Cloning of Bacillus subtilis phytase gene construct in Escherichia coli

Background and Objectives: Phytase has a hydrolysis function of phytic acid, which yields inorganic phosphate. Bacillus species can produce thermostable alkaline phytase. The aim of this study was to isolate and clone a Phytase gene (Phy) from Bacillus subtilis in Escherichia coli. Materials and Methods: In this study, the extracellular PhyC gene was isolated from Bacillus subtilis Phytase C. After purification of the bands, DNA fragment of Phy gene was cloned by T/A cloning technique, and the clone was transformed into Escherichia coli. Afterward, the pGEM-Phy was transferred into E. coli Top-10 strain and the recombinants were plated on LB agar containing 100 µg/ml ampicillin. The colonization of 1171 bp of gene Phytase C was confirmed by PCR. The presence of gene-targeting in vector was confirmed with enzymatic digestion by XhoI and XbaI restriction enzymes. Results: The Phytase gene was successfully cloned in E. coli. The result of cloning of 1171 bp Phytase gene was confirmed by PCR assay. Conclusion: Our impression of this article is that several methods, such as using along with microbial, plant phytase reproduction, or low-phytic acid corn may be the better way from a single phytase.

Network Inference of Transcriptional Regulation in Germinating Low Phytic Acid Soybean Seeds

The low phytic acid (lpa) trait in soybeans can be conferred by loss-of-function mutations in genes encoding myo-inositol phosphate synthase and two epistatically interacting genes encoding multidrug-resistance protein ATP-binding cassette (ABC) transporters. However, perturbations in phytic acid biosynthesis are associated with poor seed vigor. Since the benefits of the lpa trait, in terms of end-use quality and sustainability, far outweigh the negatives associated with poor seed performance, a fuller understanding of the molecular basis behind the negatives will assist crop breeders and engineers in producing variates with lpa and better germination rate. The gene regulatory network (GRN) for developing low and normal phytic acid soybean seeds was previously constructed, with genes modulating a variety of processes pertinent to phytic acid metabolism and seed viability being identified. In this study, a comparative time series analysis of low and normal phytic acid soybeans was carried out to investigate the transcriptional regulatory elements governing the transitional dynamics from dry seed to germinated seed. GRNs were reverse engineered from time series transcriptomic data of three distinct genotypic subsets composed of lpa soybean lines and their normal phytic acid sibling lines. Using a robust unsupervised network inference scheme, putative regulatory interactions were inferred for each subset of genotypes. These interactions were further validated by published regulatory interactions found in Arabidopsis thaliana and motif sequence analysis. Results indicate that lpa seeds have increased sensitivity to stress, which could be due to changes in phytic acid levels, disrupted inositol phosphate signaling, disrupted phosphate ion (Pi) homeostasis, and altered myo-inositol metabolism. Putative regulatory interactions were identified for the latter two processes. Changes in abscisic acid (ABA) signaling candidate transcription factors (TFs) putatively regulating genes in this process were identified as well. Analysis of the GRNs reveal altered regulation in processes that may be affecting the germination of lpa soybean seeds. Therefore, this work contributes to the ongoing effort to elucidate molecular mechanisms underlying altered seed viability, germination and field emergence of lpa crops, understanding of which is necessary in order to mitigate these problems.

Agronomic Performance in Low Phytic Acid Field Peas

Field pea is a pulse that delivers high protein content, slowly digestible starch and fiber, and many vitamins and minerals, including iron. Naturally occurring plant phytic acid molecules bind iron, lowering its availability for absorption during digestion. Two low phytic acid (lpa) pea lines, 1-2347-144 and 1-150-81, developed by our group had 15% lower yield and 6% lower seed weight relative to their progenitor cultivar. Subsequently, we crossed the two lpa lines and two cultivars, and derived 19 promising lpa pea breeding lines; here we document their agronomic performance based on 10 replicated field trials in Saskatchewan. Seventeen of these lpa lines yielded greater than 95% of the check mean (associated cultivars) and 16 were above 98% of the check mean for 1000 seed weight. The 19 lpa lines showed 27 to 55% lower phytic acid concentration than the check mean. Iron concentrations were similar in all the lpa lines and cultivars, yet the Caco-2 human cell culture assay revealed 14 of the 19 lpa lines had 11 to 55% greater iron bioavailability than check means. Thus, a single round of plant breeding has allowed for closing the gap in performance of low phytic acid pea.

