Identification and Expression Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Peanut (Arachis hypogaea L.)

Phosphoenolpyruvate carboxylase （PEPC） is widely distributed in plants and bacteria, and catalyzes the carboxylation of phosphoenolpyruvate to form oxaloacetate and inorganic phosphate. To investigate the molecular mechanisms of the regulation and control of peanut oil, with the degenerated primers and RACE-PCR approach, five PEPC genes were cloned from peanut, and designated as AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5, respectively. The structure and phylogenetic analysis of PEPC protein indicated that AhPEPC1-4 genes encoded a typical plant-type PEPC-enzyme, and AhPEPC5 a bacterial-type. By real-time quantitative RT-PCR approach the expression pattern of each gene was detected in various tissues of normal and high oil-content peanut varieties. It was found that there was a lower expression level of AhPEPCs genes except for the AhPEPC2 in high-oil peanut than normal-oil peanut line. The results provide some fundamental information for the further investigation of plant PEPC proteins and their role in regulation of oil-content in peanut seeds.

Functional Analysis of the Phosphoenolpyruvate Carboxylase on the Lipid Accumulation of Peanut(Arachis hypogaea L.) Seeds

Phosphoenolpyruvate carboxylase（PEPC;EC 4.1.1.31） catalyses phosphoenolpyruvate（PEP） to yield oxaloacetate,which is involved in protein biosynthesis.Pyruvate kinase（PK;EC 2.7.1.40） catalyzes PEP to yield pyruvate,which is involved in fatty acid synthesis.In this study,five PEPC genes（AhPEPC1,AhPEPC2,AhPEPC3,AhPEPC4,and AhPEPC5） from peanut have been cloned.Using a quantitative real-time RT-PCR approach,the expression pattern of each gene was monitored during the seed development of four peanut varieties（E11,Hebeigaoyou,Naihan 1,and Huayu 26）.It was found that these five genes shared similar expression behaviors over the developmental stages of E11 with high expression levels at 30 and 40 d after pegging（DAP）;whereas these five genes showed irregular expression patterns during the seed development of Hebeigaoyou.In Naihan 1 and Huayu 26,the expression levels of the five genes remained relatively high in the first stage.The PEPC activity was monitored during the seed development of four peanut varieties and seed oil content was also characterized during whole period of seed development.The PEPC activity followed the oil accumulation pattern during the early stages of development but they showed a significantly negative correlation thereafter.These results suggested that PEPC may play an important role in lipid accumulation during the seed development of four peanut varieties tested.

Photosynthetic Characteristics and Heterosis in Transgenic Hybrid Rice with Maize Phosphoenolpyruvate Carboxylase (pepc) Gene

Three F3 hybrids derived from the sterile rice lines Gang 46A, 776A and 2480A and the improved restorer line Shuhui 881 containing maize phosphoenolpyruvate carboxylase （pepc） gene were used to analyze the effect of pepc gene on the heterosis and photosynthetic characteristics, while the F3 obtained by crossing Shuhui 881 with the above three sterile lines served as controls. The dynamics of photosynthetic characteristics in leaves of three F1 with pepc gene and their controls were determined at the initial-tillering, maxium-tillering, elongation, initial-heading, heading, maturity stages, and other different times after flag leaf fully expanded. The PEPCase activities of the three F1 with pepc gene increased significantly as compared with control plants during the whole developmental stages. Moreover, the net photosynthesis rate （Pn） also increased to certain extent. The data showed that PEPCase activity was significantly correlated to Pn with a correlation coefficient of 0.6081. The photosynthetic indexes of the three F1 with pepc gene were obviously superior to respective controls in apparent quantum efficiency, light compensation point and carboxylation efficiency, while the CO2 compensation point was lower than that of corresponding control. The Pn of the three F1 with pepc gene at light saturation point and CO2 saturation point was also higher than that of control plants. in addition, the three F1 with pepc gene had an average increase of 37.10% in grain yields per plant in comparison with control plants. The results indicated that the photosynthetic characteristics of hybrid rice containing pepc gene had been improved to some extent due to the introduction of pepc gene.

