Protein nanoparticles as potent delivery vehicles for polycytosine RNA-binding protein one

Ma et al recently reported in the World Journal of Diabetes that ferroptosis occurs in osteoblasts under high glucose conditions,reflecting diabetes pathology.This condition could be protected by the upregulation of the gene encoding polycytosine RNA-binding protein 1(PCBP1).Additionally,Ma et al used a lentivirus infection system to express PCBP1.As the authors’method of administration can be improved in terms of stability and cost,we propose delivering PCBP1 to treat type 2 diabetic osteoporosis by encapsulating it in protein nanoparticles.First,PCBP1 is small and druggable.Second,intravenous injection can help deliver PCBP1 across the mucosa while avoiding acid and enzyme-catalyzed degradation.Furthermore,incorporating PCBP1 into nanoparticles prevents its interaction with water or oxygen and protects PCBP1’s structure and activity.Notably,the safety of the protein materials and the industrialization techniques for large-scale production of protein nanoparticles must be comprehensively investigated before clinical application.

RGS4 promotes the progression of gastric cancer through the focal adhesion kinase/phosphatidyl-inositol-3-kinase/protein kinase B pathway and epithelial-mesenchymal transition

BACKGROUND Regulator of G protein signaling(RGS)proteins participate in tumor formation and metastasis by acting on theα-subunit of heterotrimeric G proteins.The speci-fic effect of RGS,particularly RGS4,on the progression of gastric cancer(GC)is not yet clear.AIM To explore the role and underlying mechanisms of action of RGS4 in GC develop-ment.METHODS The prognostic significance of RGS4 in GC was analyzed using bioinformatics based public databases and verified by immunohistochemistry and quantitative polymerase chain reaction in 90 patients with GC.Function assays were employed to assess the carcinogenic impact of RGS4,and the mechanism of its possible influence was detected by western blot analysis.A nude mouse xenograft model was established to study the effects of RGS4 on GC growth in vitro.RESULTS RGS4 was highly expressed in GC tissues compared with matched adjacent normal tissues.Elevated RGS4 expression was correlated with increased tumor-node-metastasis stage,increased tumor grade as well as poorer overall survival in patients with GC.Cell experiments demonstrated that RGS4 knockdown suppressed GC cell proliferation,migration and invasion.Similarly,xenograft experiments confirmed that RGS4 silencing significantly inhibited tumor growth.Moreover,RGS4 knockdown resulted in reduced phosphorylation levels of focal adhesion kinase,phosphatidyl-inositol-3-kinase,and protein kinase B,decreased vimentin and N-cadherin,and elevated E-cadherin.CONCLUSION High RGS4 expression in GC indicates a worse prognosis and RGS4 is a prognostic marker.RGS4 influences tumor progression via the focal adhesion kinase/phosphatidyl-inositol-3-kinase/protein kinase B pathway and epithelial-mesenchymal transition.

Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

Protein arginine methyltransferase-6 participates in a range of biological functions,particularly RNA processing,transcription,chromatin remodeling,and endosomal trafficking.However,it remains unclear whether protein arginine methyl transferase-6 modifies neuropathic pain and,if so,what the mechanisms of this effect.In this study,protein arginine methyltransferase-6 expression levels and its effect on neuropathic pain were investigated in the spared nerve injury model,chronic constriction injury model and bone cancer pain model,using immunohistochemistry,western blotting,immunoprecipitation,and label-free proteomic analysis.The results showed that protein arginine methyltransferase-6 mostly co-localized withβ-tubulinⅢin the dorsal root ganglion,and that its expression decreased following spared nerve injury,chronic constriction injury and bone cancer pain.In addition,PRMT6 knockout(Prmt6~(-/-))mice exhibited pain hypersensitivity.Furthermore,the development of spared nerve injury-induced hypersensitivity to mechanical pain was attenuated by blocking the decrease in protein arginine methyltransferase-6 expression.Moreover,when protein arginine methyltransferase-6 expression was downregulated in the dorsal root ganglion in mice without spared nerve injury,increased levels of phosphorylated extracellular signal-regulated kinases were observed in the ipsilateral dorsal horn,and the response to mechanical stimuli was enhanced.Mechanistically,protein arginine methyltransferase-6 appeared to contribute to spared nerve injury-induced neuropathic pain by regulating the expression of heterogeneous nuclear ribonucleoprotein-F.Additionally,protein arginine methyltransfe rase-6-mediated modulation of hete rogeneous nuclear ribonucleoprotein-F expression required amino atids 319 to 388,but not classical H3R2 methylation.These findings indicated that protein arginine methyltransferase-6 is a potential therapeutic target fo r the treatment of peripheral neuro pathic pain.

