High-throughput screening system of citrus bacterial cankerassociated transcription factors and its application to the regulation of citrus canker resistance

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

Characteristics and expression of the TCP transcription factors family in Allium senescens reveal its potential roles in drought stress responses

Allium senescens,is an important economic and ecological grassland plant with drought-resistant characteristics.A TCP protein transcription factor is important in the regulation of plant development and adverse responses.However,the mechanism by which TCP transcription functions in drought resistance in Allium senescens is still not clear.Here,we obtained a total of 190,305 transcripts with 115,562 single gene clusters based on RNA-Seq sequencing of Allium senescens under drought stress.The total number of bases was 97,195,096 bp,and the average length was 841.06 bp.Furthermore,we found that there were eight genes of the TCP family that showed an upregulated expression trend under drought stress in Allium senescens.We carried out an investigation to determine the evolution and function of the AsTCP family and how they produce an effect in drought resistance.The 14 AsTCP genes were confirmed and divided into class I and class II containing CIN and CYC/TBI subfamilies,respectively.We also found that the expression of AsTCP17 was remarkably upregulated with drought treatment.Besides,the transformation of AsTCP17 in Arabidopsis revealed that the protective enzymes,namely polyphenol oxidase(POD)and superoxide dismutase(SOD),were increased by 0.4 and 0.8 times,respectively.Chlorophyll content was also increased,while the H2O2 and malondialdehyde(MDA)contents were decreased.Staining assays with 3,3′-diaminobenzidine(DAB)also suggested that the AsTCP17 downregulates reactive oxygen species(ROS)accumulation.In addition,overexpression of the AsTCP17 affected the accumulation of drought-related hormones in plants,and the synthesis of ABA.The expression of AtSVP and AtNCED3,related ABA synthesis pathway genes,indicated that the level of expression of AtSVP and AtNCED3 was obviously enhanced,with the overexpression of line 6 showing a 20.6-fold and 7.0-fold increase,respectively.Taken together,our findings systematically analyze the AsTCPs family at the transcriptome expression level in Allium senescens,and we also demonstrated that AsTCP17 protein,as a positive regulator,was involved in drought resistance of Allium senescens.In addition,our research contributes to the comprehensive understanding of the drought stress defense mechanism in herbaceous plants.

Comparative transcriptome analysis of the climacteric of apple fruit uncovers the involvement of transcription factors affecting ethylene biosynthesis

Apple(Malus domestica)fruit generally undergoes a climacteric.During its ripening process,there is a peak in ethylene release and its firmness simultaneously decreases.Although more in-depth research into the mechanism of climacteric-type fruit ripening is being carried out,some aspects remain unclear.In this study,we compared the transcriptomes of 0-Pre and 15-Post(pre-and post-climacteric fruit),and 15-Post and 15-MCP[fruit treated with 1-MCP(1-methylcyclopropene)].Various transcription factors,such as MADS-box,ERF,NAC,Dof and SHF were identified among the DEGs(differential gene expressions).Furthermore,these transcription factors were selected for further validation analysis by qRT-PCR.Moreover,yeast one hybrid(Y1H),β-glucuronidase(GUS)transactivation assay and dual-luciferase reporter assay showed that MdAGL30,MdAGL104,MdERF008,MdNAC71,MdDof1.2,MdHSFB2a and MdHSFB3 bound to MdACS1 promoter and directly regulated its transcription,thereby regulating ethylene biosynthesis in apple fruit.Our results provide useful information and new insights for research on apple fruit ripening.

Identification of the target genes of AhTWRKY24 and AhTWRKY106 transcription factors reveals their regulatory network in Arachis hypogaea cv.Tifrunner using DAP-seq

WRKY transcription factors(TFs)have been identified as important core regulators in the responses of plants to biotic and abiotic stresses.Cultivated peanut(Arachis hypogaea)is an important oil and protein crop.Previous studies have identified hundreds of WRKY TFs in peanut.However,their functions and regulatory networks remain unclear.Simultaneously,the AdWRKY40 TF is involved in drought tolerance in Arachis duranensis and has an orthologous relationship with the AhTWRKY24 TF,which has a homoeologous relationship with AhTWRKY106 TF in A.hypogaea cv.Tifrunner.To reveal how the homoeologous AhTWRKY24 and AhTWRKY106 TFs regulate the downstream genes,DNA affinity purification sequencing(DAP-seq)was performed to detect the binding sites of TFs at the genome-wide level.A total of 3486 downstream genes were identified that were collectively regulated by the AhTWRKY24 and AhTWRKY106 TFs.The results revealed that W-box elements were the binding sites for regulation of the downstream genes by AhTWRKY24 and AhTWRKY106 TFs.A gene ontology enrichment analysis indicated that these downstream genes were enriched in protein modification and reproduction in the biological process.In addition,RNA-seq data showed that the AhTWRKY24 and AhTWRKY106 TFs regulate differentially expressed genes involved in the response to drought stress.The AhTWRKY24 and AhTWRKY106 TFs can specifically regulate downstream genes,and they nearly equal the numbers of downstream genes from the two A.hypogaea cv.Tifrunner subgenomes.These results provide a theoretical basis to study the functions and regulatory networks of AhTWRKY24 and AhTWRKY106 TFs.