Promotive Effect of Low Concentrations of NaHSO3 on Photophosphorylation and Photosynthesis in Phosphoenolpyruvate Carboxylase Transgenic Rice Leaves

Spraying a 1–2 mmol/L solution of NaHSO3 on the leaves of wild-type rice (Oryza sativa L.) Kitaake (WT), phosphoenolpyruvate carboxylase (PEPC) transgenic (PC) rice and PEPC+phosphate dikinase (PPDK) transgenic rice (PC+PK), in which the germplasm was transformed with wild-type Kitaake as the gene receptor, resulted in an enhancement of the net photosynthetic rate by 23.0%, 28.8%, and 34.4%, respectively, for more than 3 d. It was also observed that NaHSO3 application caused an increase in the ATP content in leaves. Spraying PMS (a cofactor catalysing the photophosphorylation cycle) and NaHSO3 separately or together on leaves resulted in an increase in photosynthesis with all treatments. There was no additional effect on photosynthetic rate when the mixture was applied, suggesting that the mechanism by which NaHSO3 promotes photosynthesis is similar to the mechanism by which PMS acts and that both of compounds enhanced the supply of ATP. After spraying a solution of NaHSO3 on leaves, compared with the WT Kitaake rice, a greater enhancement of net photosynthetic rate was observed in PEPC transgenic (PC) and PEPC+PPDK transgenic (PC+PK) rice, with the greatest increase being observed in the latter group. Therefore ATP supply may become the limiting factor that concentrates CO2 in rice leaves transformed with an exogenous PEPC gene and exogenous PEPC+PPDK genes.

CO2 Exchange and Chlorophyll Fluorescence of Phosphoenolpyruvate Carboxylase Transgenic Rice Pollen Lines

To elucidate the photosynthetic physiological characteristics and the physiological inherited traits of rice （Oryza sativa L.） hybrids and their parents, physiological indices of photosynthetic CO2 exchange and chlorophyll fluorescence parameters were measured in leaves of the maize phosphoenolpyruvate carboxylase （PEPC） transgenic rice as the male parent, sp. japonica rice cv. 9516 as the female parent, and the stable JAAS45 pollen line. The results revealed that the PEPC gene could be stably inherited and trans- ferred from the male parent to the JAAS45 pollen line. Moreover, the JAAS45 pollen line exhibited high levels of PEPC activity, manifesting higher saturated photosynthetic rates, photosynthetic apparent quantum yield （AQY）, photochemical efficiency of photosystem II and photochemical and non-photochemical quenching, which indicated that the JAAS45 pollen line has a high tolerance to photo-inhibition/photooxidation under strong light and high temperature. Furthermore, JAAS45 was confirmed to still be a C3 plant by δ^13C carbon isotope determination and was demonstrated to have a limited photosynthetic C4 microcycle by feeding with exogenous C4 primary products, such as oxaloacetate or malate, or phosphoenolpyruvate. The present study explains the physiological inherited properties of PEPC transgenic rice and provides an expectation for the integration of traditional breeding and biological technology.

Exchange of a nuclear corepressor between NF-kB and CREB mediates inhibition of phosphoenolpyruvate carboxykinase transcription by NF-kB

