GbLMI1 over-expression improves cotton aboveground vegetative growth

Leaves are the main organ for photosynthesis and organic synthesis in cotton.Leaf shape has important effects on photosynthetic efficiency and canopy formation,thereby affecting cotton yield.Previous studies have shown that LMI1(LATE MERISTEM IDENTITY1)is the main gene regulating leaf shape.In this study,the LMI1 gene was inserted into the 35S promoter expression vector,and cotton plants overexpressing LMI1(OE)were obtained through genetic transformation.Statistical analysis of the biological traits of the T_(1) and T_(2) populations showed that compared to the wild type(WT),OE plants had significantly larger leaves,thicker stems and significantly greater dry weight.Furthermore,plant sections of the main vein and petiole showed that the numbers of cells in those tissues of OE plants were significantly greater.In addition,RNA-seq analysis revealed the differential expression of genes related to gibberellin synthesis and NAC gene family(genes containing the NAC domain)between the OE and WT plants,suggesting that LMI1 is involved in secondary wall formation and cell proliferation,which promotes stem thickening.Moreover,Gene Ontology(GO)analysis revealed enrichment in the terms of calcium ion binding,and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed enrichment in the terms of fatty acid degradation,phosphatidylinositol signal transduction system,and c AMP(cyclic adenosine monophosphate)signal pathway.These results suggested that LMI1 OE plants are responsive to gibberellin hormone signals,and have altered messenger signals(c AMP,Ca^(2+))which amplify this function,to promote stronger aboveground vegetative growth.This study found the LMI1 greatly increased the vegetative growth in cotton,which is the basic requirement for higher yield.

The tumor-selective over-expression of the human Hsp 70 gene is attributed to the aberrant controls at both initiation and elongation levels of transcription

The tumor selective over-expression of the human Hsp70 gene has been well documented in human tumors,linked to the poor prognosis,being refractory to chemo-and radio-therapies as well as the advanced stage of tumorous lesions in particular.However,both the nature and details of aberrations in the control of the Hsp70 expression in tumor remain enigmatic.By comparing various upstream segments of the Hsp70 gene for each''s ability to drive the luciferase reporter genes in the context of the tumor cell lines varying in their p53 status and an immortal normal liver cell line,we demonstrated in a great detail the defects in the control mechanisms at the both initiation and elongation levels of transcription being instrumental to the tumor selective profile of its expression.Our data should not only offer new insights into our understanding of the tumor specific over-expression of the human Hsp70 gene,but also paved the way for the rational utilization of the tumor selective mechanism with the Hsp70 at the central stage fortargeting the therapeutic gene expression to human tumors.

SIMULTANEOUS OVER-EXPRESSION OF INSULIN-LIKE GROWTH FACTOR- Ⅱ (IGF- Ⅱ ) AND IGF- Ⅱ RECEPTOR(IGF- Ⅱ R) GENES IN HUMAN PRIMARY CANCER-IMPLICATION OF AUTOCRINE AND PARACRINE MECHANISM IN AUTONOMOUS GROWTH OF HEPATIC CANCER

This is first report about the simultaneous over-expression of both Insulin-like growth factor (IGF- I ) and its receptor (IGF- I R) at mRNA level in human primary hepatic Cancer (PHC). In 10 PHC samples from China, IGF-I and IGF- I R were both over-expressed, whereas only a background signal was detected in normal liver. In 5 pairs of PHC and its non- tumorous adjacent liver tissues from South Africa, IGF- I and IGF- I R were also over-expressed in PHC. mRNA expression of IGF- I in all 5 cases and IGF- I R in 4 of 5 cases were higher in cancer than non- tumorous adjacent liver tissues. These results strongly implicate that an autocrine and/ or paracrine mechanism might be Involved in formation and progression of PHC.

Protocol for Artificial MicroRNA Mediated Over-Expression of miR820 in Indica Rice

In the present study, we illustrate the strategy and protocol required to generate rice transgenics over-expressing the 21-nt form of Osa-miR820. The miR exists in two size variants of 21-nt and 24-nt so the natural precursor cannot be employed for the purpose of miR over-expression as the cellular machinery can process both size variants thereby masking the role of PTGS regulation. Hence, we adopted the artificial miR technology to specifically over-express the 21-nt species in the transgenics. During the course of experiments it was observed that the amiR constructs probably interfered with the regeneration of the transformed callus, necessitating protocol modifications. The results indicate the successful over-expression of the 21-nt miR species. These plants can serve as a useful source for the functional dissection of the role played by the 21-nt Osa-miR820 species. They will also be valuable in highlighting the importance for the existence of a dual mode of miR mediated target regulation.

