed the close relatedness of the proteins. Since the core proteins were found not to be glycosylated variants of each other, and certain unique peptides were observed, it was concluded that they probably are products of separate genes. The partial peptide maps of the charge isomers of both core protein A and B were shown to be nearly identical. In the A series, charge isomers A1 to A6 are probably post-translational modifications of one protein. The B series of charge isomers B1 to B5 were concluded to be post-translational variants of one or possibly two proteins. It was not possible to identify the nature of the post-synthetic modification responsible for the charge isomers

Paired growth of cultivated and halophytic wild rice under salt stress induces bacterial endophytes and gene expression responses

IntroductionUtilizing salt-affected marginal lands in coastal regions can help meet the growing demand for rice. We explored a nature-based solution involving wild halophytic rice (O. coarctata, Oc) and commercial rice BRRI Dhan 67 (O. sativa, Os) grown in close proximity to each other under salt stress.MethodsThis was to investigate whether a paired planting strategy could help complement rice growth and yield under stress. We also investigated the gene expression and endophytic bacterial profiles of both Os and Oc in unpaired and paired conditions without and with salt.ResultsPaired plants exhibited lower salt damage indicators such as smaller reduction in plant height, electrolyte leakage and chlorophyll loss, as well as higher K+/Na+ ratio under saline stress. Some of the 39 endophytic bacteria in the mutualism experiment were unique to Oc and transferred to Os when paired. Differentially expressed genes in leaves of paired Os versus unpaired Os were 1097 (994 up-regulated, 101 down-regulated) without salt and 893 (763 up-regulated, 130 down-regulated) under salt stress. The presence of Oc plants under salt stress influenced major biological processes in Os, including oxidative stress; chitinase activity; phenylalanine catabolic process and response to ABA. Protein binding and serine/threonine kinase activity were primarily affected in molecular function. The downregulated WRKY transcription factor 22 in paired conditions under salt stress played a role in the MAPK signaling pathway, reducing respiratory cell death. The upregulated auxin-responsive protein IAA18 gene, involved in hormone signaling and cell enlargement, was present only in paired plants.DiscussionOur findings therefore, offer insights into developing more effective cultivation strategies for sustainable rice production

Salt tolerance QTLs of an endemic rice landrace, \u3ci\u3eHorkuch\u3c/i\u3e at seedling and reproductive stages

Salinity has a significant negative impact on production of rice. To cope with the increased soil salinity due to climate change, we need to develop salt tolerant rice varieties that can maintain their high yield. Rice landraces indigenous to coastal Bangladesh can be a great resource to study the genetic basis of salt adaptation. In this study, we implemented a QTL analysis framework with a reciprocal mapping population developed from a salt tolerant landrace Horkuch and a high yielding rice variety IR29. Our aim was to detect genetic loci that contributes to the salt adaptive responses of the two different developmental stages of rice which are very sensitive to salinity stress. We identified 14 QTLs for 9 traits and found that most are unique to specific developmental stages. In addition, we detected a significant effect of the cytoplasmic genome on the QTL model for some traits such as leaf total potassium and filled grain weight. This underscores the importance of considering cytoplasm-nuclear interaction for breeding programs. Finally, we identified QTLs co-localization for multiple traits that highlights the possible constraint of multiple QTL selection for breeding programs due to different contributions of a donor allele for different traits

Characterizing the Saltol quantitative trait locus for salinity tolerance in rice