Background NF-KB p65 was shown to inhibit transcription of phosphoenolpyruvate carboxykinase （PEPCK）, a rate-limiting enzyme in gluconeogenesis in the liver. To understand the mechanism of action of NF-KB p65, we investigated the nuclear receptor corepressor in the regulation of PEPCK transcription. Methods Rat H411E cells, human hepatoma HepG2 cells and human embryo kidney （HEK） 293 cells were used in this study. The transcriptional activity of a rat PEPCK gene promoter （-490/＋100） was analyzed in HepG2 cells, a HepG2 super suppressor IkBa （sslkBa） stable cell line, and HEK 293 cells. The effects of p65 and sslkBa on a rat PEPCK gene promoter were observed using the PEPCK luciferase reporter system. The interaction of the cAMP-response- element-binding （CREB） protein, histone deacetylase 3 （HDAC3） and silencing mediator for retinoic and thyroid hormone receptors （SMRT） with the PEPCK gene promoter were investigated using the chromatin immunoprecipitation （CHIP） assay, p65 cotransfection and RNAi-mediated gene knockdown were used to determine the corepressor involved in the inhibition of PEPCK by NF-KB p65 and the transcriptional regulation of CREB by NF-KB p65. Results NF-KB p65 inhibited PEPCK expression and the inhibition was blocked by sslkBa. The inhibitory effect of p65 was completely blocked in a HepG2 stable cell line in which sslkBa was expressed. HDAC3 or SMRT knockdown led to a significant up-regulation of PEPCK reporter activity in the presence of p65 cotransfection. In the ChIP assay the interaction of HDAC3 and SMRT with the PEPCK gene promoter was induced by p65 activation, but the CREB signal was reduced. Transcriptional activity of CREB was inhibited by NF-kB p65 cotransfection. The inhibitory effect of NF-kB p65 was blocked by HDAC3 RNAi or SMRT RNAi. Conculsions The study showed that the inhibition of PEPCK by NF-kB p65 was dependent on HDAC3 and SMRT, which form a nuclear corepressor complex for transcriptional inhibition. The transcription factors NF-kB p65 and CREB share the same corepressor HDAC3-SMRT, and the corepressor exchange leads to inhibition of PEPCK gene transcription by NF-kB p65.

Enhanced tolerance to drought in transgenic rice plants overexpressing C_4 photosynthesis enzymes

Maize-specific pyruvate orthophosphate dikinase(PPDK) was overexpressed in rice independently or in combination with the maize C4-specific phosphoenolpyruvate carboxylase(PCK). The wild-type(WT) cultivar Kitaake and transgenic plants were evaluated in independent field and tank experiments. Three soil moisture treatments,well-watered(WW), moderate drought(MD) and severe drought(SD), were imposed from 9d post-anthesis till maturity. Leaf physiological and biochemical traits, root activities,biomass, grain yield, and yield components in the untransformed WT and two transgenic rice lines(PPDK and PCK) were systematically studied. Compared with the WT, both transgenic rice lines showed increased leaf photosynthetic rate: by 20%–40% under WW, by45%–60% under MD, and by 80%–120% under SD. The transgenic plants produced 16.1%,20.2% and 20.0% higher grain yields than WT under the WW, MD and SD treatments,respectively. Under the same soil moisture treatments, activities of phosphoenolpyruvate carboxylase(PEPC) and carbonic anhydrase(CA) in transgenic plants were 3–5-fold higher than those in WT plants. Compared with ribulose-1,5-bisphosphate carboxylase, activities of PEPC and CA were less reduced under both MD and SD treatments. The transgenic plants also showed higher leaf water content, stomatal conductance, transpiration efficiency, and root oxidation activity and a stronger active oxygen scavenging system than the WT under all soil moisture treatments, especially MD and SD. The results suggest that drought tolerance is greatly enhanced in transgenic rice plants overexpressing C4photosynthesis enzymes. This study was performed under natural conditions and normal planting density to evaluate yield advantages on a field basis. It may open a new avenue to droughttolerance breeding via overexpression of C4enzymes in rice.

Marker-assisted Selection of ZmC_4Ppc in Rice Breeding and Yield Trait Performances of Advanced Lines