Over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector

Background Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells （ADSCs） are available. Therefore we intended to construct a lentiviral VEGF165 expression vector and then infect the ADSCs to produce therapeutic seed cells.Methods EHS1001-68950485313912 clone was mutated by PCR method to produce consensus fragment of VEGF165 transcript （NM_001025368）. Lentivirus was enveloped with pGC-FU, pHelper 1.0 and pHelper 2.0 plasmids in 293T cells.And then the ADSCs （multiplicity of infection=20） were transfected with the vectors after titer determination. Stable expression of VEGF165 in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis.Results DNA sequencing and 293T transfection verified VEGF165 was linked to the GFP fused vector. The virus titer is up to 2x10a determined by quantitative PCR. VEGF165 transduced cells could show green fluorescence confirmed by immunofluorescence staining （almost 95%）. ELISA analyses could detect out the density of VEGF was 850.86-1202.13pg/ml （mean （923.00±31.22） pg/ml） in the supernatant of VEGF16s-transduced cells but not detected in the GFP-transduced cells （P 〈0.001） and the Western blotting analyses also confirmed VEGF165 expression in VEGF165-transduced cells.Conclusions The VEGF165 over-expression ADSCs were obtained and may be used as a cell therapeutic tool and may be applied for vascular regeneration, especially in the treatment of erectile dysfunction.

Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis

The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway,the HMGR gene from Glycyrrhiza uralensis Fisch.(GuHMGR)was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC.The results showed that all the recombinant P.pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains.However,as the copy number increased,the content of ergosterol showed an increasing–decreasing–increasing pattern.This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G.uralensis.

Over-expression of CD163, CD169, and CD151 is not sufficient to improve the susceptibility to porcine reproductive and respiratory syndrome virus infection in transgenic mice

Porcine reproductive and respiratory syndrome virus （PRRSV） is a major pathogen that causes reproductive failure and respiratory disease in pigs, resulting in devastating economic losses worldwide. Porcine alveolar macrophages （PAMs） are the primary target cells of PRRSV , and the putative receptors, including CD163, CD169, and CD151, play key roles during infection . However, the understanding of PRRSV infection and pathogenesis is limited by its narrow host range. Pig is the unparalleled animal susceptible to PRRSV, but not well-suited for the study of long-term chronic infection or immune response in vivo because of their long breed- ing cvcle, size, high cost, and lack of biological materials.

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.

Improving the accumulation of 18α-and 18β-glycyrrhizins by over-expressing GuHMGR, GuSQS1, and GuBAS genes in Glycyrrhiza uralensis

Objective:To study the influence of over-expression of three functional genes involved in GC biosynthetic pathway,GuHMGR,GuSQS1,and GuBAS on GC production.Methods:Three plant expression vectors were constructed and transformed into Agrobacterium tumefaciens EHA105,which were used to infect Glycyrrhiza uralensis hypocotyls explants.After induction,selection,differentiation,culture,and transplantation,12,15,and 5 regenerated plants over-expressing GuHMGR,GuSQS1,and GuBAS,were obtained,respectively.Results:RT-PCR analysis showed these transgenic regenerated G.uralensis plants had 2-6 copies of GuHMGR,GuSQS1,or GuBAS.HPLC analysis showed the contents of 18α-and 18β-GC in all transgenic regenerated samples were both higher than that in the blank control.With the increase of copy numbers of GuHMGR,GuSQS1,and GuBAS,the contents of 18α-and 18β-GC were both increased in most samples.The highest 18α-and 18β-GC contents in transgenic regenerated plants were about 3.05 times and 2.80 times higher than that in the blank control,respectively.Conclusion:Over-expression of the GuHMGR,GuSQS1,and GuBAS genes enhance the accumulation of 18α-and 18β-GC in the roots and rhizomes of G.uralensis.We hope this work can lay a foundation for the molecular breeding research of G.uralensis and improving the quality of the roots and rhizomes of G.uralensis cultivars.

