Manuela Malatesta, University of Verona scholar, published a number of papers that feeding animals with genetically modified agricultural products caused health damage. She is one of the international influential scholars in researching the safety of Genetically Modified Organisms using experimental methods. Based on Malatesta's literature from Web of Science data, the research contents, methods and conclusions of 10 papers which hold toxicology view of Genetically Modified agricultural products were investigated. According to its related tracking evidence and academia evaluation, its reliability of research methods and conclusions were discussesd. The results showed that Malatesta's literature, which hold unsafe view of Genetically Modified agricultural products, were denied by the academia and follow-up similar studies using normative research methods since Malatesta's research materials and methods had problems.
Stacked transgenic plant which harboring two or more foreign genes, can meet the diverse needs of the growers and bring multiple benefits, has become an important trend in the current development of genomic modified crops. Different countries of the world take the different safety assessment model for stacked transgenic plant according to its own regulation idea and the view on genetic stability and gene interaction. The application of the stacked transgenic plant, safety assessment model of different countries were reviewed, and the suggestions on safety assessment of stacked transgenic plant in China were made also.
The industrialization situation of genetically modified crop on a global scale was analyzed first. And then the necessity of promoting the industrialization of genetically modified corn in China from the consumption presents of the domestic market of genetically modified corn, the technical requirements in maize production,the research status of transgenic maize and so on were discussed. Finally, the problem of promoting the industrialization of genetically modified corn in our country in science and management level were analyzed. In order to promote the industrialization of genetically modified corn, some suggestions for the researcher and government staff in research and safety supervision of transgenic maize were put forward.
As the core of modern biotechnology, genetic modification shows great potential in resolving food and resource scarcity, and environmental contamination. China and many countries in the world regard the technology as a strategic choice to seize the commanding point of science and technology,and to enhance the competitiveness of agriculture. Aim to help people better understand the technology under a scientific view, the general development situation of agricultural breeding technology of GMOs, the causes of public opinion environment, application trends and essential characteristics of genetic modification, as well as the safety administration of agricultural genetically modified organisms were introduced. In addition, it makes five suggestions on promoting development and industrialization of genetic modification technology.
Heterologous recombinant expression and purification of SARS coronavirus main protease (SARS-CoV Mpro) , Then utilized it as the target, used drug screening model in vitro based on fluorescence resonance energy transfer (FRET), evaluated inhibition activity of 96 compounds from the protease inhibitor focused library, and explored the inhibition of compounds to SARS-CoV Mpro from the perspective of kinetics. The result show that 5 compounds are obtained of which inhibition rate >80%, quenching rate <20% by screened, they are P-1-08,P-1-19,P-2-24,P-2-28 and P-2-54. Their half effective inhibitory concentration (IC50) are 0.69±0.05μmol/L、1.19±0.41μmol/L、0.14±0.01μmol/L、1.36±0.07μmol/L、0.36±0.03μmol/L, respectively. The inhibition of P-1-08, P-1-19, P-2-24 and P-2-54 to SARS-CoV Mpro is irreversible. P-2-28 is reversible inhibition, according to Dixon diagram and Lineweaver-Burk curve found it is competitive inhibition for SARS-CoV Mpro, the inhibition constant Ki is 0.81μmol/L. Through the study about relationship of substrate concentration, IC50 and Ki, validated P-2-28 was competitive inhibition. The discovery of this inhibitor set up the foundation of resource to SARS-CoV Mpro inhibitors, and provided a lead compound for the development of anti-SARS virus drug.
To achieve a persistent in vivo efficacy, based on the characteristic that ciliary neurotrophic factor has a free cysteine residue while transferrin does not, transferrin was coupled to ciliary neurotrophic factor by using NHS-PEG5k-MAL. Combining with the purification method of each module, transferrin-PEG5k-CNTF conjugate was successfully prepared with a purity of above 90%. High performance size exclusion chromatography and dynamic light scattering analysis showed the apparent molecular volume of Tf-PEG5k-CNTF was larger than CNTF and transferrin together. The survival activity of TF-1(CN5α-1) cells showed that the bioactivity of Tf-PEG5k-CNTF was decreased to 65.8% of the CNTF. Pharmacokinetics study showed that the in vivo half-life of Tf-PEG5k-CNTF conjugate extended from about 28 minutes to 8.2 hours, increasing by about 17 times compared with CNTF. Pharmacodynamics showed that Tf-PEG5k-CNTF impacted more significantly on reducing food intake and losing weight in mice with twice weekly administration of 1.0 mg/kg drugs. Thus, transferrin coupling technique could be used for long-acting protein drugs delivery to the brain.