Exploring the interaction between the gut microbiota and cyclic adenosine monophosphate-protein kinase A signaling pathway:a potential therapeutic approach for neurodegenerative diseases

The interaction between the gut microbiota and cyclic adenosine monophosphate(cAMP)-protein kinase A(PKA)signaling pathway in the host's central nervous system plays a crucial role in neurological diseases and enhances communication along the gut–brain axis.The gut microbiota influences the cAMP-PKA signaling pathway through its metabolites,which activates the vagus nerve and modulates the immune and neuroendocrine systems.Conversely,alterations in the cAMP-PKA signaling pathway can affect the composition of the gut microbiota,creating a dynamic network of microbial-host interactions.This reciprocal regulation affects neurodevelopment,neurotransmitter control,and behavioral traits,thus playing a role in the modulation of neurological diseases.The coordinated activity of the gut microbiota and the cAMP-PKA signaling pathway regulates processes such as amyloid-β protein aggregation,mitochondrial dysfunction,abnormal energy metabolism,microglial activation,oxidative stress,and neurotransmitter release,which collectively influence the onset and progression of neurological diseases.This study explores the complex interplay between the gut microbiota and cAMP-PKA signaling pathway,along with its implications for potential therapeutic interventions in neurological diseases.Recent pharmacological research has shown that restoring the balance between gut flora and cAMP-PKA signaling pathway may improve outcomes in neurodegenerative diseases and emotional disorders.This can be achieved through various methods such as dietary modifications,probiotic supplements,Chinese herbal extracts,combinations of Chinese herbs,and innovative dosage forms.These findings suggest that regulating the gut microbiota and cAMP-PKA signaling pathway may provide valuable evidence for developing novel therapeutic approaches for neurodegenerative diseases.

Spastin and alsin protein interactome analyses begin to reveal key canonical pathways and suggest novel druggable targets

Developing effective and long-term treatment strategies for rare and complex neurodegenerative diseases is challenging. One of the major roadblocks is the extensive heterogeneity among patients. This hinders understanding the underlying disease-causing mechanisms and building solutions that have implications for a broad spectrum of patients. One potential solution is to develop personalized medicine approaches based on strategies that target the most prevalent cellular events that are perturbed in patients. Especially in patients with a known genetic mutation, it may be possible to understand how these mutations contribute to problems that lead to neurodegeneration. Protein–protein interaction analyses offer great advantages for revealing how proteins interact, which cellular events are primarily involved in these interactions, and how they become affected when key genes are mutated in patients. This line of investigation also suggests novel druggable targets for patients with different mutations. Here, we focus on alsin and spastin, two proteins that are identified as “causative” for amyotrophic lateral sclerosis and hereditary spastic paraplegia, respectively, when mutated. Our review analyzes the protein interactome for alsin and spastin, the canonical pathways that are primarily important for each protein domain, as well as compounds that are either Food and Drug Administration–approved or are in active clinical trials concerning the affected cellular pathways. This line of research begins to pave the way for personalized medicine approaches that are desperately needed for rare neurodegenerative diseases that are complex and heterogeneous.

