Carboxylase Gene Research Articles

CRISPR/Cas9-based genome engineering in the filamentous fungus Rhizopus oryzae and its application to L-lactic acid production.

The filamentous fungus Rhizopus oryzae is one of the main industrial strains for the production of a series of important chemicals such as ethanol, lactic acid, and fumaric acid. However, the lack of efficient gene editing tools suitable for R. oryzae makes it difficult to apply technical methods such as metabolic engineering regulation and synthetic biology modification. A CRISPR-Cas9 system suitable for efficient genome editing in R. oryzae was developed. Firstly, four endogenous U6 promoters of R. oryzae were identified and screened with the highest transcriptional activity for application to sgRNA transcription. It was then determined that the U6 promoter mediated CRISPR/Cas9 system has the ability to efficiently edit the genome of R. oryzae through NHEJ and HDR-mediated events. Furthermore, the newly constructed CRISPR-Cas9 dual sgRNAs system can simultaneously disrupt or insert different fragments of the R. oryzae genome. Finally, this CRISPR-Cas9 system was applied to the genome editing of R. oryzae by knocking out pyruvate carboxylase gene (PYC) and pyruvate decarboxylase gene (pdcA) and knocking in phosphofructokinase (pfkB) from Escherichia coli and L-lactate dehydrogenase (L-LDH) from Heyndrickxia coagulans, which resulted in a substantial increase in L-LA production. In summary, this study showed that the CRISPR/Cas9-based genome editing tool is efficient for manipulating genes in R. oryzae.

Draft genome of an anaerobic nitrate-reducing, benzene-degrading member of the order Thermincolales.

We present a metagenome assembled genome (MAG) of an anaerobic bacterium from a nitrate-reducing, benzene-degrading enrichment culture (NRBC). The draft Thermincolales genome consists of 20 contigs with a total length of 4.09 Mbp and includes putative carboxylase genes likely involved in benzene activation.

Effective enhancement of the ability of Monascus pilosus to produce lipid-lowering compound Monacolin K via perturbation of metabolic flux and histone acetylation modification

Monacolin K (MK), also known as lovastatin, is a polyketide compound with the ability to reduce plasma cholesterol levels and many other bio-activities. Red yeast rice (also named Hongqu) rich in MK derived from Monascus fermentation has attracted widespread attention due to its excellent performance in reducing blood lipids. However, industrial Monascus fermentation suffers from the limitations such as low yield of MK, long fermentation period, and susceptibility to contamination. In this study, we firstly blocked the competitive pathway of MK biosynthesis to create polyketide synthase gene pigA (the key gene responsible for the biosynthesis of Monascus azaphilone pigments) deficient strain A1. Then, based on the strategies to increase precursor supply for MK biosynthesis, acetyl-CoA carboxylase gene acc overexpression strains C1 and C2 were constructed with WT and A1 as the parent, respectively. Finally, histone deacetylase gene hos2 overexpression strain H1 was constructed by perturbation of histone acetylation modification. HPLC detection revealed all these four strains significantly increased their abilities to produce MK. After 14 days of solid-state fermentation, the MK yields of strains A1, C1, C2, and H1 reached 2.03 g/100 g, 1.81 g/100 g, 2.45 g/100 g and 2.52 g/100 g, which increased by 28.5 %, 14.7 %, 43.9 % and 36.1 % compared to WT, respectively. RT-qPCR results showed that overexpression of hos2 significantly increased the expression level of almost all genes responsible for MK biosynthesis after 5-day growth. Overall, the abilities of these strains to produce MK has been greatly improved, and MK production period has been shortened to 14 days from 20 days, providing new approaches for efficient production of Hongqu rich in MK.