This study characterized Pokkali-derived quantitative trait loci (QTLs) for seedling stage salinity tolerance in preparation for use in marker-assisted breeding. An analysis of 100 SSR markers on 140 IR29/Pokkali recombinant inbred lines (RILs) confirmed the location of the Saltol QTL on chromosome 1 and identified additional QTLs associated with tolerance. Analysis of a series of backcross lines and near-isogenic lines (NILs) developed to better characterize the effect of the Saltol locus revealed that Saltol mainly acted to control shoot Na +/K + homeostasis. Multiple QTLs were required to acquire a high level of tolerance. Unexpectedly, multiple Pokkali alleles at Saltol were detected within the RIL population and between backcross lines, and representative lines were compared with seven Pokkali accessions to better characterize this allelic variation. Thus, while the Saltol locus presents a complex scenario, it provides an opportunity for markerassisted backcrossing to improve salt tolerance of popular varieties followed by targeting multiple loci through QTL pyramiding for areas with higher salt stress

Modification of Recombination-based GATEWAY TM Binary Destination Vector with Novel Promoter for Agrobacterium-mediated Transformation of Rice

The GATEWAY TM Binary Destination Vector pH7WG2 is available for easy insertion of genes for transformation into plants. The gene of interest integrates downstream of the Cauliflower Mosaic Virus Promoter CaMV 35S by recombination. The CaMV 35S promoter is however not suitable for transformation and expression of genes in monocots like rice. We isolated and cloned a ~1100 bp upstream region from two rice (Pokkali and IR64) Na/H antiporter genes into the GATEWAY TM promoter cloning vector pHGWFS7. The Pokkali promoter expressed the β-glucuronidase or GUS gene ~25-fold more efficiently than the CaMV 35S promoter in rice calli, while that of IR64 was 7-fold more. The IR64 promoter however showed efficient expression in transgenic rice leaves. The promoter from Pokkali Na/H antiporter was used to replace the CaMV 35S sequence in pH7WG2. The CaMV 35S region was cut out and the linear vector fragment blunted and T-tailed. After amplification of the promoter from Pokkali rice DNA, it was A-tailed and ligated to the modified T-vector. The resultant vector, named pH7WG3, following the nomenclature at the gateway site, www.plantgenetics.rug.ac.be/gatewayT, can now be used for recombination of any genes for efficient rice transformation

Enhanced salt tolerance conferred by the complete 2.3 kb cDNA of the rice vacuolar Na+/H+antiporter gene compared to 1.9 kb coding region with 5' UTR in transgenic lines of Rice

Soil salinity is one of the most challenging problems that restricts the normal growth and production of rice worldwide. It has therefore become very important to produce more saline tolerant rice varieties. This study shows constitutive over-expression of the vacuolar Na+/H+ antiporter gene (OsNHX1) from the rice landrace (Pokkali) and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5ʹ UTR with CDS and the other of 2.3 kb, including 5ʹ UTR, CDS and the 3ʹ UTR regions were performed. The transgenic plants with these two different constructs were advanced to the T3 generation and physiological and molecular screening of homozygous plants was conducted at seedling and reproductive stages under salinity (NaCl) stress. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5ʹ and 3ʹ UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3′ UTR. At the seedling stage at 12 dS/m stress, the chlorophyll content was significantly higher (P 1.9 kb> and WT lines. Yield in g/plant in the best line from the 2.3 kb plants was significantly more (P<0.01) compared respectively to the best 1.9 kb line and WT plants at stress of 6 dS/m. Transformation with the complete transcripts rather than the CDS may therefore provide more durable level of tolerance

Overexpression of a DEAD box helicase, PDH45, confers both seedling and reproductive stage salinity tolerance to rice (Oryza sativa L.)