AAV mediated carboxyl terminus of Hsp70 interacting protein overexpression mitigates the cognitive and pathological phenotypes of APP/PS1 mice

The E3 ubiquitin ligase,carboxyl terminus of heat shock protein 70(Hsp70)interacting protein(CHIP),also functions as a co-chaperone and plays a crucial role in the protein quality control system.In this study,we aimed to investigate the neuroprotective effect of overexpressed CHIP on Alzheimer’s disease.We used an adeno-associated virus vector that can cross the blood-brain barrier to mediate CHIP overexpression in APP/PS1 mouse brain.CHIP overexpression significantly ameliorated the performance of APP/PS1 mice in the Morris water maze and nest building tests,reduced amyloid-βplaques,and decreased the expression of both amyloid-βand phosphorylated tau.CHIP also alleviated the concentration of microglia and astrocytes around plaques.In APP/PS1 mice of a younger age,CHIP overexpression promoted an increase in ADAM10 expression and inhibitedβ-site APP cleaving enzyme 1,insulin degrading enzyme,and neprilysin expression.Levels of HSP70 and HSP40,which have functional relevance to CHIP,were also increased.Single nuclei transcriptome sequencing in the hippocampus of CHIP overexpressed mice showed that the lysosomal pathway and oligodendrocyte-related biological processes were up-regulated,which may also reflect a potential mechanism for the neuroprotective effect of CHIP.Our research shows that CHIP effectively reduces the behavior and pathological manifestations of APP/PS1 mice.Indeed,overexpression of CHIP could be a beneficial approach for the treatment of Alzheimer’s disease.

Regulator of G protein signaling 6 mediates exercise-induced recovery of hippocampal neurogenesis,learning,and memory in a mouse model of Alzheimer’s disease

Hippocampal neuronal loss causes cognitive dysfunction in Alzheimer’s disease.Adult hippocampal neurogenesis is reduced in patients with Alzheimer’s disease.Exercise stimulates adult hippocampal neurogenesis in rodents and improves memory and slows cognitive decline in patients with Alzheimer’s disease.However,the molecular pathways for exercise-induced adult hippocampal neurogenesis and improved cognition in Alzheimer’s disease are poorly understood.Recently,regulator of G protein signaling 6(RGS6)was identified as the mediator of voluntary running-induced adult hippocampal neurogenesis in mice.Here,we generated novel RGS6fl/fl;APP_(SWE) mice and used retroviral approaches to examine the impact of RGS6 deletion from dentate gyrus neuronal progenitor cells on voluntary running-induced adult hippocampal neurogenesis and cognition in an amyloid-based Alzheimer’s disease mouse model.We found that voluntary running in APP_(SWE) mice restored their hippocampal cognitive impairments to that of control mice.This cognitive rescue was abolished by RGS6 deletion in dentate gyrus neuronal progenitor cells,which also abolished running-mediated increases in adult hippocampal neurogenesis.Adult hippocampal neurogenesis was reduced in sedentary APP_(SWE) mice versus control mice,with basal adult hippocampal neurogenesis reduced by RGS6 deletion in dentate gyrus neural precursor cells.RGS6 was expressed in neurons within the dentate gyrus of patients with Alzheimer’s disease with significant loss of these RGS6-expressing neurons.Thus,RGS6 mediated voluntary running-induced rescue of impaired cognition and adult hippocampal neurogenesis in APP_(SWE) mice,identifying RGS6 in dentate gyrus neural precursor cells as a possible therapeutic target in Alzheimer’s disease.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

F-Actin Visualization in Generative and Sperm Cells of Living Pollen of Rice Using a GFP-Mouse Talin Fusion Protein

Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels the actin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryza sativa L.) plant, A005-G-T-1-2. Observations were made on pollen at four major developmental stages, viz. I. uni-nucleate microspore stage; II. early bi-cellular pollen stage; III. late bi-cellular pollen stage; and IV. tri-cellular pollen stage. At each of these developmental stages vegetative nucleus, generative nucleus/ cell, and sperm cells were seen undergoing continuous and coordinated motion and migration. These movements seemed to be influenced by associated microfilament networks existing in the pollen. Based on these observations we propose that it is the interaction between the microfilament networks (usually one existing in the central cytoplasm and another in the cortex) that controls the dynamic movement of the vegetative nucleus, generative nucleus/cell and sperm cells. Furthermore, we have also observed that there is an array of microfilaments (oriented mostly parallel to the long axis of the cell) existing in the generative and sperm cells. As far as we are aware, this is the first report showing the existence of microfilaments in living generative and sperm cells of rice pollen. The implication and significance of the existence of microfilaments in generative and sperm cells in rendering self-propelled motion of these cells in relation to their passage and movement in the pollen tube and embryo sac for fertilization were discussed.

Actin Visualization in Living Immature Pollen of Rice Using a GFP-Mouse Talin Fusion Protein

Green fluorescent protein (GFP) fused to the F_actin binding domain of mouse talin labels the actin cytoskeleton in the immature pollen of stable transformed rice (Oryza sativa L.) plants. Actin microfilaments could be visualized only in the late_developmental stage of the immature pollen. During this developmental stage, microfilaments, initially composed of very short fibrils, develop into a very complex and novel network that sometimes totally and sometimes partially encloses the vegetative nucleus and the spherical shaped generative cell in the central cytoplasm of the immature pollen. The behavior of the actin microfilamentous structure throughout the late_developmental stage of the immature pollen is extremely dynamic, and the likelihood of this structure in generating forces for vegetative nucleus and generative cell movement in the immature pollen has been discussed. No actin filaments were visualized in the spherical generative cells.

Transformation of Arabidopsis by Rice OsWRKY78::GFP Fusion Gene and Subcellular Localization of OsWRKY78 Protein

[Objective] The study was to understand the subcellular localization of OsWRKY78 protein in plants. [Method] Primers specific for OsWRKY78 gene were designed according to the OsWRKY78 full length sequence in Genbank. The gene was cloned by RT-PCR method. The gene was then recombined into a plasmid expression vector carrying green fluorescent protein (GFP) gene, pBinGFP. The recombinant was confirmed by PCR and enzyme digestion. The recombinant plasmid pBinGFP-OsWRKY was transformed into Arabidopsis through Agrobacterium tumefaciens strain GV3101 and transgenic plants were obtained. [Result] Measured by fluorescence microscopy, the expression of OsWRKY78 and GFP fusion protein in root tip cells was localized in the nucleus. [Conclusion] This study laid the foundation for further investigating the function of OsWRKY78 gene and its role in related signal transduction and provided theoretical basis for exploring the relation between OsWRKY78 gene and brown planthoppers.

Localization of Two GFP_tagged Tobacco Plastid Division Protein NtFtsZs in Escherichia coli

Two plastid division genes, NtFtsZ1 and NtFtsZ2 isolated from Nicotiana tabacum L. were fused with gfp and expressed in Escherichia coli . The regular localizations of full length NtFtsZs∶GFP along the filamentous bacteria indicated that the NtFtsZs could recognize the potential division sites in E. coli and be polymerized with heterogeneous FtsZ from bacteria. The overexpression of NtFtsZs ∶ gfp inhibited the division of host strain cells and resulted in the long filamentous bacterial morphology. These results suggested that eukaryotic ftsZs have similar function to their prokaryotic homologs. Meanwhile, the different deletions of motifs of NtFtsZs are also employed to investigate the functions of these proteins in E. coli . The results showed that the C_terminal domains of NtFtsZs were related to the correct localization of NtFtsZs in E. coli and the N_terminal domains of NtFtsZs were responsible for the polymerization of homogeneous and heterogeneous FtsZ proteins. The significance of these results in understanding the functions of NtFtsZs in plastid division were discussed.

Soluble Expression and Rapid Quantification of GFP-hepA Fusion Protein in Recombinant Escherichia coli

To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C terminus of a green fluorescent protein mutant （GFPmutl）. As a result, not only was the functional recombinant expression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluorescence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.