Molecular Identification Validates Morphological Identification of Javanese Cardamom from Banyumas in Central Java, Indonesia

Cardamom is one of the prominent plants with significant economic value in the Banyumas Regency, Central Java, Indonesia. Although Javanese cardamom is traditionally categorized under Amomum compactum, the morphological variations observed create ambiguity about its exact species status. DNA barcoding using the maturase K (matK) and ribulose-bisphosphate carboxylase (rbcL) genes is proven as a reliable technique to elucidate the taxonomic status of morphological variable plant cultivars. This study aimed to characterize cardamom from Banyumas Regency using morphological and molecular approaches for taxonomic status identification and genetic diversity evaluation. The matK and rbcL genes were selected as genetic markers and sequenced using a bidirectional sequencing technique. Morphological examination showed significant color variations at the cardamom stem base. All samples had high genetic identities to reference species in databases and were supported by high query cover and zero e-values. Therefore, molecular characterization, alongside geographic distribution assessment, established that this plant belongs to a single species, Amomum compactum. Additionally, the analysis conducted showed a low level of genetic diversity, as evidenced by haplotype and nucleotide diversity. Low-level genetic diversity provides additional data to convince that cardamon in Banyumas Regency belongs to a single species. These results are essential data in seed selection for further cultivation.

Effect of Guanidinoacetic Acid on Production Performance, Serum Biochemistry, Meat Quality and Rumen Fermentation in Hu Sheep.

Guanidinoacetic acid (GAA) can effectively improve the metabolism of energy and proteins by stimulating creatine biosynthesis. We present a study exploring the impact of GAA on production performance, serum biochemistry, meat quality and rumen fermentation in Hu sheep. A total of 144 weaned male Hu sheep (body weight 16.91 ± 3.1 kg) were randomly assigned to four groups with three replicates of twelve sheep in each group. The diets were supplemented with 0 (CON), 500 (GAA-1), 750 (GAA-2) and 1000 mg/kg (GAA-3) of GAA (weight of feed), respectively. After a comprehensive 90-day experimental period, we discovered that the supplementation of GAA had a remarkable impact on various muscle parameters. Specifically, it significantly enhanced the average daily growth (ADG) of the animals and improved the shear force and fiber diameter of the muscle, while also reducing the drip loss and muscle fiber density. Furthermore, the addition of GAA to the feed notably elevated the serum concentrations of high-density lipoprotein cholesterol (HDL-C), total protein (TP) and globulin (GLB), as well as the enzyme activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Concurrently, there was a decrease in the levels of triglycerides (TG) and malondialdehyde (MDA) in the serum. In addition, GAA decreased the pH and the acetate-to-propionate ratio and increased the total volatile fatty acids (TVFA) and ammoniacal nitrogen (NH3-N) levels of rumen fluid. Additionally, GAA upregulated acetyl-CoA carboxylase (ACC) gene expression in the Hu sheep's muscles. In conclusion, our findings suggest that GAA supplementation not only enhances muscle quality but also positively affects serum biochemistry and ruminal metabolism, making it a potential candidate for improving the overall health and performance of Hu sheep.

Enhanced Herbicide Metabolism and Target Site Mutation Enabled the Multiple Resistance to Cyhalofop-butyl, Florpyrauxifen-benzyl, and Penoxsulam in Echinochloa crus-galli.

This study investigated the multiple herbicide resistance (MHR) mechanism of one Echinochloa crus-galli population that was resistant to florpyrauxifen-benzyl (FPB), cyhalofop-butyl (CHB), and penoxsulam (PEX). This population carried an Ala-122-Asn mutation in the acetolactate synthase (ALS) gene but no mutation in acetyl-CoA carboxylase (ACCase) and transport inhibitor response1 (TIR1) genes. The metabolism rate of PEX was 2-fold higher, and the production of florpyrauxifen-acid and cyhalofop-acid was lower in the resistant population. Malathion and 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) could reverse the resistance, suggesting that cytochrome P450 (CYP450) and glutathione S-transferase (GST) contribute to the enhanced metabolism. According to RNA-seq and qRT-PCR validation, two CYP450 genes (CYP71C42 and CYP71D55), one GST gene (GSTT2), two glycosyltransferase genes (rhamnosyltransferase 1 and IAAGLU), and two ABC transporter genes (ABCG1 and ABCG25) were induced by CHB, FPB, and PEX in the resistant population. This study revealed that the target mutant and enhanced metabolism were involved in the MHR mechanism in E. crus-galli.

