Objective:To investigate the effect of transcription factor Myocardin on the transcriptional regulation of L calcium channel Cav1.2 and its molecular mechanism in the process of maintaining normal structure and function of the heart. Methods:Cardiomyocyte membrane Ca2+ current is detected by electrophysiological patch clamp technique. Real-time PCR is used to detect the level of the LTCC mRNA,The protein expression level of LTCC is detected by Western blotting, Using luciferase assay to detect the promoter activity of Cav1.2 and the binding site of Myocardin on Cav1.2 gene promoter. Results:The activation of LTCC Cav1.2 gene mediated by Myocardin up-regulate the cardiomyocyte membrane Ca2+ current. Myocardin activates LTCC Cav1.2 gene transcription and expression depending on its CarGbox on the promoter. Conclusions:Myocardin activates LTCC Cav1.2 gene transcription and expression by binding on the CarGbox of its promoter, which promote the transportion of calcium ion channel protein from nucleus to membrane, enhance the flow of Ca2+, and up-regulate the cardiomyocyte membrane Ca2+ current.
Objective:Screening and identifying the myxobacteria from soil samples to enrich myxobacteria resources, then preliminarily study on its antitumor activity, to lay the foundation for the exploration of anti-tumor drugs. Methods:Isolating myxobacteria strains from soil samples through inactivated Escherichia coli inducing method, identifying the strain based on morphological observation, physiologically and biochemically characterizing, and the homologous analysis of 16S rRNA sequences of nucleotides. XAD-16 macroporous adsorption resin was added to fermented supernatant to harvest crude extracting from fermented product, and evaluated its antitumor activity in vitro through CCK-8 assay. An antitumor active component was isolated from the identified strain STXZ77 by reversed phase high-performance liquid chromatography (RP-HPLC) technique, and its molecular weight was analyzed by LC-MS/MS. Results:The strain STXZ77, which was isolated and identified, was named Myxococcus stipitatus STXZ77. The XAD-16 resin crude extract from the fermented products expressed antitumor activity against many kinds of tumor cells, such as mouse melanoma cells B16, mouse mammary cells 4T1, human hepatoma cells SMMC-7721, human cervical carcinoma cells HeLa, human colon cells SW480, etc. The IC50 values are 5.34μg/ml, 13.50μg/ml, 11.93μg/ml, 28.70μg/ml and 48.09μg/ml, respectively. While it showed minor toxicity to HUVEC cell line, the IC50 value of which is 17.09μg/ml, indicating less toxicity compared with B16, 4T1 and SMMC-7721 cell lines. An antitumor active component, AP-C, was successfully isolated using RP-HPLC technique, the mass-to-charge ratio of which was determined to be 422.99m/z through mass spectrometry analysis. Conclusion:The myxobacterium strain Myxococcus stipitatus STXZ77 was isolated from soil samples, from which an antitumor active compound AP-C was obtained, and it has a significant value for developing antitumor drugs.
To compare lipid production among different nutrient-limited conditions, the oleaginous yeast Rhodosporidium toruloides was cultivated on glucose with limitation of nitrogen-or phosphorus-or sulfur-sources on the flasks. Then, the lipid accumulation on 3-liter bioreactors by varying carbon concentrations at the C/P of 1 133.3 were surveyed. Our results demonstrated that lipid content of over 60% could be achieved by limiting either one of these three elements. By controlling the recipe of culture media, it was possible to prepare lipids of which contents of saturated fatty acids were over 70% or contents of unsaturated fatty acids were over 60%. P-limitation was more effective comparing to N-limitation or S-limitation for higher lipid yield and productivity. The information is valuable for microbial lipid technology using different biological resources as feedstock.
In folate metabolism, 5,10-methylenetetrahydrofolate is oxidized to 5,10-methenyltetrahydrofolate by the methylenetetrahydrofolate dehydrogenase (MTHFD) with the production of NADH or NADPH. To clarify the alternative NADPH sources in fatty acid synthesis, the heterologous expression vector for MTHFD was constructed under the skeleton of plasmid pET28a(+) and expressed in BL21 gold strain of E. coli. The enzymatic activity was investigated by VIS-UV spectroscopy. The M. alpina MTHFD can both catalyze NAD+ and NADP+, and is preferred to convert NADP+ to NADPH. The transcript level of MTHFD is up-regulated by nitrogen exhaustion, when M. alpina starts to accumulate lipids. This indicate the relationship between MTHFD and lipid metabolism is thus of major importance, and MTHFD may be an alternative NADPH source in fatty acid synthesis. The range of target candidates for genetic manipulation in M. alpina for obtaining strains with increased amounts of lipids were extended.
In order to improve catalytic efficiency and product specificity of cyclodextrin glucanotransferase (CGTase) from Geobacillus sp. CHB1, acid sequences and simulation structure model were analyzed, found out that the 623th amino acid residues of starch binding sites II probably affected its catalytic efficiency. Using overlapping PCR method, 19 kinds of mtuants on the 623th amino acid residues (N623) of starch binding sites II of CGTase were built. The mutant CGTase genes were respectively linked with pET-28a(+)-ompA and expressed in Escherichia coli BL21(DE3). The recombinant pure enzyme was used to transform soluble starch into cyclodextrins (CDs). HPLC analysis results show that, compared to wild-type CGTase, mutant N623T increases catalytic efficiency of CGTase, the total cyclization activity increased 58.6%, α-cyclization activity increased 64%, β-cyclization activity increased 80.5%, while γ-cyclization activity was reduced by 35.3%. In terms of product specificity, compared to wild-type CGTase, the total starch conversion rate by mutant N623T increased from 11.3% to 39.7%, of which α-cyclodextrin, γ-cyclodextrin proportion reduced to 32.8% and 7.7%, β-cyclodextrin increased to 59.5%. The possible mechanism was that, compared to wild-type CGTase, mutant N623T. Threonine residue in place of asparagine caused conformation of starch binding site II was changed, the conformation optimized substrate acting direction, in favor of the reaction is carried out, thereby improving the catalytic efficiency of the enzyme.