To improve the salinity tolerance of rice, a DEAD-box helicase gene isolated from pea with a CaMV35S promoter was transformed into the Bangladeshi rice variety Binnatoa through Agrobacterium-mediated transformation. The transgenic seedlings showed significantly higher chlorophyll content, but decreased root length compared to wild type (WT) under normal physiological conditions. Their status was confirmed by polymerase chain reaction (PCR), semi-quantitative reverse-transcription PCR and Southern blot hybridization for positive integration of the transgene. The T2 progenies from three independent transformation events were characterized for salinity tolerance both at seedling and reproductive stages. Compared to the WT plants, the average decrease in chlorophyll content and dry weight of seedling leaves was lower by 20 and 12% respectively at 12 deciSiemens per meter (dS/m) NaCl stress in hydroponics. A higher leaf K+/Na+ ratio of 0.346 was maintained by the transgenic lines compared to the WT ratio of 0.157, which indicated induced ion homeostasis. At the reproductive stage, transgenic rice plants expressing PDH45 showed better fertility and produced higher grain yield by 16% compared to WT plants under continuous stress of 6 dS/m from 30 days till maturity. One of the transformed lines, PDH45-P3, outperformed the others, and replicated data in reproductive stage soil stress of 12 dS/m NaCl showed its enhanced fertility and yield by 46 and 29% over WT, respectively

Type 2 diabetes linked FTO gene variant rs8050136 is significantly associated with gravidity in gestational diabetes in a sample of Bangladeshi women: Meta-analysis and case-control study.

ObjectiveGestational diabetes mellitus (GDM) is a growing public health concern that has not been extensively studied. Numerous studies have indicated that a variant (rs8050136) of the fat mass-associated gene, FTO, is associated with both GDM and Type 2 diabetes mellitus(T2DM). We conducted a meta-analysis on the association between the FTO single nucleotide polymorphism (SNP) rs8050136 and T2DM, followed by a case-control study on the association of the said SNP and GDM in a sample of Bangladeshi women.MethodA total of 25 studies were selected after exploring various databases and search engines, which were assessed using the Newcastle-Ottawa Scale (NOS). The MetaGenyo web tool was used to conduct this meta-analysis. A case-control study was performed on 218 GDM patients and 284 controls to observe any association between FTO rs8050136 and GDM. Genotyping was performed using the tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS) method, and statistical analyses were performed using various statistical softwares.ResultsIn the meta-analysis 26231 cases and 43839 controls were examined. Pooled association analyses revealed a statistically significant relationship between the FTO rs8050136 polymorphism and an elevated risk of T2DM under all genetic models (PConclusionOur meta-analysis revealed a significant association between SNP rs8050136 of FTO with T2DM, and this variant was substantially associated with an increased risk of GDM in a sample of Bangladeshi multigravida women

Profiling Bangladeshi rice diversity based on grain size and amylose content using molecular markers

Bangladesh lies within the origin of rice biodiversity and is home to many genotypes with varying amylose content and grain size. There is however no precise data on their amylose content and correlation with grain size and type. Yet these classifications governs the taste, cooking and nutritional quality of rice, ultimately determining its consumer preference and market price. Correlating grain size with amylose content will be useful information for Diabetics, particularly due to the unproven assumption that long grains correlate with high amylose content and vice versa. In this study, 131 long, medium and short grain Bangladeshi rice cultivars were analyzed and classified into high, intermediate, low or very low (H, I, L or VL) amylose slender or bold grain types. Both short and medium grain cultivars have mostly H or I amylose. The maximum amount of L and VL amylose was found among the long grain cultivars which also contain H and I amylose-containing rice. Therefore we could clearly show that long grains are associated with L and VL amylose, rather than medium and short grain. The microsatellite marker (RM190) and CAPS marker (RM190F-GBSSW2R/AccI) are linked to the gene responsible for amylose content and can be used for distinguishing variants. Thirty-five cultivars with all grain sizes were found to have four RM190 alleles (1–4: 100, 110, 116 and 120bp). Cultivars having VL amylose were identified by RM190F-GBSSW2R/AccI cleavage pattern. High amylose-containing rice was found mostly associated with class 1 and 2 alleles. The intermediate amylose group was characterized by the absence of the class 1 allele. Therefore we could distinguish VL, H and I amylose rice with simple tests with the rest belonging to the L group. The study thus demonstrated the use of simple tests in the categorization of rice, based on amylose groups. Keywords: Amylose content, CAPS, Single nucleotide polymorphism (SNP), Restriction enzyme (AccI