The role of water channel proteins and nitric oxide signaling in rice seed germination

Previous studies have demonstrated the possible role of several aquaporins in seed germination. But systematic investigation of the role ofaquaporin family members in this process is lacking. Here, the developmental regulation of plasma membrane intrinsic protein （PIP） expression throughout germination and post-germination processes in rice embryos was analyzed. The expression patterns of the PIPs suggest these aquaporins play different roles in seed germination and seedling growth. Partial silencing of the water channel genes, OsPIP1;1 and OsPIP1：3, reduced seed germination while over-expression of OsPIP1：3 promoted seed germination under water-stress conditions. Moreover, spatial expression analysis indicates that OsPIP1：3 is expressed predominantly in embryo during seed germination. Our data also revealed that the nitric oxide （NO） donors, sodium nitroprusside （SNP） and S-nitrosoglutathione （GSNO）, promoted seed germination; furthermore, the NO scavenger, 2-（4-carboxyphenyl）-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibited germination and reduced the stimulative effects of SNP and GSNO on rice germination. Exogenous NO stimulated the transcription of OsPIP1：1, OsPIP1：2, OsPIP1：3 and OsPIP2：8 in germinating seeds. These results suggest that water channels play an important role in seed germination, acting, at least partly, in response to the NO signaling pathway.

Characterization of NPR1 Genes from Norton and Cabernet Sauvignon Grapevine

Non-expressor of pathogenesis-related genes 1 （NPR1） plays a significant role in the defense responses of plants to pathogens by regulating the expression of defense-related genes. In the present study, we isolated two NPR1 genes from Vitis aestivalis cv. Norton and Vitis vinifera cv. Cabernet Sauvignon, which were referred to as VaNPR1.1 and VvNPR1. 1-CS, respectively. They encode a protein of 584 amino acids with a predicted molecular weight of 64.8 kDa and a theoretical isoelectric point （pI） of 5.74. The predicted amino acid sequences of VaNPR1.1 and VvNPR1.1-CS differ by only one amino acid. Over-expression of VaNPR1.1 gene in Arabidopsis npr1-1 mutant plants restores the transcriptional expression of AtPR-1 gene, though not to the full scale. This result demonstrated that a grapevine VaNPR1.1 possesses a similar function to the Arabidopsis NPR1 in the regulation of defense-related genes. Over-expression of VaNPR1.1 in transgenic Arabidopsis plant increased tolerance to salinity, but had no effect on the drought tolerance. We conclude that VaNPR1.1 is a functional ortholog of AtNPR1 and also involved in grapevine＇s response to the salt stress.

Lentivirus-mediated Persephin overexpression in Parkinson's disease rats

Persephin, together with glial cell line-derived neurotrophic factor and neurturin, has a neurotrophic effect and promotes the survival of motor neurons cultured in vitro. In this study, dopaminergic neurons in the substantia nigra of rats were transfected with the Persephin gene. One week later 6-hydroxydopamine was injected into the anterior medial bundle to establish a Parkinson＇s disease model in the rats. Results found that the number of dopaminergic neurons in the substantia nigra increased, tyrosine hydroxylase expression was upregulated and concentrations of dopamine and its metabolites in corpus striatum were increased after pretreatment with Persephin gene. In addition, the rotating effect of the induced Parkinson＇s disease rats was much less in the group pretreated with the Persephin gene. Persephin has a neuroprotective effect on the 6-hydroxydopamine-induced Parkinson＇s disease through protecting dopaminergic neurons.

Bzw2 Promotes Proliferation and Lactation of Mammary Epithelial Cell in Dairy Goat

Mitosis of mammary epithelial cell is foundation of mammal lactation. We developed a strategy of combined application of generation of longer cDNA fragments from the serial analysis of gene expression （SAGE） tags for gene identification （GLGI） to screen and identify genes influencing lactating ability of mammary epithelial cell in dairy goat. GLGI as a new tag identification technique was brought about with SAGE. Bzw2 was found as a candidate gene related to lactation by screening Long-SAGE library of mammary gland in dairy goat. Bzw2 cDNA was synthesized by switching mechanism at 5＂-end of RNA transcript （SMART） technology. The mRNA level of Bzw2 was relatively higher in early lactation than in other development stages of mammary gland. The proliferation of mammary epithelial cell was inhibited by transfecting specific shRNA of Bzw2. The mRNA levels of Stat5, Csn2 and Prlr were also down-regulated, suggesting the lactating ability of mammary epithelial cell was attenuated after Bzw2 RNAi. The reduction of mammary epithelial cell growth and lactation by Bzw2 RNAi was rescued through over-expression of Bzw2. These results revealed that Bzw2 might play an important role in lactation though the molecular mechanism was still unclear.