The full-length of intact Zea mays gene for phosphoenolpyruvate carboxylase gene （ZmC4Ppc） is 6 781 bp. The products of PCR for this gene were not clear with poor repeatability, resulting in that it was difficult for marker-assisted selection （MAS） both in rice and maize. For selecting the markers for MAS, sequences presented only in maize rather than in rice were identified by BLAST, and used for primer design using Primer Premier 5.0. A pair of specific primer termed MRpc （Forward： 5＇ AAGCAGGGAAGCGAGACG 3＇, Reverse： 5＇ GATTGCCGCCAGCAGTAG 3＇） was used for selection of transformed rice, and ZmC4Ppc could be highly and constitutively expressed at each tested developmental stages in the transformed rice selected by using MRpc. Thus, MRpc was used for MAS of progenies carrying ZmC4Ppc gene in rice and some restorer lines with ZmC4Ppc （e.g. FPM881） derived from ZmC4Ppc-transformed Kitaake backcrossed with a restorer line Shuhui 881 were obtained. The analyses on genetic background, PEPCase activity, net photosynthetic rate, general combining ability （GCA） and specific combining ability （SCA） of FPM881 showed that similarity of genetic background reached above 95%, the PEPCase and net photosynthetic rate were higher than those of the control, and some of the progenies carrying ZmC4Ppc gene had better GCA and SCA for grain yield per plant, number of panicles per plant, and 1000-grain weight than those of the control. This suggested that the introduction of maize ZmC4Ppc gene via MAS and its stable expression could increase grain yield of rice and would likely provide a pathway for rice varietal improvement.

Co-ordinated combination of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway in Escherichia coli to promote L-tryptophan production

In this study,phosphoenolpyruvate and erythrose-4-phosphate are efficiently supplied by collaborative design of Embden-Meyerhof-Parnas(EMP)pathway and pentose phosphate(PP)pathway in Escherichia coli,thus increasing the L-tryptophan production.Firstly,the effects of disrupting EMP pathway on L-tryptophan production were studied,and the results indicated that the strain with deletion of phosphofructokinase A(i.e.,E.coli JW-5ΔpfkA)produced 23.4±2.1 g/L of L-tryptophan production.However,deletion of phosphofructokinase A and glucosephosphate isomerase is not conducive to glucose consumption and cell growth,especially deletion of glucosephosphate isomerase.Next,the carbon flux in PP pathway was enhanced by introduction of the desensitized glucose-6-phosphate dehydrogenase(zwf)and 6-phosphogluconate dehydrogenase(gnd)and thus increasing the L-tryptophan production(i.e.,26.5±3.2 g/L vs.21.7±1.3 g/L)without obviously changing the cell growth(i.e.,0.41 h^(-1) vs.0.44 h^(-1))as compared with the original strain JW-5.Finally,the effects of co-modifying EMP pathway and PP pathway on L-tryptophan production were investigated.It was found that the strain with deletion of phosphofructokinase A as well as introduction of the desensitized zwf and gnd(i.e.,E.coli JW-5 zwf243 gnd361ΔpfkA)produced 31.9±2.7 g/L of L-tryptophan,which was 47.0%higher than that of strain JW-5.In addition,the glucose consumption rate of strain JW-5 zwf243 gnd361ΔpfkA was obviously increased despite of the bad cell growth as compared with strain JW-5.The results of this study have important reference value for the following application of metabolic engineering to improve aromatic amino acids producing strains.

The LncRNA FEZF1-AS1 promotes tumor proliferation in colon cancer by regulating the mitochondrial protein PCK2

LncRNAs and metabolism represents two factors involved in cancer initiation and progression.However,the interaction between lncRNAs and metabolism remains to be fully explored.In this study,lncRNA FEZF1-AS1(FEZF1-AS1)was found upregulated in colon cancer after screening all the lncRNAs of colon cancer tissues deposited in TCGA,the result of which was further confirmed by RNAscope staining on a colon tissue chip.The results obtained using FEZF1-AS1 knockout colon cancer cells(SW480 KO and HCT-116 KO)constructed using CRISPR/Cas9 system confirmed the proliferation,invasion,and migration-promoting function of FEZF1-AS1 in vitro.Mechanistically,FEZF1-AS1 associated with the mitochondrial protein phosphoenolpyruvate carboxykinase(PCK2),which plays an essential role in regulating energy metabolism in the mitochondria.Knockdown of FEZF1-AS1 greatly decreased PCK2 protein levels,broke the homeostasis of energy metabolism in the mitochondria,and inhibited proliferation,invasion,and migration of SW480 and HCT-116 cells.PCK2 overexpression in FEZF1-AS1 knockout cells partially rescued the tumor inhibitory effect on colon cancer cells both in vitro and in vivo.Moreover,PCK2 overexpression specifically rescued the abnormal accumulation of Flavin mononucleotide(FMN)and succinate,both of which play an important role in oxidative phosphorylation(OXPHOS).Overall,these results indicate that FEZF1-AS1 is an oncogene through regulating energy metabolism of the cell.This research reveals a new mechanism for lncRNAs to regulate colon cancer and provides a potential target for colon cancer diagnosis and treatment.