Porcine circovirus type 2(PCV2)Cap gene has been used as target gene for developing genetic engineering vaccine. However, it is usually expressed as the form of inclusion body in E.coli which having low protective efficacy against PCV2 challenge. The PCV2 ORF2 gene condons was optimized and fused with MPG gene. Then, they were cloned into expression vector pET28a and induced for expression in E.coli BL21(DE3) individually. SDS-PAGE and Western blot results confirmed that they could be expressed with the production of soluble proteins. After purification, the two kinds of proteins were mixed with GEL01, ISA206 and ISA15A adjuvant respectively. Mice experiment results showed that the GEL01 groups induced the highest levels of antibodies against PCV2 with ELISA and neutralization antibody assay. Following challenge with PCV2, the PCV2 virus loads in spleen in immunized groups except the ISA15A were significantly lower than challenge control group. It indicated that the vaccine prepared by the two recombinant proteins and GEL01 and ISA206 could induce immune protection in mice against PCV2 infection, which laid a foundation for the development of PCV2 subunit vaccine.
Objective: To investigate the potentiation effect of RNA silenced Runx2 on bone morphogenetic protein 2 (BMP2) induced chondrogenic differentiation of mesenchymal stem cells (MSCs). Method: C3H10T1/2 cells were treated with adenovirus: Ad-BMP2, Ad-SiRunx2 or Ad-GFP. The sulfated glycosaminoglycan of cellular matrix was detected by Alcian Blue Staining. The mRNA level of Runx2, Col2a1, aggrecan , and Col10a1 were detected by RT-PCR,and the protein level of BMP2, Col2a1,Col10a1 were detected by Western blot .Results: The BMP2+SiRunx2 group (C3H10T1/2 cells were treated with AdBMP2 and AdSiRunx2) was more blue than the BMP2 group on day 10 proven by Alcian blue staining. The BMP2+SiRunx2 group showed a higher mRNA level of Col2a1 and aggrecan (P<0.05) and a lower protein level of Col10a1 (P<0.05) than the BMP2 group on day 7 and 14. The BMP2+SiRunx2 group showed a higher protein level of Col2a1(P<0.05) and a lower protein level of Col10a1(P<0.05) than the BMP2 group on day 10. Conclusion: The siRNA targeting Runx2 promotes BMP2-induced stem cell chondrogenic differentiation and supresses the chondrocyte hypertrophy and maturation.
The double-stranded RNA of KAT gene was synthesized for RNA interference (RNAi)in bees. After RNAi, the expression of KAT gene and the synthesis of 10-HDA were detected respectively by semi-quantitative RT-PCR and HPLC in order to investigate the relationship between the silencing of KAT gene and the amount of 10-HDA. These results showed that the bees which were injected with 2μl dsKAT had the highest survival rate. And when the bees were injected for five days,the KAT mRNA levels seemed the lowest and the secretion of 10-HDA in honeybee workers showed a significant decrease(P<0.05)relative to the control group which was injected with DEPC water.And after knockdown of KAT, the glands of the bees MGs appeared to be wizened and loose .The KAT gene in bees was silenced successfully,which speculated that KAT might be involved in the biosynthetic pathway of 10-HDA and described the relationship between KAT gene and the synthesis of 10-HDA. It provides a reference for analysing the function of other genes in honeybees by RNAi, and has laid a foundation for the further perfection of 10-HDA biosynthetic pathway. In addition, it provides the basis for improving the yield of 10-HDA by increasing the expression of genes.