Age-related driving mechanisms of retinal diseases and neuroprotection by transcription factor EB-targeted therapy

Retinal aging has been recognized as a significant risk factor for various retinal disorders,including diabetic retinopathy,age-related macular degeneration,and glaucoma,following a growing understanding of the molecular underpinnings of their development.This comprehensive review explores the mechanisms of retinal aging and investigates potential neuroprotective approaches,focusing on the activation of transcription factor EB.Recent meta-analyses have demonstrated promising outcomes of transcription factor EB-targeted strategies,such as exercise,calorie restriction,rapamycin,and metformin,in patients and animal models of these common retinal diseases.The review critically assesses the role of transcription factor EB in retinal biology during aging,its neuroprotective effects,and its therapeutic potential for retinal disorders.The impact of transcription factor EB on retinal aging is cell-specific,influencing metabolic reprogramming and energy homeostasis in retinal neurons through the regulation of mitochondrial quality control and nutrient-sensing pathways.In vascular endothelial cells,transcription factor EB controls important processes,including endothelial cell proliferation,endothelial tube formation,and nitric oxide levels,thereby influencing the inner blood-retinal barrier,angiogenesis,and retinal microvasculature.Additionally,transcription factor EB affects vascular smooth muscle cells,inhibiting vascular calcification and atherogenesis.In retinal pigment epithelial cells,transcription factor EB modulates functions such as autophagy,lysosomal dynamics,and clearance of the aging pigment lipofuscin,thereby promoting photoreceptor survival and regulating vascular endothelial growth factor A expression involved in neovascularization.These cell-specific functions of transcription factor EB significantly impact retinal aging mechanisms encompassing proteostasis,neuronal synapse plasticity,energy metabolism,microvasculature,and inflammation,ultimately offering protection against retinal aging and diseases.The review emphasizes transcription factor EB as a potential therapeutic target for retinal diseases.Therefore,it is imperative to obtain well-controlled direct experimental evidence to confirm the efficacy of transcription factor EB modulation in retinal diseases while minimizing its risk of adverse effects.

Transcription factor OsSPL10 interacts with OsJAmyb to regulate blast resistance in rice

Transcription factors(TFs)play essential roles in transcriptional reprogramming during activation of plant immune responses to pathogens.OsSPL10(SQUAMOSA promoter binding protein-like10)is an important TF regulating trichome development and salt tolerance in rice.Here we report that knockout of OsSPL10 reduces whereas its overexpression enhances rice resistance to blast disease.OsSPL10 positively regulates chitin-induced immune responses including reactive oxygen species(ROS)burst and callose deposition.We show that OsSPL10 physically associates with OsJAmyb,an important TF involved in jasmonic acid(JA)signaling,and positively regulates its protein stability.We then prove that OsJAmyb positively regulates resistance to blast.Our results reveal a molecular module consisting of OsSPL10 and OsJAmyb that positively regulates blast resistance.

The BEL1-like transcription factor GhBLH5-A05 participates in cotton response to drought stress

Drought stress impairs crop growth and development.BEL1-like family transcription factors may be involved in plant response to drought stress,but little is known of the molecular mechanism by which these proteins regulate plant response and defense to drought stress.Here we show that the BEL1-like transcription factor GhBLH5-A05 functions in cotton(Gossypium hirsutum)response and defense to drought stress.Expression of GhBLH5-A05 in cotton was induced by drought stress.Overexpression of GhBLH5-A05 in both Arabidopsis and cotton increased drought tolerance,whereas silencing GhBLH5-A05 in cotton resulted in elevated sensitivity to drought stress.GhBLH5-A05 binds to cis elements in the promoters of GhRD20-A09 and GhDREB2C-D05 to activate the expression of these genes.GhBLH5-A05 interacted with the KNOX transcription factor GhKNAT6-A03.Co-expression of GhBLH5-A05 and GhKNAT6-A03 increased the transcription of GhRD20-A09 and GhDREB2C-D05.We conclude that GhBLH5-A05 acts as a regulatory factor with GhKNAT6-A03 functioning in cotton response to drought stress by activating the expression of the drought-responsive genes GhRD20-A09 and GhDREB2C-D05.