Effects of RNA Interference with Acetyl-CoA Carboxylase Gene on Expression of Fatty Acid Metabolism-Related Genes in Macrobrachium rosenbergii under Cold Stress

Macrobrachium rosenbergii is a warm water species, and low temperature is a limiting factor for its growth and survival. In order to explore the role of the acetyl-CoA-carboxylase (ACC) gene in response to the cold stress of M. rosenbergii, we investigated the effects of RNA interference (RNAi) with the ACC gene on the expression of fatty acid metabolism-related genes and the mortality of M. rosenbergii under cold stress. The results showed that different siRNA sequences and different injection concentrations had different inhibiting effects on ACC gene expression, and siRNA-III with an injection concentration of 2.0 μg/g (siRNA/prawn body weight) had the best interference effect. With the optimal siRNA and the optimal concentration under cold stress, the expressions of three fatty acid metabolism-related genes, FabD, echA, and ACOT, were generally significantly down-regulated. Compared to negative (scrambled-siRNA) and blank (PBS) control groups, the expression of FabD in the interference group was extremely significantly down-regulated at 12 h in the hepatopancreas and at 18 h in the muscles and gills; EchA was highly significantly down-regulated at 6 and 12 h in the muscles and gills; and ACOT was extremely significantly down-regulated and kept declining in the gills. Within 6–18 h after injection under cold stress, the mortality rate of the siRNA interference group (75%) was much lower than that of the negative (95%) or blank control group (97.5%), and all prawns died after 24 h. In conclusion, RNA interference with the ACC gene inhibited the expression of some fatty acid metabolism-related genes, and could partly improve the tolerance of M. rosenbergii to cold stress, indicating that the ACC gene might play an important role in the response of M. rosenbergii to cold stress.

Anti-obesity potentiality of Lactiplantibacillus plantarum E2_MCCKT isolated from a fermented beverage, haria: a high fat diet-induced obese mice model study.

Obesity is a growing epidemic worldwide. Several pharmacologic drugs are being used to treat obesity but these medicines exhibit side effects. To find out the alternatives of these drugs, we aimed to assess the probiotic properties and anti-obesity potentiality of a lactic acid bacterium E2_MCCKT, isolated from a traditional fermented rice beverage, haria. Based on the 16S rRNA sequencing, the bacterium was identified as Lactiplantibacillus plantarum E2_MCCKT. The bacterium exhibited in vitro probiotic activity in terms of high survivability in an acidic environment and 2% bile salt, moderate auto-aggregation, and hydrophobicity. Later, E2_MCCKT was applied to obese mice to prove its anti-obesity potentiality. Adult male mice (15.39 ± 0.19g) were randomly divided into three groups (n = 5) according to the type of diet: normal diet (ND), high-fat diet (HFD), and HFD supplemented with E2_MCCKT (HFT). After four weeks of bacterial treatment on the obese mice, a significant reduction of body weight, triglyceride, and cholesterol levels, whereas, improvements in serum glucose levels were observed. The bacterial therapy led to mRNA up-regulation of lipolytic transcription factors such as peroxisome proliferator-activated receptor-α which may increase the expression of fatty acid oxidation-related genes such as acyl-CoA oxidase and carnitine palmitoyl-transferase-1. Concomitantly, both adipocytogenesis and fatty acid synthesis were arrested as reflected by the down-regulation of sterol-regulatory element-binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase genes. In protein expression study, E2_MCCKT significantly increased IL-10 expression while decreasing pro-inflammatory cytokine (IL-1Ra and TNF-α) expression. In conclusion, the probiotic Lp. plantarum E2_MCCKT might have significant anti-obesity effects on mice.

Physio-biochemical and DNA methylation analysis of the defense response network of wheat to drought stress