Combination of High Zn Density and Low Phytic Acid for Improving Zn Bioavailability in Rice (Oryza stavia L.) Grain

Background Zn deficiency is one of the leading public health problems in the world. Staple food crop, such as rice, cannot provide enough Zn to meet the daily dietary requirement because Zn in grain would chelate with phytic acid, which resulted in low Zn bioavailability. Breeding new rice varieties with high Zn bioavailability will be an effective, economic and sustainable strategy to alleviate human Zn deficiency. Results The high Zn density mutant LLZ was crossed with the low phytic acid mutant Os-lpa-XS110–1, and the contents of Zn and phytic acid in the brown rice were determined for the resulting progenies grown at different sites. Among the hybrid progenies, the double mutant always displayed significantly higher Zn content and lower phytic acid content in grain, leading to the lowest molar ratio of phytic acid to Zn under all environments. As assessed by in vitro digestion/Caco-2 cell model, the double mutant contained the relatively high content of bioavailable Zn in brown rice. Conclusions Our findings suggested pyramiding breeding by a combination of high Zn density and low phytic acid is a practical and useful approach to improve Zn bioavailability in rice grain.

Impact of cross-breeding on the metabolites of the low phytic acid rice mutant Os-lpa-MH86-1.

Phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate), the major storage form of phosphorus in cereals, is considered as an antinutrient in food and feed. During the past few years, various cereals have been subjected to mutation breeding for generating low phytic acid (lpa) crops. Recently, it was demonstrated that reduction of phytic acid in the rice mutant Os-lpa-MH86-1 obtained by gamma irradiation was due to a disruption of OsSULTR3;3, an orthologue of the sulfate transporter family group 3 genes. The application of a GC/MS-based metabolite profiling approach revealed that the reduction of phytic acid was accompanied by changes in concentrations of metabolites from different classes in the Os-lpa-MH86-1 mutant.Lpa mutant lines often exhibit lower grain yield and seed viability compared with their wild-type parents. To improve the agronomic performance of the Os-lpa-MH86-1 mutant, cross-breeding with a commercial cultivar was performed. The resulting progenies were genotyped using molecular markers to identify homozygous wildtype and lpa mutants from generations F4 to F7. The objectives of this study were: (i) to observe the consistent metabolic changes in Os-lpa-MH86-1 lpa mutants by following their composition over several independent field trials; (ii) to investigate the impact of cross-breeding on the phytic acid content and the metabolic phenotype of the homozygous lpa mutant; and (iii) to assess the stability of the mutation-specific metabolite signature in the lpa progenies over several generations. Statistical assessment of the data via multivariate and univariate approaches demonstrated that the lpa trait and the mutation-induced metabolite signature in the lpa progenies were comparable to the progenitor Os-lpa-MH86-1 mutant and consistently expressed over generations. These findings extend the basis for implementing mutation breeding in the generation of lpa rice cultivars.

An Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase 1 Mutant with a 33-nt Deletion Showed Enhanced Tolerance to Salt and Drought Stress in Rice

OsIPK1 encodes inositol 1,3,4,5,6-pentakisphosphate 2-kinase, which catalyzes the conversion of myo-inositol-1,3,4,5,6-pentakisphosphate to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) in rice. By clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas9)-mediated mutagenesis in the 3rd exon of the gene, three OsIPK1 mutations, i.e., osipk1_1 (a 33-nt deletion), osipk1_2 (a 1-nt deletion), and osipk1_3 (a 2-nt deletion) were identified in T0 plants of the rice line Xidao #1 (wild type, WT). A transfer DNA free line with the homozygous osipk1_1 mutation was developed; however, no homozygous mutant lines could be developed for the other two mutations. The comparative assay showed that the osipk1_1 mutant line had a significantly lower level of phytic acid (PA, IP6; −19.5%) in rice grain and agronomic traits comparable to the WT. However, the osipk1_1 mutant was more tolerant to salt and drought stresses than the WT, with significantly lower levels of inositol triphosphate (IP3), reactive oxygen species (ROS) and induced IP6, and higher activities of antioxidant enzymes in seedlings subjected to these stresses. Further analyses showed that the transcription of stress response genes was significantly upregulated in the osipk1_1 mutant under stress. Thus, the low phytic acid mutant osipk1_1 should have potential applications in rice breeding and production.