Sugarcane transcription factor ScWRKY4 negatively regulates resistance to pathogen infection through the JA signaling pathway

WRKY transcription factors,transcriptional regulators unique to plants,play an important role in defense response to pathogen infection.However,the resistance mechanisms of WRKY genes in sugarcane remain unclear.In the present study,gene ontology(GO)enrichment analysis revealed that WRKY gene family in sugarcane was extensively involved in the response to biotic stress and in defense response.We identified gene ScWRKY4,a classⅡc member of the WRKY gene family,in sugarcane cultivar ROC22.This gene was induced by salicylic acid(SA)and methyl jasmonate(MeJA)stress.Interestingly,expression of ScWRKY4 was down-regulated in smut-resistant sugarcane cultivars but up-regulated in smutsusceptible sugarcane cultivars infected with Sporisorium scitamineum.Moreover,stable overexpression of the ScWRKY4 gene in Nicotiana benthamiana enhanced susceptibility to Fusarium solani var.coeruleum and caused down-regulated expression of immune marker-related genes.Transcriptome analysis indicated suppressed expression of most JAZ genes in the signal transduction pathway.ScWRKY4 interacted with ScJAZ13 to repress its expression.We thus hypothesized that the ScWRKY4 gene was involved in the regulatory network of plant disease resistance,most likely through the JA signaling pathway.The present study depicting the molecular involvement of ScWRKY4 in sugarcane disease resistance lays a foundation for future investigation.

Molecular mechanisms of the suppression of axon regeneration by KLF transcription factors

Molecular mechanisms of the Kruppel-like family of transcription factors （KLFs） have been studied more in proliferating cells than in post-mitotic cells such as neurons. We recently found that KLFs regulate intrinsic axon growth ability in central nervous system （CNS） neurons in- cluding retinal ganglion cells, and hippocampal and cortical neurons. With at least 15 of 17 KLF family members expressed in neurons and at least 5 structurally unique subfamilies, it is import- ant to determine how this complex family functions in neurons to regulate the intricate genetic programs of axon growth and regeneration. By characterizing the molecular mechanisms of the KLF family in the nervous system, including binding partners and gene targets, and comparing them to defined mechanisms defined outside the nervous system, we may better understand how KLFs regulate neurite growth and axon regeneration.

High-throughput sequencing of highbush blueberry transcriptome and analysis of basic helix-loop-helix transcription factors

The highbush blueberry（Vaccinium corymbosum）,Duke,was used to construct a de novo transcriptome sequence library and to perform data statistical analysis.Mega 4,CLC Sequence Viewer 6 software,and quantitative PCR were employed for bioinformatics and expression analyses of the basic helix-loop-helix（BHLH）transcription factors of the sequencing library.The results showed that 28.38 gigabytes of valid data were obtained from transcriptome sequencing and were assembled into 108 033 unigenes.Functional annotation showed that 32 244 unigenes were annotated into Clusters of Orthologous Groups（COG）and Gene Ontology（GO）databases,whereas the rest of the 75 789 unigenes had no matching information.By using COG and GO classification tools,sequences with annotation information were divided into 25 and 52 categories,respectively,which involved transport and metabolism,transcriptional regulation,and signal transduction.Analysis of the transcriptome library identified a total of 59 BHLH genes.Sequence analysis revealed that 55 genes of that contained a complete BHLH domain.Furthermore,phylogenetic analysis showed that BHLH genes of blueberry（Duke）could be divided into 13 sub-groups.PCR results showed that 45 genes were expressed at various developmental stages of buds,stems,leaves,flowers,and fruits,suggesting that the function of BHLH was associated with the development of different tissues and organs of blueberry,Duke.The present study would provided a foundation for further investigations on the classification and functions of the blueberry BHLH family.