In the present work, physio-biochemical and DNA methylation analysis were conducted in wheat (Triticum aestivum L.) cultivars “Bolani” (drought-tolerant) and “Sistan” (drought-sensitive) during drought treatments: well-watered (at 90% field capacity (FC)), mild stress (at 50% FC, and severe stress (at 25% FC). During severe stress, O2•− and H2O2 content in cultivar Sistan showed significant increase (by 1.3 and 2.5-fold, respectively) relative to cultivar Bolani. In Bolani, the increased levels of radical scavenging activity (by 32%), glycine betaine (GB) (by 11.44%), proline (4-fold), abscisic acid (by 63.76%), and more stability of relative water content (RWC) (2-fold) were observed against drought-induced oxidative stress. Methylation level significantly decreased from 70.26% to 60.64% in Bolani and from 69.06% to 59.85% in Sistan during stress, and higher decreased tendency was related to CG and CHG in Bolani but CG in Sistan under severe stress. Methylation patterns showed that the highest polymorphism in Bolani was mainly as CG. As the intensity of stress increased, the enhanced physio-biochemical responses of Bolani cultivar were accompanied by a more decrease in the number of unchanged bands. According to heat map analysis, the highest difference (84.38%) in methylation patterns was observed between control and severe stress. Multivariate analysis using principal component analysis (PCA) showed a cultivar-specific methylation during stress and that methylation changes between cultivars are much higher than that of within a cultivar. Higher methylation to demethylation in Bolani (30.06 vs. 22.12%) compared to that of cultivar Sistan (23.21 vs. 30.15%) indicated more demethylation did not induce tolerance responses in Sistan. Sequencing differentially methylated fragments along with qRT-PCR analysis showed the efficient role of various DNA fragments, including demethylated fragments such as phosphoenol pyruvate carboxylase (PEPC), beta-glucosidase (BGlu), glycosyltransferase (GT), glutathione S-transferase (GST) and lysine demethylase (LSD) genes and methylated fragments like ubiquitin E2 enzyme genes in the development of drought tolerance. These results suggested the specific roles of DNA methylation in development of drought tolerance in wheat landrace.

Topical rhubarb charcoal-crosslinked chitosan/silk fibroin sponge scaffold for the repair of diabetic ulcers improves hepatic lipid deposition in db/db mice via the AMPK signalling pathway

BackgroundType 2 diabetes mellitus (T2DM) is closely linked to metabolic syndrome, characterised by insulin resistance, hyperglycaemia, abnormal lipid metabolism, and chronic inflammation. Diabetic ulcers (DUs) comprise consequential complications that arise as a result of T2DM. To investigate, db/db mice were used for the disease model. The findings demonstrated that a scaffold made from a combination of rhubarb charcoal-crosslinked chitosan and silk fibroin, designated as RCS/SF, was able to improve the healing process of diabetic wounds in db/db mice. However, previous studies have primarily concentrated on investigating the impacts of the RSC/SF scaffold on wound healing only, while its influence on the entire body has not been fully elucidated.Material and methodsThe silk fibroin/chitosan sponge scaffold containing rhubarb charcoal was fabricated in the present study using a freeze-drying approach. Subsequently, an incision with a diameter of 8 mm was made on the dorsal skin of the mice, and the RCS/SF scaffold was applied directly to the wound for 14 days. Subsequently, the impact of RCS/SF scaffold therapy on hepatic lipid metabolism was assessed through analysis of serum and liver biochemistry, histopathology, quantitative real-time PCR (qRT-PCR), immunohistochemistry, and Western blotting.ResultsThe use of the RCS/SF scaffold led to an enhancement in the conditions associated with serum glucolipid metabolism in db/db mice. An assessment of hepatic histopathology further confirmed this enhancement. Additionally, the qRT-PCR analysis revealed that treatment with RCS/SF scaffold resulted in the downregulation of genes associated with fatty acid synthesis, fatty acid uptake, triglyceride (TG) synthesis, gluconeogenesis, and inflammatory factors. Moreover, the beneficial effect of the RCS/SF scaffold on oxidative stress was shown by assessing antioxidant enzymes and lipid peroxidation. Additionally, the network pharmacology analysis verified that the adenosine monophosphate-activated protein kinase (AMPK) signalling pathway had a vital function in mitigating non-alcoholic fatty liver disease (NAFLD) by utilizing R. officinale. The measurement of AMPK, sterol regulatory element binding protein 1 (SREBP1), fatty acid synthase (FASN), and acetyl CoA carboxylase (ACC) gene and protein expression provided support for this discovery. Furthermore, the molecular docking investigations revealed a robust affinity between the active components of rhubarb and the downstream targets of AMPK (SREBP1 and FASN).ConclusionBy regulating the AMPK signalling pathway, the RCS/SF scaffold applied topically effectively mitigated hepatic lipid accumulation, decreased inflammation, and attenuated oxidative stress. The present study, therefore, emphasises the crucial role of the topical RCS/SF scaffold in regulating hepatic lipid metabolism, thereby confirming the concept of "external and internal reshaping".