Jatropha curcas, grown in many harsh environment, has been widely regarded as an excellent source of renewable biofuels, but their molecular regulation mechanism in response to cadmium (Cd) stress is not yet clear. Analyzing whole expression pattern would be very important to effectively screen the key regulation genes, to reveal molecular regulatory network of Cd stress response as well as to promote molecular breeding. Illumina sequencing technology has been applied in leaf of Cd treated Jatropha curcas (100μmol/L) and control plants(CK), 50 448 unigenes obtained from the two libraries after de novo assembly. 2 551 differentially expressed genes (DEGs) found between the two libraries, 539 up-regulated, 2 012 down-regulated. Large amounts of annotation information suggested that Cd stress in J. curcas caused change of genes involved in photosynthesis, carbon metabolism, plant hormone signal transduction and plant-pathogen interaction. DAVID annotation indicated Cd stress significantly affect the homeostasis of sodium and iron in J. curcas. Transcription factor analysis demonstrated WRKY, ZIP play vital role in Cd exposed J. curcas. QRT-PCR analysis of 5 randomly selected DEGs showed that the expression patterns were highly accordant with the results of RNA-seq. This useful information for the molecular mechanisms of Cd exposed J. curcas could be practical applied in genetic engineering and phytoremediation.
Objective: To clone and identify Echinococcus granulosus egG1Y162 gene and analysis the protein expression, adaptive evolution and identification.of antigenicity. Methods: According to the gene sequence of emy162 , a pair of primers was designed from Echinococcus granulosus protoscolices, cystic wall students send layer, adults and eggs of four developmental stage respectively , extract the genomic DNA and total RNA, mRNA was reverse transcribed into cDNA,amplified egG1Y162 gene via PCR with genomic DNA and cDNA . Construct the recombinant plasmid of PUCm-T/EgG1Y162 and identified by PCR, enzyme digestion and sequencing to determine its correctness. The characteristics of EgG1Y162 gene were analyzed by DNAman software and MEGA4 software, and construct the phylogenetic tree of EgG1Y162 nucleic acid to further explore its homology. Fluorescence quantitative PCR detection egG1Y162 gene at four different developmental stages including Echinococcus granulosus protoscolices, cystic wall students expression layer, adults and eggs. The EgG1Y162 gene was cloned into prokaryotic expression plasmid PET-41a by using the directed cloning technology, and the positive clones were identified by the restriction analysis and PCR identification, and the sequence was determined by sequencing. IPTG was initially induced and expressed by EgG1Y162-GST recombinant proteins and identified by SDS-PAGE and Western blot assay. Results: EgG1Y162 gene were cloned from both two different developmental stages of Echinococcus granulosus, from total DNA clone obtained length for 1 680bp fragment , from a cDNA clone obtained length for 459bp fragment .The similarity of the EgG1Y162 gene was 91%, while the similarity of emY162 gene cDNA and EgG1Y162 was 95%. Further analysis showed that the sequence of EgG1Y162 gene was composed of 3 exons and 2 introns, the exon regions were 1~70,1064~1380and 1577~1648. The amino acids of 1~16 amino acids in the hydrophobic side constitute the EgG1Y162 signal peptide sequence, and 35~115 amino acids form a large fibronectin splicing FN3133~152 amino acids which constitute the carboxyl terminal transmembrane region. The result of DNA sequencing showed that the length of EgG1Y162 gene was 360bp and 120 amino acids were encoded.. By fluorescence quantitative PCR detection found egG1Y162 in adults, germinal layer, protoscolex and the egg stage had different degrees of expression. But the majority egG1Y162 expression in the adult stage, at most a relative value is 19.526 difference was statistically significant (P< 0.01), secondly germinal layer, 5.122, again in protoscolex stage, relative value 5.083, in the egg stage at least for 1.6588. The prokaryotic expression plasmid of PET-41a/EgG1Y162 was induced by SDS-PAGE, and the IPTG assay showed that the recombinant protein of EgG1Y162-GST was successfully expressed and expressed in the relative molecular weight of 44KDa. Western blot analysis showed positive blot bands, the molecular weight of 44kda, egG1Y162 recombinant protein can with Echinococcus granulosus infection after 40 days of canine sera react; serum from patients with hydatid disease also have positive reaction. Conclusion: Successfully cloned egG1Y162 antigen gene, sequence comparison analysis showed high similarity with cDNA of the egG1Y162 cDNA and emy162, differences in gene mainly exists in the intron region, the egG1Y162 antigen gene is a new gene. The expression of EgG1Y162 in adult was the most. The recombinant protein was successfully induced, and the recombinant protein of EgG1Y162 has significant antigenicity.