CPTA treatment reveals potential transcription factors associated with carotenoid metabolism in flowers of Osmanthus fragrans

Osmanthus fragrans is one of the top ten traditional flowers in China.It is divided into three different groups according to its color.α-Carotene and β-carotene are the main determinants to distinguish the color differences between three groups.However,the dominant genes and transcription factors involved in carotenoid metabolism remain unclear.CPTA treatment(0.7mmol·L−1)remarkably promoted lycopene,α-carotene and β-carotene contents in flowers.Transcriptome sequencing analysis revealed that CPTA treatment could trigger chain reactions in carotenoid metabolism pathway genes.Four up-regulated and 10 down-regulated transcription factors which have close association with carotenoid variation were significantly induced by CPTA treatment.The up-regulated TFs such as MYB43,MYB123,HSF,were further subjected to transcript expression determination in different cultivars with drastic colors.Among them,transcript expression of four up-regulated TFs coincided with the carotenoid accumulation in different cultivars.We selected up-regulated OfMYB43 to verify its function,which is related to stress tolerance and transcriptional regulation.Transient overexpression of OfMYB43 in O.fragrans flowers showed that it could remarkably promote the expression of PDS,ZISO,LCYE and CCD4,leading to increased accumulation of β-branch carotenoids.OfMYB43 was a potential positive regulator of carotenoid biosynthesis in O.fragrans flowers.This study provides insight into the molecular mechanism of carotenoid metabolism in O.fragrans.

Genome-Wide Identification of Zn_(2)Cys_(6 ) Class Fungal-Specific Transcription Factors(ZnFTFs)and Functional Analysis of UvZnFTFI in Ustilaginoidea virens

Transcription factors(TFs)orchestrate the regulation of cellular gene expression and thereby determine cell functionality.In this study,we analyzed the distribution of TFs containing domains,which named as ZnFTFs,both in ascomycete and basidiomycete fungi.We found that ZnFTFs were widely distributed in these fungal species,but there was more expansion of the ZnFTF class in Ascomycota than Basidiomycota.We identified 40 ZnFTFs in Ustilaginoidea virens,and demonstrated the involvement of UvZnFTF1 in vegetative growth,conidiation,pigment biosynthesis and pathogenicity.RNA-Seq analysis suggested that UvZnFTF1 may regulate different nutrient metabolism pathways,the production of secondary metabolites,and the expression of pathogen-host interaction genes and secreted protein-encodi ng genes.Analysis of the distributi on of differe nt fungal TFs in U.virens further dem on strated that UvZnFTFs make up a large TF family and may play essential biological roles in U.virens.

Genome-wide analyses on transcription factors and their potential microRNA regulators involved in maize male fertility

Anther development is a programmed biological process crucial to plant male reproduction. Genomewide analyses on the functions of transcriptional factor(TF) genes and their microRNA(miRNA) regulators contributing to anther development have not been comprehensively performed in maize. Here, using published RNA-Seq and small RNA-Seq(sRNA-Seq) data from maize anthers at ten developmental stages in three genic male-sterility(GMS) mutants(ocl4, mac1, and ms23) and wild type W23, as well as newly sequenced maize anther transcriptomes of ms7-6007 and lob30 GMS mutants and their WT lines, we analyzed and found 1079 stage-differentially expressed(stage-DE) TF genes that can be grouped into six(premeiotic, meiotic, postmeiotic, premeiotic-meiotic, premeiotic-postmeiotic, and meiotic-postmeiotic clusters) expression clusters. Functional enrichment combined with cytological and physiological analyses revealed specific functions of genes in each expression cluster. In addition, 118 stage-DE miRNAs and99 miRNA-TF gene pairs were identified in maize anthers. Further analyses revealed the regulatory roles of zma-miR319 and zma-miR159 as well as ZmMs7 and ZmLOB30 on ZmGAMYB expression. Moreover,ZmGAMYB and its paralog ZmGAMYB-2 were demonstrated as novel maize GMS genes by CRISPR/Cas9 knockout analysis. These results extend our understanding on the functions of miRNA-TF gene regulatory pairs and GMS TF genes contributing to male fertility in plants.

Genome-wide analysis of the B3 transcription factors reveals that RcABI3/VP1 subfamily plays important roles in seed development and oil storage in castor bean(Ricinus communis)

The B3 transcription factors(TFs)in plants play vital roles in numerous biological processes.Although B3 genes have been broadly identified in many plants,little is known about their potential functions in mediating seed development and material accumulation.Castor bean(Ricinus communis)is a non-edible oilseed crop considered an ideal model system for seed biology research.Here,we identified a total of 61 B3 genes in the castor bean genome,which can be classified into five subfamilies,including ABI3/VP1,HSI,ARF,RAV and REM.The expression profiles revealed that RcABI3/VP1 subfamily genes are significantly up-regulated in the middle and later stages of seed development,indicating that these genes may be associated with the accumulation of storage oils.Furthermore,through yeast one-hybrid and tobacco transient expression assays,we detected that ABI3/VP1 subfamily member RcLEC2 directly regulates the transcription of RcOleosin2,which encodes an oil-body structural protein.This finding suggests that RcLEC2,as a seed-specific TF,may be involved in the regulation of storage materials accumulation.This study provides novel insights into the potential roles and molecular basis of B3 family proteins in seed development and material accumulation.