Identification and Analysis of PEPC Gene Family Reveals Functional Diversification in Orchidaceae and the Regulation of Bacterial-Type PEPC.

Phosphoenolpyruvate carboxylase (PEPC) gene family plays a crucial role in both plant growth and response to abiotic stress. Approximately half of the Orchidaceae species are estimated to perform CAM pathway, and the availability of sequenced orchid genomes makes them ideal subjects for investigating the PEPC gene family in CAM plants. In this study, a total of 33 PEPC genes were identified across 15 orchids. Specifically, one PEPC gene was found in Cymbidium goeringii and Platanthera guangdongensis; two in Apostasia shenzhenica, Dendrobium chrysotoxum, D. huoshanense, Gastrodia elata, G. menghaiensis, Phalaenopsis aphrodite, Ph. equestris, and Pl. zijinensis; three in C. ensifolium, C. sinense, D. catenatum, D. nobile, and Vanilla planifolia. These PEPC genes were categorized into four subgroups, namely PEPC-i, PEPC-ii, and PEPC-iii (PTPC), and PEPC-iv (BTPC), supported by the comprehensive analyses of their physicochemical properties, motif, and gene structures. Remarkably, PEPC-iv contained a heretofore unreported orchid PEPC gene, identified as VpPEPC4. Differences in the number of PEPC homolog genes among these species were attributed to segmental duplication, whole-genome duplication (WGD), or gene loss events. Cis-elements identified in promoter regions were predominantly associated with light responsiveness, and circadian-related elements were observed in each PEPC-i and PEPC-ii gene. The expression levels of recruited BTPC, VpPEPC4, exhibited a lower expression level than other VpPEPCs in the tested tissues. The expression analyses and RT-qPCR results revealed diverse expression patterns in orchid PEPC genes. Duplicated genes exhibited distinct expression patterns, suggesting functional divergence. This study offered a comprehensive analysis to unveil the evolution and function of PEPC genes in Orchidaceae.

The effect of aerobic exercise and ethanolic extract of rice bran on the expression of Acetyl-CoA Carboxylase and HMGCR genes in the liver tissue of rats fed with a high-fat diet

The effect of aerobic exercise and ethanolic extract of rice bran on the expression of Acetyl-CoA Carboxylase and HMGCR genes in the liver tissue of rats fed with a high-fat diet

Rhodophyta DNA Barcoding: Ribulose-1, 5-Bisphosphate Carboxylase Gene and Novel Universal Primers.

Red algae (Rhodophyta) are a heterogeneous group of marine algal species that have served as a source of high-value molecules, including antioxidants and scaffolds, for novel drug development. However, it is challenging to identify Rhodophytes through morphological features alone, and in most instances, that has been the prevailing approach to identification. Consequently, this study undertook the identification of red algae species in Kenton-on-Sea, South Africa, as a baseline for future research on red algae biodiversity and conservation. The identification was achieved by designing, analysing, and using a set of universal primers through DNA barcoding of the rbcL gene. The PCR products of the rbcL gene were sequenced, and 96% of the amplicons were successfully sequenced from this set and matched with sequences on BOLD, which led to these species being molecularly described. Amongst these species are medicinally essential species, such as Laurencia natalensis and Hypnea spinella, and potential cryptic species. This calls for further investigation into the biodiversity of the studied region. Meanwhile, the availability of these primers will ease the identification process of red algae species from other coastal regions.

Overexpression of phosphoenol pyruvate carboxylase gene of Flaveria trinervia in transgenic mulberry (Morus spp.) leads to improved photosynthesis rate and tolerance to drought and salinity stresses

Overexpression of phosphoenol pyruvate carboxylase gene of Flaveria trinervia in transgenic mulberry (Morus spp.) leads to improved photosynthesis rate and tolerance to drought and salinity stresses

Optimization of Aerobic Synthesis of Succinic Acid from Glucose by Recombinant <i>Escherichia coli</i> Strains Through the Variant Tricaboxylic Acid Cycle Mediated by the Action of 2-ketoglutarate-decarboxylase