Transient expression of the enhanced green fluorescent protein(egfp) gene in Sargassum horneri

Sargassum horneri is a macroalga widespread in North Asia-Pacific region, and these years its bloom has caused huge damage to the environment and the economic in China. To make up the blank on genetic engineering research, a transient transformation system for the multicellular marine brown alga S . horneri was established in this research. The algae used in this research were collected from the Yellow Sea of China and verified as a same species S . horneri with analysis of molecular markers. The S . horneri parietal leaves were transformed with the enhanced green fluorescent gene as the reporter by micro-particle bombardment. The results show that green fluorescent protein (GFP) is an eff ective transgene reporter for S . horneri and that particle bombardment is a suitable method for transformation of S . horneri . Through selection of four diff erent promoters for EGFP and six groups’ bombardment characters, the highest transformation efficiency approximately 1.31% was got with the vector pEGFP-N1 at bombardment characters 900 spi and 6 cm distance. This research paves a way for the further research and application of S . horneri .

Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the control of promoters： RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae： RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ. In addition, a threefold increase of GFP in Tran-Ⅱ was induced by heat shock at 40℃. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

Expression and Purification of Recombinant MP-GFP Protein in Escherichia coli

Guided pesticide is an unique compound resulted from the conjugation with carrier （amino acid, protein, sugars, etc） and the active pesticide ingredient. One of the attributes of the guided pesticide is its potential to accumulate at the site of the damaged points caused by pest or at the site of entry to the target pests, such as via inhalation, cuticular penetration, and oral digestion. Movement protein （MP） is a kind of protein coded by plant virus. A genetic fusion between green fluorescent protein （GFP） and movement protein resulted in the expression of a fluorescent fusion MP-GFP protein, which was fully biologically active in mediating the cell-to-cell spread of virus. In order to obtain a suitable carrier for a pesticide, fluorescent carrier MP-GFP was constructed. It was found that the recombinant MP-GFP protein was the inclusion body. The results indicated that optimized cultural condition for expression of recombinant MP-GFP protein was incubation at 37°C for 2 h and induction with 0.2 mmol L-1 IPTG （isopropyl-b-dthiogalactopyranoside） at 25°C for 4 h. MP-GFP protein was purified by using Ni-NTA resin. The expressed recombinant MP-GFP protein had both the fluorescence character of report GFP gene and moving character of movement protein. It could provide a guided carrier for studying the guided pesticide. It could also provide convenience for studying the delivery and distribution of the guided pesticide ingredients in the plant.

绿色荧光蛋白(GFP)标记杧果细菌性角斑病病原菌及其定殖规律

为研究杧果细菌性角斑病病菌在杧果不同器官组织中的定殖规律。利用电击转化法将质粒pBBR1MCS2-Tac-EGFP导入野油菜黄单胞菌杧果致病变种(Xanthomonas citri pv.mangiferaeindicae)菌株Xcm003中,通过转化子生长表型和外源基因的PCR鉴定,成功获得带绿色荧光蛋白(GFP)标记的转化子Xcm003-EGFP,采用室内刺伤和盆栽苗灌根接种试验,结合组织学观察法,追踪该病原菌在不同杧果器官组织中的定殖规律。结果表明,病原菌通过伤口侵入杧果果实果皮外层细胞层,随着侵染时间推移,向内层果皮细胞层垂直扩散并定殖,后期病原菌在伤口处大量聚集,出现隆起开裂状病斑。在杧果叶片中,病原菌从伤口侵入叶片上表皮细胞层,随后迁移到叶肉细胞间隙,侵染后期,病原菌在气孔和伤口定殖,造成气孔和伤口堵塞,导致接种点出现黑褐色水浸病斑。盆栽苗灌根后发现,该病原菌可在土壤中定殖,入侵杧果根部,但并不扩散至其他杧果组织,不会为害整株幼苗。说明杧果细菌性角斑病病菌可在不同杧果组织中定殖,但仅表现为局部侵染特性。