An engineered <i>Phlebia radiata</i>manganese peroxidase: expression, refolding, purification and preliminary characterization

Manganese peroxidases (MnPs) are interesting enzymes in protein engineering, aimed at maximizing industrial bioprocesses such as lignin degradation and biofuel production. cDNA of the secreted short-type of MnP from Phlebia radiata (Pr-MnP3) has been successfully engineered and amplified by polymerase chain reaction (PCR). Five mutant genes (E40H, E44H, E40H/E44H, D186H and D186N) of recombinant Phlebia radiata MnP3 (rPr-MnP3) were generated. The wild-type and the mutant genes were expressed in Escherichia coli (W3110 strain) and the resultant body proteins were lysed, purified and refolded into active enzymes. 6% - 7% recovery of pure and fully active rPr-MnP3 for wild-type and mutants were obtained and the availability of rPr-MnP3 enzymes will greatly facilitate its structure-function relationships studies. rPr-MnP3 mass was characterised using SDS-PAGE and MALDI-TOF mass spectrometry. Molecular weight of both the wild-type and mutant rPr-MnP3 enzymes was approximately 36 kDa. This describes the spectral characterization of the wild-type and mutant rPr-MnP3 enzymes with are very close similarities;substantially high spin haem enzymes. Therefore we report the engineering, cloning, expression, refolding/activation of MnP3 genes and preliminary characterization of the wild-type and mutant Phlebia radiata MnP3 enzymes.

Overexpression of aspen sucrose synthase gene promotes growth and development of transgenic Arabidopsis plants

In plants, sucrose synthase (SUS) enzymes catalyze conversion of sucrose into fructose and UDP-glucose in the presence of UDP. To investigate the impact of overexpression of heterologous SUS on the growth and development of Arabidopsis, we transformed Arabidopsis plants with an overexpression vector containing an aspen SUS gene (PtrSUS1). The genomic PCR confirmed the successful integration of PtrSUS1 transgene in the Arabidopsis genome. PtrSUS1 expression in transgenic Arabidopsis plants was confirmed by RT-PCR. The SUS activity was dramatically increased in all transgenic lines examined. The three selected transgenic PtrSUS1 lines exhibited faster growth and flowered about 10 days earlier compared to untransformed controls, and also possessed 133%, 139%, and 143% SUS activity compared to controls. Both fresh weights and dry biomass yields of the whole plants from these three selected transgenic lines were significantly increased to 125% of the controls. Transgenic PtrSUS1 lines also had a higher tolerance to higher concentration of sucrose which was reflective of the increased SUS activity in transgenic versus wild-type plants. The growth differences between wild-type and transgenic plants, either in root and hypocotyl length or in fresh and dry weight of whole plant, became more pronounced on the media containing higher sucrose concentrations. Taken together, these results showed that the early flowering, faster growth and increased tolerance to higher sucrose in transgenic lines were caused by the genome integration and constitutive expression of the aspen PtrSUS1 gene in transgenic Arabidopsis.

Characterization of flounder (Paralichthys olivaceus) FoxD5 and its function in regulating myogenic regulatory factor

As one member of winged helix domain transcription factors, FoxD5 was reported to be a trunk organizer. Recent study showed that zebrafish foxd5 is expressed in the somites. To further understand the function of FoxD5 in fish muscle development, the FoxD5 gene was isolated from flounder. Its expression pattern was analyzed by in situ hybridization, while its function in regulating myogenic regulatory factor, MyoD, was analyzed by ectopic expression. It showed that flounder FoxD5 was firstly expressed in the tailbud, adaxial cells, and neural plate of the head. In flounder embryo, FoxD5 is expressed not only in forebrain but also in somite cells that will form muscle in the future. When flounder FoxD5 was over-expressed in zebrafish by microinjection, the expression of zebrafish MyoD in the somites was reduced, suggesting that FoxD5 is involved in myogenesis by regulating the expression of MyoD.