The biosynthesis of succinic acid from glucose by the previously engineered E. coli strain SUC1.0 (pMW119-kgd) (MG1655 ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, PLglk, PtacgalP, ∆aceBAK, ∆glcB, ∆sdhAB, pMW119-kgd) was optimized. The yield of the target substance was increased, upon the activation in the strain of the variant tricarboxylic acid cycle, mediated by the action of heterologous 2-ketoglutarate decarboxylase, due to the intensification of the anaplerotic formation of oxaloacetic acid. Inactivation of the nonspecific thioesterase YciA in the strain did not considerably change the biosynthetic characteristics of the producer. The enhancement of the expression of native phosphoenolpyruvate carboxylase led to an increase in the yield of the target compound by the recombinant synthesizing succinic acid via the reactions of the native tricarboxylic acid cycle from 25 to 42%, and from 67 to 75% upon the induced expression of Mycobacterium tuberculosis 2-ketoglutarate decarboxylase. Expression in the strain of pyruvate carboxylase gene from Bacillus subtilis resulted in an increase in the yield of succinic acid up to 84%. Functioning in whole-cell biocatalyst mode, the engineered strain SUC1.0 PL-pycA (pMW119-kgd) demonstrated a substrate to target product conversion ratio reaching 93%, approaching the corresponding theoretical maximum.

Effect of Tea Polyphenols, α-Lipoic Acid and Their Joint Use on the Antioxidant and Lipid Metabolism Performance of Hybrid Grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatu) Fed with High-Lipid Diets.

This study investigated tea polyphenols (TP), α-lipoic acid (ALA) and their joint use on the antioxidant and lipid metabolic performance of hybrid grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatu) took food with high-fat diets. Six high-lipid diets with isonitrogen (50% of dry matter) and isolipid (17% of dry value) were designed, in which a total content of 1,000 mg/kg additives were added to each group except for the control group (FL). The additives addition ratios in each group were ALA (AL), TP (PL), ALA : TP = 1 : 1 (EL), ALA : TP = 1 : 2 (OL), ALA : TP = 2 : 1 (TL). Each diet was divided into three repeat groups with 30 tails (6.84 ± 0.01 g) in each group and fed for 8 weeks. The consequences were as follows: (1) the highest weight gain rate, specific growth rate, as well as the lowest feed conversion ratio and ingestion rate were discovered in the OL team, which were opposite to the TL group. (2) The body fat content and muscle fat content in the fish oil group were the lowest (P < 0.05), while those of the TL group were the highest. (3) Serum catalase, glutathione peroxidase, total antioxidant capacity, and superoxide dismutase activities were the highest, and the content of reactive oxygen species was the lowest in the OL group. (4) The OL group has the highest hepatic lipase activity and the lowest very low-density lipoprotein content of the liver. In contrast, the TL group had the highest fatty acid synthetase (FAS) activity (P < 0.05). (5) The oil-red aspects of liver tissue displayed lipid particles in other groups were reduced to different degrees compared with FL group, and the OL group showed the best lipid-lowering effect. (6) Compared with the FL group, the relative expressions of FAS, acetyl-CoA carboxylase (acc), and apolipoprotein b-100 (apoB100) genes in the liver were decreased. The relative expressions of lipoprotein lipase (lpl) and peroxisome proliferators-activated receptors-α (pparα) genes related to lipid catabolism were increased, among which the OL group had the most significant change (P < 0.05). (7) According to the 7-day challenge test of Vibrio alginolyticus, the OL group had the highest survival rate. To sum up, both ALA and TP have positive effects on relieving the lipid metabolism disorder of hybrid grouper. If they are jointly used, adding ALA : TP in a ratio of 1 : 2 (OL) may have the best effect, and an addition ratio of 2 : 1 (TL) may inhibit the hybrid grouper growth and increase the feeding cost.

Expression of Acetyl Coa Carboxylase Gene from Cleome Viscosa L. and Its Seed Oil