Mapping of liver-enriched transcription factors in the human intestine

AIM: To investigate the gene expression pattern of hepatocyte nuclear factor 6 (HNF6) and other liverenriched transcription factors in various segments of the human intestine to better understand the differentiation of the gut epithelium. METHODS: Samples of healthy duodenum and jejunum were obtained from patients with pancreatic cancer whereas ileum and colon was obtained from patients undergoing right or left hemicolectomy or (recto)sigmoid or rectal resection. All surgical specimens were subjected to histopathology. Excised tissue was shock-frozen and analyzed for gene expression of liver-enriched transcription factors by semiquantitative reverse transcription polymerase chain and compared to the human colon carcinoma cell line Caco-2. Protein expression of major liver-enriched transcription factors was determined by Western blotting while the DNA binding of HNF6 was investigated by electromobility shift assays. RESULTS: The gene expression patterning of liverenriched transcription factors differed in the various segments of the human intestine with HNF6 gene expression being most abundant in the duodenum (P < 0.05) whereas expression of the zinc finger protein GATA4 and of the HNF6 target gene ALDH3A1 was most abundant in the jejunum (P < 0.05). Likewise, expression of FOXA2 and the splice variants 2 and 4 of HNF4α were most abundantly expressed in the jejunum (P < 0.05). Essentially, expression of transcription factors declined from the duodenum towards the colon with the most abundant expression in the jejunum and less in the ileum. The expression of HNF6 and of genes targeted by this factor, i.e. neurogenin 3 (NGN3) was most abundant in the jejunum followed by the ileum and the colon while DNA binding activity of HNF4α and of NGN3 was conf irmed by electromobility shift assays to an optimized probe. Furthermore, Western blotting provided evidence of the expression of several liver-enriched transcription factors in cultures of colon epithelial cells, albeit at different levels. CONCLUSION: We describe significant local and segmental differences in the expression of liver-enriched transcription factors in the human intestine which impact epithelial cell biology of the gut.

Polyploidy events shaped the expansion of transcription factors in Cucurbitaceae and exploitation of genes for tendril development

Cucurbitaceae is one of the most important plant families distributed worldwide.Transcription factors(TFs)regulate plant growth at the transcription level.Here,we performed a systematic analysis of 42641 TFs from 63 families in 14 Cucurbitaceae and 10 non-cucurbit species.Whole-genome duplication(WGD)was the dominant event type in almost all Cucurbitaceae plants.The TF families were divided into 1210 orthogroups(OGs),of which,112 were unique to Cucurbitaceae.Although the loss of several gene families was detected in Cucurbitaceae,the gene families expanded in five species that experienced a WGD event comparing with grape.Our findings revealed that the recent WGD events that had occurred in Cucurbitaceae played important roles in the expansion of most TF families.The functional enrichment analysis of the genes that significantly expanded or contracted uncovered five gene families,AUX/IAA,NAC,NBS,HB,and NF-YB.Finally,we conducted a comprehensive analysis of the TCP gene family and identified 16 tendril-related(TEN)genes in 11 Cucurbitaceae species.Interestingly,the characteristic sequence changed from CNNFYFP to CNNFYLP in the TEN gene(Bhi06M000087)of Benincasa hispida.Furthermore,we identified a new characteristic sequence,YNN,which could be used for TEN gene exploitation in Cucurbitaceae.In conclusion,this study will serve as a reference for studying the relationship between gene family evolution and genome duplication.Moreover,it will provide rich genetic resources for functional Cucurbitaceae studies in the future.