Overexpression of the PdpapERF109 gene enhances resistance of Populus davidiana×P.alba var.pyramidalis to Fusarium oxysporum infection

The key transcription factor gene PdP apE RF109 was cloned from Populus davidiana×P.alba var.pyramidalis(Pdpap),and after overexpression of P dP ap ERF109 in transformants,the gene functions in the resistance response to Fusarium oxysporum infection.Compared with the wild Pdpap,after inoculation with F.oxysporum,the physiological and biochemical characteristics,including relative fresh weight,peroxidase activity,and the percentage of electrolyte leakage showed that,after overexpression of the PdPapERF109 gene,the transformants grew well and displayed significant resistance to F.oxysporum infection.By comparing the reactive oxygen species scavenging capacity of Pdpap plants after pathogen infection,the P dPapERF109-overexpressing plants had significantly better reactive oxygen species scavenging ability than the wild plants.Comprehensive analysis of plant morphology and various physiological and biochemical parameters showed that the overexpression of the P dpapERF109 gene significantly improved the resistance of Pdpap plants to F.oxysporum root rot.Therefore,increasing the expression of the homologous ERF109 gene can be an effective strategy to increase disease resistance in hybrid poplars.

Cyr61/CTGF/Nov family proteins in gastric carcinogenesis

Gastric cancer(GC)is the second leading cause of cancer-related death.The poor survival rate may reflect the relatively aggressive tumor biology of GC.Recently,the importance of the tumor microenvironment in carcinogenesis has emerged.In the tumor microenvironment,tumor cells and the surrounding stromal cells aberrantly secrete matricellular proteins capable of modulating carcinogenesis and regulating metastasis.The Cyr61/CTGF/Nov(CCN)proteins are a family of matricellular proteins with variable roles in many physiological and pathological processes.The evidence suggests that CCN family proteins contribute to GC carcinogenic processes.Here,we briefly review recent research on the effects of CCN family proteins in GC carcinogenesis and the development of new targeted agents in this field.

Improve Ethanol Yield Through Minimizing Glycerol Yield in Ethanol Fermentation of Saccharomyces cerevisiae

In ethanol fermentation of Saccharomyces cerevisiae （S. cerevisiae）, glycerol is one of the main by-products. The purpose of this investigation was to increase ethanol yield through minimizing glycerol yield by using mutants in which FPS1 encoding a channel protein that mediates glycerol export and GPD2 encoding one of glycerol-3-phosphate dehydrogenase were knocked-out using one-step gene replacement. GLT1 and GLN1 that encode glutamate synthase and glutamine synth.etase, respectively,were overexpressed using two-step gene replacment in fpsl△gpd2△ mutant.The fermentation properties of ZAL69（fpsl△：：LEU2 gpd2△：：URA3） and ZAL808 （fps1△：：LEU2 gpd2△：：URA3 PPGK1-GLT1 PPGK1-GLN1） under microaerobic conditions were investigated and compared with those of wild type（DC124）. Consumption of glucose, yield of ethanol, yield of glycerol, acetic acid, and pyruvic acid were monitored. Compared with wild type, the ethanol yield of ZAL69 and ZAL808 were improved by. 13.17% and 6.66 %, respectively, whereas glycerol yield decreased by 37.4 % and 41.7 %. Meanwhile, acetic acia yield and pyruvic acid yield aecreasea aramatlcally comparea to wild type. Our results indicate that FPS1 and GPD2 deletion of S. cerevisiae resulted in reduced glycerol yield and increased ethanol yield, but simultaneous overexpression of GLT1 and GLN1 infps1△gpd2△ mutant did not have a higher ethanol yield thanfps1△gpd2△ mutant.

Can overexpression of TGF-β1 gene change the sex ratio in transgenic mice?

Mouse TGF-β1 gene was microinjected into male pronuclei of F2 hybrid fertilized eggs obtained by mating CSJLF1 and C57BL/6J inbred strains to generate transgenic mice with over-expressed TGF-β1 gene. The rate of founder production is 31% and Southern blot analysis of founder mice tail DNAS gave an integration efficiency of 33%. TGF-β1 gene could be stably integrated to the chromosomes of transgenic mice and transmitted to their progeny at a rate of 33% in the second generation. Dot blot analysis of tail RNA of some transgenic mice indicated a moderate expression of the transgene. The most interesting finding of the present work is the striking deviation from the normal male:female sex ratio in transgenic mice,with an average ratio of 6.7:1. The possible nature of the predominance of male sex in transgenic mice overexpressing TGF-β1 is discussed.