Overexpression of PtPEPCK1 gene promotes nitrogen metabolism in poplar

To understand the function of phosphoenolpyruvate carboxylase kinase,we introduced PtPEPCK1 gene under the control of 35S promoter into 84K poplar(Populus alba×P.glandulosa).PtPEPCK1 gene is well-known for its role in gluconeogenesis.However,our data confi rmed that it has signifi cant eff ects on amino acid biosynthesis and nitrogen metabolism.Immunohistochemistry and fl uorescence microscopy indicate that PtPEPCK1 is specifi cally expressed in the cytoplasm of the spongy and palisade tissues.Overexpression of PtPEPCK1 was characterized through transcriptomics and metabolomics.The metabolites concentration of the ornithine cycle and its precursors also increased,of which N-acetylornithine was up-regulated almost 50-fold and ornithine 33.7-fold.These were accompanied by a massive increase in levels of several amino acids.Therefore,overexpression of PtPEPCK1 increases amino acid levels with urea cycle disorder.

Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice

Endogenous reference genes (ERGs) provide vital information regarding genetically modified organisms (GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified (GM) ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase (SPS), phospholipase D (PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs, a new ERG gene, phosphoenolpyruvate carboxylase (PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R2 of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection, which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.

Concomitant Increases of the Developing Seed Phosph<i>oenol</i>pyruvate Carboxylase Ac-tivity and the Seed Protein Content of Field-Grown Wheat with Nitrogen Supply

Wheat seed storage protein is of great importance for human food. To increase the contents of storage proteins effectively, nitrogen fertilizer at flowering stages is commonly applied. In our previous study, rice phosphoenolpyruvate carboxylase (PEPCase) activity in developing seeds was observed in response to nitrogen application at a flowering stage and was positively correlated to the response of the protein content in seeds of six cultivars. This observation might indicate that the seeds have a biological system for accepting nitrogen in seeds by using PEPCase. To test whether this physiological event occurs in wheat, we examined the PEPCase activity and protein content in field-grown wheat seeds under different nitrogen supply conditions. With only basal dressing, seeds showed lower PEPCase activity and protein content (both 0.90-fold) compared to seeds without basal fertilizer. With ammonium sulfate application at 8.3 and 25 g/m2 at a flowering stage, seeds showed higher PEPCase activity (1.08- and 1.17-fold, respectively) and protein content (1.15- and 1.42-fold, respectively), depending on the nitrogen level. We investigated the relationship between PEPCase activity and protein content in the seeds among four conditions. The effect of the nitrogen supply on PEPCase activity during grain-filling stages was validated by the results of a hydroponic culture experiment. Together the results demonstrate that our hypothesis seems to apply to field-grown wheat.

Hepatic retinaldehyde deficiency is involved in diabetes deterioration by enhancing PCK1-and G6PC-mediated gluconeogenesis