SOX transcription factors and glioma stem cells:Choosing between stemness and differentiation

Glioblastoma(GBM)is the most common,most aggressive and deadliest brain tumor.Recently,remarkable progress has been made towards understanding the cellular and molecular biology of gliomas.GBM tumor initiation,progression and relapse as well as resistance to treatments are associated with glioma stem cells(GSCs).GSCs exhibit a high proliferation rate and self-renewal capacity and the ability to differentiate into diverse cell types,generating a range of distinct cell types within the tumor,leading to cellular heterogeneity.GBM tumors may contain different subsets of GSCs,and some of them may adopt a quiescent state that protects them against chemotherapy and radiotherapy.GSCs enriched in recurrent gliomas acquire more aggressive and therapy-resistant properties,making them more malignant,able to rapidly spread.The impact of SOX transcription factors(TFs)on brain tumors has been extensively studied in the last decade.Almost all SOX genes are expressed in GBM,and their expression levels are associated with patient prognosis and survival.Numerous SOX TFs are involved in the maintenance of the stemness of GSCs or play a role in the initiation of GSC differentiation.The fine-tuning of SOX gene expression levels controls the balance between cell stemness and differentiation.Therefore,innovative therapies targeting SOX TFs are emerging as promising tools for combatting GBM.Combatting GBM has been a demanding and challenging goal for decades.The current therapeutic strategies have not yet provided a cure for GBM and have only resulted in a slight improvement in patient survival.Novel approaches will require the fine adjustment of multimodal therapeutic strategies that simultaneously target numerous hallmarks of cancer cells to win the battle against GBM.

Regulatory Network of Transcription Factors in Response to Drought in Arabidopsis and Crops

Drought is one of the most important environmental constraints limiting plant growth, development and crop yield. Many drought-inducible genes have been identified by molecular and genomic analyses in Arabidopsis, rice and other crops. To better understand reaction mechanism of plant to drought tolerance, we mainly focused on introducing the research of transcription factors （TFs） in signal transduction and regulatory network of gene expression conferring drought. A TF could bind multiple target genes to increase one or more kinds of stress tolerance. Sometimes, several TFs might act together with a target gene. So drought-tolerance genes or TFs might respond to high-salinity, cold or other stresses. The crosstalk of multiple stresses signal pathways is a crucial aspect of understanding stress signaling.

A Nonradioactive Method for Detecting DNA-binding Activity of Nuclear Transcription Factors

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-κB were labled with DIG by terminal transferase After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8 % nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film The results showed that nuclear proteins binded specifically to the NF-κB consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-κB in PMA group was more than that in PMA+PDTC group It is suggested that detection of NF-κB by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories

Effects of the Ratio of Lysine to Digestible Energy Level in the Diet on the Expression and Activity of Transcription Factors Involved in Lipogenesis in Rongchang Pigs

This study was conducted to determine the effects of varying the ratio of lysine to digestible energy level On the activity and gene expression of the transcription factors peroxisome proliferator-activated receptor-γ （PPAR-γ） and CCAAT/enhancer-binding protein-or and -β （C/EBP-α and C/EBP-β） to better understand the regulatory mechanisms controlling adipogenesis in fat and muscle tissue of the Rongchang pig. A total of 144 castrated Rongchang pigs weighing approximately 20 kg were used in a 2 ×2 factorial design experiment. Diets were formulated to contain a high （14.22 MJ/kg） or low （13.11 MJ/kg） digesti- ble energy （DE） level. Within each energy level, pigs were fed diets containing a high lysine： DE ratio （0.67,0. 53, or 0. 42） or a low lysine ： DE ratio （0.49,0.38 ,or 0.30） during the periods from 20 to 50 kg, 50 to 80 kg, and 80 kg to slaughter, respectively. Each diet was fed to six replicate pens, each containing nine pigs. When the pigs reached average live weights of 20,35,60, and 90 kg ,one pig from each of the replicates was chosen at random and slaughtered.Samples of back fat and longissimus dorsi muscle were collected for the assessment of transcriptional factor. The results showed that feeding a high DE level significantly increased （ P 〈 0.05 ） the expression of PPAR-T at 60 and 90 kg in muscle and at 35,60, and 90 kg in back fat. Energy level also significantly increased the expression of C/EBP-fl at 35 and 60 kg in both muscle and back fat （ P 〈 0.05 ）. Higher dieta- ry lysine increased the expression of C/EBP-fl in muscle at 35 and 90 kg （ P 〈 0.05）, but decreased the expression in back fat at 35 （P = 0.03 ） and 90 kg （P = 0.09）. The lysine level increased the expression of PPAR-3~ in muscle at 60 kg only. Energy level and lysine content had no significant effects on promote the activity of PPAR-γ, C/EBP-α, or C/EBP-β either in muscle or in back fat at any level of the body weights tested. Collectively, these data indicated that dietary energy density and lysine level were equally important for lipid deposition in muscle tissue, whereas dietary energy density was more important than lysine level for fat deposition in fat tissue.