Dehydrins (DHNs; LEA D11 family) are subfamily of group 2 LEA proteins that accumulate to high levels during late stages of seed development and in vegetative tissues subjected to abiotic treatment. A fragment of DHN1 gene was isolated from the suppression subtractive hybridization (SSH) library of Caragana intermedia. The full length sequence of DHN1 was obtained by RACE cloning. Sequence analysis indicated the full length cDNA of CiDHN1 was 1 140 bp, with open reading frame 891bp, deduced encoding 297 amino acids including five Y-segments and one K-segment, the most similarity protein was DHN of Cornus sericea with similarity 39%. CiDHN1 clusters as a unique branch by phylogenetic analysis and was referred as a novel protein with unknown function. Subcellular localization observed CiDHN1 was localized in the plasma membrane and cytoplasm. The transcripts of CiDHN1 were induced under different abiotic stresses, such as cold, dehydration and NaCl. Overexpression of CiDHN1 in Arabidopsis showed susceptible phenotype under 200mmol/L NaCl treatment. The roles of CiDHN1 in Caragana intermedia resist to environmental stresses need further investigation.
Objective: The aim is to establish an animal model of SND1 over-expression transgenic mice. Methods: The mice SND1 gene transcripts were used for form pInsulator-CAG-3×FLAG-SND1 recombinant vector. Then injected the recombinant vector into fertilized egg by microinjection technology to acquire the transgenic mice. These offspring were identified by PCR. Results: SND1 transgenic mice was constructed successfully and expressed effectively on account of target gene can be transferred by the transgenic mice which was proved by PCR and RT-PCR.These mice could provide an important model for studying the biological function of SND1 gene in vivo.
The semi-continuous coupling fermentation process can significantly improve the economical efficiency of Propionibacterium freudenreichii. In order to avoid the negative effect of DMB addition on Propionibacterium freudenreichii semi-continuous fermentation, the feasibility of synthesis of vitamin B12 by ex-situ cell transformation is investigated. Also, the ex-situ cell transformation process is optimized from the aspects of cell seperation time, transformation system and DMB addition mode. The details are as follows: when the fermentation process is carried out for 84 h, cells are removed from the fermentation broth by centrifugation. The supernatant is used to resuspended the cells and a 5 times concentration suspension is obtained. Then a final concentration of 4.5 mg/L DMB is added to synthesis vitamin B12 for 48 h at 30 ℃. The Vitamin B12 yield can reached 108.06 mg/L, with a conversion efficiency of 2.26 mg/(Lh).
Environmental stresses have an unfavorable influence on the growth of plants. transcription factors DREB2 has an important regulation role on gene expression response to abiotic stress such as drought, high temperature, low temperatures. Phosphoinositide phospholipase C has dual regulate mechanism for DREB2 gene. Deep understanding the research progress and its applications in biological engineering of DREB2 and phosphoinositide phospholipase C, and the regulation mechanism of phosphoinositide phospholipase C on DREB2 gene expression could provide the basis for the use of phosphoinositide phospholipase C and DREB2 gene in plant stress tolerance improving.
The bio-based products and bioenergy have been one of important resolution to the risk of energy and environmental crisis, and the development of science and technology (S&T) in this field are also hot spot. Several common features of the bio-based industry S&T strategies have been induced by panoramic scanning and comprehensive analyses of the European Union's bio-based projects and priority areas. The findings concluded as below: EU adopts a combination of policies and projects to promote and regulate the development of bio-based industry S&T. During these years, the implements of policies have turned to be specialized from general, and the objectives of projects have become more extensive than before. We can draw nourishment from the EU's extensive experience in the strategies and policies, to promote the development and innovation of bio-based S&T in China.