Type 2 diabetes(T2D) is often accompanied with an induction of retinaldehyde dehydrogenase 1(RALDH1 or ALDH1A1) expression and a consequent decrease in hepatic retinaldehyde(Rald)levels. However, the role of hepatic Rald deficiency in T2D progression remains unclear. In this study, we demonstrated that reversing T2D-mediated hepatic Rald deficiency by Rald or citral treatments, or liverspecific Raldh1 silencing substantially lowered fasting glycemia levels, inhibited hepatic glucogenesis,and downregulated phosphoenolpyruvate carboxykinase 1(PCK1) and glucose-6-phosphatase(G6PC)expression in diabetic db/db mice. Fasting glycemia and Pck1/G6pc mRNA expression levels were strongly negatively correlated with hepatic Rald levels, indicating the involvement of hepatic Rald depletion in T2D deterioration. A similar result that liver-specific Raldh1 silencing improved glucose metabolism was also observed in high-fat diet-fed mice. In primary human hepatocytes and oleic acidtreated HepG2 cells, Rald or Rald + RALDH1 silencing resulted in decreased glucose production and downregulated PCK1/G6PC mRNA and protein expression. Mechanistically, Rald downregulated direct repeat 1-mediated PCK1 and G6PC expression by antagonizing retinoid X receptor a, as confirmed by luciferase reporter assays and molecular docking. These results highlight the link between hepatic Rald deficiency, glucose dyshomeostasis, and the progression of T2D, whilst also suggesting RALDH1 as a potential therapeutic target for T2D.

Improved oxidative tolerance in suspension‐cultured cells of C_4-pepctransgenic rice by H_2O_2 and Ca^(2+)under PEG-6000

To understand the molecular responses of PC （Overexpressing the maize C4‐pepc gene, which encodes phosphoenolpyruvate carboxylase （PEPC））, to drought stress at cel level, we analyzed changes in the levels of signaling molecules （hydrogen peroxide （H2O2）, calcium ion （Ca2t）, and nitric oxide （NO）） in suspension‐cultured PC and wild‐type （WT） rice （Oryza sativa L.） cel under drought stress induced by 20%polyethylene glycol 6000 （PEG‐6000）. Results demonstrated that PC improved drought tolerance by enhancing antioxidant defense, retaining higher relative water content, survival percentages, and dry weight of cel s. In addition, PEPC activity in PC under PEG treatment was strengthened by addition of H2O2 inhibitor, dimethylthiourea （DMTU） and NO synthesis inhibitor, 2‐（4‐carboxyphenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide （cPTIO）, respectively, while that in PC was weakened by addition of free calcium chelator, ethylene glycol‐bis（b‐aminoethylether）‐N,N,N0 ,N0‐tetraacetic acid （EGTA） t calcium channel outflow inhibitor, ruthenium red （RR） t plasma membrane channel blocker La（NO3）3, but EGTA t RR did not. Results also showed that NO and Ca2t was lying downstream of H2O2 in drought‐induced signaling. Calcium ion was also involved in the expression of C4‐pepc in PC. These results suggested that PC could improve oxidative tolerance in suspension‐cultured cel s and the acquisition of this tolerance required downregulation of H2O2 and the entry of extracel ular Ca2t into cel s across the plasma membrane for regulation of PEPC activity and C4‐pepc expression.

Increased hepatic peroxisome proliferator-activated receptor coactivator-la expression precedes the development of insulin resistance in offspring of rats from severe hyperglycemic mothers

Background Prenatal hyperglycaemia may increase metabolic syndrome susceptibility of the offspring. An underlying component of the development of these morbidities is hepatic gluconeogenic molecular dysfunction. We hypothesized that maternal hyperglycaemia will influence her offsprings hepatic peroxisome proliferator-activated receptor coactivator-la （PGC-la） expression, a key regulator of glucose production in hepatocytes. Method We established maternal hyperglycaemia by streptozotocin injection to induce the maternal hyperglycaemic Wistar rat model. Offspring from the severe hyperglycemia group （SDO） and control group （CO） were monitored until 180 days after birth. Blood pressure, lipid metabolism indicators and insulin resistance （IR） were measured. Hepatic PGC-la expression was analyzed by reverse transcription polymerase chain reaction and Western blotting, mRNA expression of two key enzymes in gluconeogenesis, glucose-6-phospha-tase （G-6-Pase） and phosphoenolpyruvate carboxykinase （PEPCK）, were analyzed and compared. Results In the SDO group, PGC-la expression at protein and mRNA levels were increased, so were expression of G-6-Pase and PEPCK （P〈0.05）. The above effects were seen prior to the onset of IR. Conclusion The hepatic gluconeogenic molecular dysfunction may contribute to the metabolic morbidities experienced by this population.