Genetic Analysis and Molecular Mapping of the Quantitative Trait Loci Governing Low Phytic Acid Content in a Novel LPA Rice Mutant, PLM11

Breeding rice varieties with a low phytic acid (LPA) content is an effective strategy to overcome micronutrient deficiency in a population which consume rice as a staple food. An LPA mutant, Pusa LPA Mutant 11 (PLM11), was identified from an ethyl methane sulfonate (EMS)-induced population of Nagina 22. The present study was carried out to map the loci governing the LPA trait in PLM11 using an F2:3 population derived from a cross between a high phytic acid rice variety, Pusa Basmati 6, with PLM11. The genotyping of the F2 population with 78 polymorphic SSR markers followed by the estimation of phytic acid content in the seeds harvested from 176 F2 plants helped in mapping a major QTL, qLPA8.1, explaining a 22.2% phenotypic variation on Chromosome 8. The QTL was delimited to a 1.96 cM region flanked by the markers RM25 and RM22832. Since there are no previous reports of a QTL/gene governing the LPA content in rice in this region, the QTL qLPA8.1 is a novel QTL. In silico analysis based on the annotated physical map of rice suggested the possible involvement of a locus, Os08g0274775, encoding for a protein similar to a phosphatidylinositol 3- and 4-kinase family member. This needs further validation and fine mapping. Since this QTL is currently specific to PLM11, the linked markers can be utilized for the development of rice varieties with reduced phytic acid (PA) content using PLM11 as the donor, thus enhancing the bioavailability of mineral micronutrients in humans.

Transcriptome analysis identifies differentially expressed genes involved in the metabolic regulatory network of progenies from the cross of low phytic acid GmMIPS1 and GmIPK1 soybean mutants

Lowering the phytic acid (PA) content of crop seeds will be beneficial for improving their nutritional traits. Low phytic acid (lpa) crop lines carrying more than one independent mutated gene have been shown to exhibit more pronounced reductions of PA content than mutants with a single lpa mutated gene. But little is known about the link between PA pathway intermediates and downstream regulation following mutation of these genes in soybean. Here, we performed a comparative transcriptome analysis using an advanced-generation recombinant inbred line [2mlpa (mips1/ipk1)] with low PA and a sibling line with homozygous non-mutant alleles and normal PA [2MWT (MIPS1/IPK1)]. RNA sequencing revealed differential expression levels of numerous genes between seeds of 2mlpa and 2MWT at five developmental stages. A total of 7,945 differentially expressed genes were identified. 3316 DEGs were in 128 metabolic and signal transduction pathways and 4980 DEGs were classified into 345 function terms associated with biological processes. Genes associated with PA metabolism, photosynthesis, starch and sucrose metabolism, and defense mechanisms were related to low PA in 2mlpa soybean line. Among these, 36 genes were up/down-regulated in PA metabolic processes, with 22 possibly contributing to the low PA phenotype of 2mlpa. Most of the genes (81 of 117) associated with photosynthesis were down-regulated in 2mlpa at the late seed stage. Three genes involved in sucrose metabolism were up-regulated at the late seed stage, which might explain the high sucrose content of 2mlpa soybeans. Additionally, 604 genes related to defense mechanisms were differentially expressed between 2mlpa and 2MWT. In this research, the soybean mutant 2mlpa was found to not only exhibit low PA but also have changes in multiple metabolites and secondary metabolites. The results delineate the regulation of these metabolic events by 2mlpa. Many genes associated with PA metabolism would contribute to the drastic reduction of PA and moderate accumulation of InsP3-InsP5 in 2mlpa mutant. And other regulated genes found in photosynthesis, starch and sucrose metabolism, and defense mechanisms would give us more insight into the nutritional and agronomic performance of 2mlpa.