Asian spider flower (Cleome viscosa L.) is annual herbaceous weed plant. Its seed oil has similar properties to the seed oil from Jatropha curcas L. which is the biodiesel plant. This research focused on the expression of acetyl CoA carboxylase gene in 4 stages of seeds (1-4 weeks after fruit set (WAF)) by Reverse transcriptase Polymerase Chain Reaction (RT-PCR). Acetyl CoA carboxylase (ACCase) is the essential enzyme for the first step in the biosynthesis of long-chain fatty and glycerol. The heteromeric ACCase consists of 4 subunits. accA, accB and accC gene were located in nuclear, while the accD gene was in chloroplast. The result showed that the expression of accA gene showed the most highly expressed in 3 WAF, while the expression of accC and accD showed the most highly expression in 2-3 WAF. These results indicated that co-expression of accA, accC and accD were highest in 3 WAF and down regulated in 4 WAF. In contrast, accB gene showed the high expression level in 4 weeks after fruit set. The total oil was obtained by petroleum ether soxhlet extraction. The high oil content was 3-4 WAF. These findings suggested that comparatively high expression of the Accase gene related to the persistence of oil accumulation during the late stage of seed oil formation for C. viscosa L.

Effects of exogenous melatonin on sugar and organic acid metabolism in early-ripening peach fruits.

To evaluated the effects melatonin (MT) on the sugar and acid metabolism of early-ripening peach fruits, the concentration of 150 μmol/L MT was sprayed on the leaves of peach trees. MT increased the contents of total soluble sugar and sucrose in peach fruits during the whole ripening period, and increased the contents of glucose and sorbitol at the mature stage. During the whole ripening period, MT also increased the activities of sucrose synthase, sucrose phosphate synthase, neutral invertase, and acidic invertase and the relative expression levels of sucrose synthase, sucrose phosphate synthase, neutral invertase, and acidic invertase genes, while decreased the activity of sorbitol oxidase and the relative expression level of sorbitol dehydrogenase to some extent. Moreover, MT decreased the contents of total organic acid, malic acid, and citric acid at mature stage. At mature stage, MT decreased the activities of citrate synthetase and phosphoenolpyruvate carboxylase and the relative expression levels of citrate synthetase and phosphoenolpyruvate carboxylase genes, while increased the relative expression levels of Nicotinamide adenine dinucleotide phosphate (NADP+)-malic enzyme, malate dehydrogenase, and aconitase genes. Therefore, MT promotes the sugar accumulation and organic acid degradation in early-ripening peach fruits.

Multiple highly expressed phosphoenolpyruvate carboxylase genes have divergent enzyme kinetic properties in two C4 grasses.

Phosphoenolpyruvate (PEP) carboxylase (PEPC) catalyses the irreversible carboxylation of PEP with bicarbonate to produce oxaloacetate. This reaction powers the carbon-concentrating mechanism (CCM) in plants that perform C4 photosynthesis. This CCM is generally driven by a single PEPC gene product that is highly expressed in the cytosol of mesophyll cells. We found two C4 grasses, Panicum miliaceum and Echinochloa colona, that each have two highly expressed PEPC genes. We characterized the kinetic properties of the two most abundant PEPCs in E. colona and P. miliaceum to better understand how the enzyme's amino acid structure influences its function. Coding sequences of the two most abundant PEPC proteins in E. colona and P. miliaceum were synthesized by GenScript and were inserted into bacteria expression plasmids. Point mutations resulting in substitutions at conserved amino acid residues (e.g. N-terminal serine and residue 890) were created via site-directed PCR mutagenesis. The kinetic properties of semi-purified plant PEPCs from Escherichia coli were analysed using membrane-inlet mass spectrometry and a spectrophotometric enzyme-coupled reaction. The two most abundant P. miliaceum PEPCs (PmPPC1 and PmPPC2) have similar sequence identities (>95 %), and as a result had similar kinetic properties. The two most abundant E. colona PEPCs (EcPPC1 and EcPPC2) had identities of ~78 % and had significantly different kinetic properties. The PmPPCs and EcPPCs had different responses to allosteric inhibitors and activators, and substitutions at the conserved N-terminal serine and residue 890 resulted in significantly altered responses to allosteric regulators. The two, significantly expressed C4Ppc genes in P. miliaceum were probably the result of genomes combining from two closely related C4Panicum species. We found natural variation in PEPC's sensitivity to allosteric inhibition that seems to bypass the conserved 890 residue, suggesting alternative evolutionary pathways for increased malate tolerance and other kinetic properties.

In silico Analysis of Two Novel Variants in the Pyruvate Carboxylase (PC) Gene Associated with the Severe Form of PC Deficiency

In silico Analysis of Two Novel Variants in the Pyruvate Carboxylase (PC) Gene Associated with the Severe Form of PC Deficiency