Transcriptomic changes associated with PCK1 overexpression in hepatocellular carcinoma cells detected by RNA-seq

Phosphoenolpyruvate carboxykinase 1(PCK1),a step limiting enzyme of gluconeogenesis,is downregulated in hepatocellular carcinoma(HCC).Overexpression of PCK1 has been shown to suppress hepatoma cell growth,but the underlying mechanism remains unclear.We used recombinant adenovirus overexpressing PCK1 or GFP in Huh7 cells,and the differentially expressed genes(DEGs)were identified by RNA-Seq.180 were upregulated by PCK1 overexpression,whereas 316 were downregulated.Pathway analysis illustrated that PCK1 was closely correlated with Wnt signaling pathway and TGF-beta signaling pathway.Hence,Wnt signaling pathway and its downstream component,FZD2,FZD6,FZD7 and b-catenin were confirmed by qRT-PCR and Western blot.In vivo we also observed that PCK1 had restrained tumor growth as a result of decreasing expression of b-catenin.Whole-transcriptomic profile analysis discovered that overexpression of PCK1 downregulates several oncogenic signaling pathways in HCC,providing potential therapeutic targets for improving HCC therapy.

Identification of Conserved Regulatory Elements in Mammalian Promoter Regions:A Case Study Using the PCK1 Promoter

A systematic phylogenetic footprinting approach was performed to identify conserved transcription factor binding sites （TFBSs） in mammalian promoter regions using human, mouse and rat sequence alignments. We found that the score distributions of most binding site models did not follow the Gaussian distribution required by many statistical methods. Therefore, we performed an empirical test to establish the optimal threshold for each model. We gauged our computational predictions by comparing with previously known TFBSs in the PCK1 gene promoter of the cytosolic isoform of phosphoenolpyruvate carboxykinase, and achieved a sensitivity of 75% and a specificity of approximately 32% Almost all known sites overlapped with predicted sites, and several new putative TFBSs were also identified. We validated a predicted SP1 binding site in the control of PCK1 transcription using gel shift and reporter assays. Finally, we applied our computational approach to the prediction of putative TFBSs within the promoter regions of all available RefSeq genes. Our full set of TFBS predictions is freely available at http：//bfgl.anri.barc.usda.gov/tfbsConsSites.

Improve gamma‑aminobutyric acid production in Corynebacterium glutamicum by optimizing the metabolic fux

Gamma-aminobutyric acid is an important nonprotein amino acid and has been extensively applied in pharmaceuticals,livestock,food additives,and so on.It is important to develop Corynebacterium glutamicum strains that can efciently produce gamma-aminobutyric acid from glucose.In this study,production of gamma-aminobutyric acid in C.glutamicum CGY700 was improved by construction of CO_(2) anaplerotic reaction and overexpression of citrate synthase.The co-expression of ppc encoding phosphoenolpyruvate carboxylase and gltA encoding citrate synthase was constructed and optimized in the chromosome to compensate carbon loss and conquer metabolic bottleneck.The expression of ppc and gltA were controlled by promoters P_(tac) and P_(tacM),and the optimal mode of P_(tacM-ppc)-P_(tac)-gltA was determined.Simultaneously,the genes pknG encoding serine/threonine protein kinase G and ldh encoding l-lactate dehydrogenase were deleted,and glnA2 encoding glutamine synthase was overexpressed in the chromosome.The fnal strain CGY-PG-304 constructed in this study could produce 41.17 g/L gamma-aminobutyric acid in shake fask cultivation and 58.33 g/L gamma-aminobutyric acid via FedBatch fermentation with a yield of 0.30 g/g glucose.CGY-PG-304 was constructed by genome editing;therefore,it is stable and not necessary to add any antibiotics and inducer during fermentation.