Constructing retroviral vector carrying green fluorescent protein(GFP)and investigating the expression of GFP in primary rat myoblast

Objective： To construct green fluorescent protein（GFP） retroviral vector（pLgXSN）, and to investigate the expression of GFP in primary rat myoblast. Methods： GFP cDNA was subcloned into the plasmid pLgXSN, and the recombinant vector was transfected into packaging cell PT67. G418 was used to select positive colony. Myoblasts were infected by a high-titer viral supernatant. The recombinant retroviral plasmid vector was identified by restriction endonuclease analysis and DNA sequence analysis. Confocal microscopy and flow cytometry were used to detect the expression of GFP. Results： The GFP cDNA sequence was identical to that of GenBank. Recombinant retroviral plasmid vector pLgGFPSN was constructed successfully. The titer of the packaged recombinant retrovirus was 1×10^6 cfu/ml. Bright green fluorescence of the transfected cells was observed under confocal microscope 48 h after transfection. The transfection rate was 33%. The effective expression of GFP in myoblast infected by recombinant retrovirus lasted for 6 weeks. Conclusion： GFP gene could be effectively and stably expressed in myoblast, which suggests that GFP could act as a marker for studies on myoblast.

Jellyfish Green Fluorescent Protein(GFP) as a Reporter for Fusarium gramminearum Development on Wheat

The plasmid pGPDGFP under the control ofpgpdA promotor was used together with vector pAN7-1 containing the hygromycin resistance cassette to co-transform protoplasts of HG1, Fusarium graminearum from Hubei Province, China. Twelve out of 14 hygromycin-resistant transformants showed green signal under the UV light and contained one or several copies ofgfp, as indicated by Southern analysis of genomic DNA digested with different restriction enzymes and hybridized to the gfp probe. A single gfp copy transformant （HG1C5） was selected for further evaluation of 80 Chinese wheat cultivars or advanced lines. The results showed different resistance type to F. graminearum were observed. GFP signals observed in the rachis and adjacent spikes of 70 Chinese wheat lines such as Chuanchongzu 104 indicated both type I （host resistance to the initial infection by the fungus） and type II （resistance to the spread of FHB symptoms within an infected spike） were not observed. While other 10 lines showed type II resistance to F. graminearum with GFP signals only in inoculated spikelets. Development of the mycelium can be intuitively observed and the resistance of wheat to F. graminearum can be identified at 7 days post inoculation （dpi） in this way. The results showed no differences were evaluated between the transformed HG1C5 and the non-transgene artificial inoculation by SAS paired chi-square test and McNernar＇s test （P=-0.0625）.

PERFORMANCE AND PERSISTENCE OF GREEN FLUORESCENT PROTEIN (gfp) MARKED AZOTOBACTER CHROOCOCCUM IN STERILIZED AND UNSTERILIZED WHEAT RHIZOSPHERIC SOIL

The persistence and performance (growth promoting potential) of green fluorescent protein (gfp) marked Azotobacter chroococcum strain ABR 4G were studied in sterilized and unsterilized wheat rhizospheric soil. The gfp was integrated via Tn 5 transposition into A. chroococcum chromosome and the resultant gfp marked colonies were identified by green fluorescent emission under UV light. The gfp was stably maintained in A. chroococcum and the gfp insertion had no apparent adverse effect on the growth promoting properties of the marked soil isolate ABR 4G. The growth promoting properties (nitrogen fixation, ammonia excretion, phosphate solubilization and IAA production) of the parent soil isolate and the gfp marked strain were found to be almost the same. All the quantitative wheat plant traits were significantly influenced by inoculation of A. chroococcum ABR 4G strain in sterilized and unsterilized soil. Inoculated bacterial counts increased gradually in wheat rhizosphere, reached maximum on 60 th d and declined on 80 th d. Fertility levels also affected survival of marked strain and the survival was comparable in sterilized and unsterilized soil. The growth promoting properties were also determined from the marked strain reisolated from wheat rhizosphere in both types of soil. Fig 1, Tab 2, Ref