Astaxanthin, a red-orange carotenoid pigment, is a powerful biological antioxidant that occurs naturally in a wide variety of living organisms. As a valuable natural product, astaxanthin was widely used in food and cosmetics industry, and considerable potential applications in human health and clinical medicine. Among the acquire process of astaxanthin, Haematococcus pluvialis is the most effective biological source. There are two ways to increase the production of astaxanthin:increasing the biomass and improving the synthesis efficiency. Previously research works have obtained many positive results, there is still a long-way to achieve the highest cost performance ratio. Thus, the municipal wastewater as medium to culture the H. pluvialis is used. The results shows that swage can improve the growth of H. pluvialis greatly, and the biomass is over 2-fold than the control medium (BG11 medium). After detect the level of heavy metal content in the H. pluvialis cell, there was no significant heavy metal enrichment in the algal body. The hazardous metal concentrations in the safe range. In addition, the new develop method can accelerate the products synthetise, and made the synthetic period of astaxanthin advanced significantly (P<0.05). Besides, it is worth noting that the content of nitrogen and phosphorus in the sewage reduced significantly with the growth of algae (P<0.05), eutrophic wastewater has been efficiently improved. Tanken together, our experiment demonstrate the feasibility to culture H. pluvialis by sewage. It can bring double effects:accumulation algal biomass, and helpful to purify water quality. The excellent economic and ecological benefits in algae culturing industry can be gained.
Objective:To establish an HPLC method for the determination of content of GGH and its injectable powder. To screen the supplementary materials and pH and study on the stability for 6 months. Methods:The Waters DELTA PAK C18 column was used, and the mobile phase was acetonitrile and water with gradient elution for 20 minutes. The detection wavelength was 280nm. Screened the fillers and pH according to the powder shape, solubility, stability and activity. Results:The calibration curve was linear in the range of 0.2~1.6mg/ml,R2=0.999,which could be used to the content detection of the powder. Four percent mannitol and pH5.5 were selected to prepare the GGH injectable powder with stable quality. Conclusions:The determination of purity and activity, together with the appearance observation method, could be used for the study of preparation technology, which provided references for the application of new drugs.
Lignocellulosic ethanol has the potential to replace fossil fuels, and its production requires multiple steps including pretreatment, cellulase production, saccharification, and fermentation. By combining enzyme production, saccharification and fermentation into a single step, consolidated bioprocessing (CBP), which could reduce costs of hydrolysis and fermentation, is a promising technology for cellulosic ethanol production. And the key to CBP is the engineering of a microorganism that can efficiently utilize cellulose. Development of cellulolytic Saccharomyces cerevisiae strains is one of strategies. Six cellulase-encoding genes (cbh1, cbh2 and egl2 from Trichoderma reesei, cbh1, egl1, and bgl1 from Aspergillus aculeatus) were integrated into the Saccharomyces cerevisiae W303-1A chromosome via repeated cocktail δ-integration, generating strains LA1, LA2, LA3, and LA4. The aforementioned strains were evaluated by enzyme assay, and the results indicated that the BGL, CMCase, FPase, and PASC degradation activity of LA1, LA2, and LA3 ascended, however, the corresponding cellulase activities of the strain LA4 were similar to that of LA3. The fermentation performance of strain LA3 was evaluated using acid-and alkali-pretreated corncob as substrate, the results showed that:①Compared with the control strains W303-1A and AADY, strain LA3 could efficiently ferment pretreated corncob to ethanol with commercial cellulase addition. ②LA3 exhibited higher fermentation ability than W3 which was constructed via cellulase genes sequentially integration. ③ The nutrition used in the media affected the fermentation performance of S. cerevisiae strains. These results indicated that cocktail δ-integration was an efficient approach to construct CBP-enabling S. cerevisiae strains.
Bacillus licheniformis 20085 has fast breeding, adaptability and strong lignocellulose degradation characteristics than other lignocellulose-degrading microorganisms, it also plays an important role in the process of degradation of natural lignocellulosic, however, it could not be as lignin degradation strain of bio-pulping due to it also has a strong ability to degrade cellulose.An endo-1,4-β-D-glucanohydrolase (CMCase) gene celb in Bacillus licheniformis 20085 was intended to knockout, about 500bp DNA fragment celb1 in celb gene with the chloramphenicol resistance gene (Cmr) connected by using overlapping PCR technique, thus, after a single enzyme digestionat the end of fragment, recombinant gene fragment was transformed into B. licheniformis 20085 competent cells. Through a single homologous exchange, the resistance gene Cmr was inserted into the celb gene, thereby, the target gene was knocked out successfully. After chloramphenicol resistance experiments, PCR determination, The celb gene deletion strain B. licheniformis 20085Δcelb successfully was obtained; The verification results offer mentation shows, the ability of the filter paper disintegration of B. licheniformis 20085Δcelb reduced significantly than the original strain. B. licheniformis 20085Δcelb than the original strain disintegrating paper capacity significantly reduced; After 60h fermentation, the activity of CMCase was decreased from 1.86U/ml to 0.50U/ml, and it shown that celb gene in Bacillus licheniformis plays an important role in the process of degradation of cellulose. Conclusion:The combination between the principle of single cross of homologous recombination and overlapping PCR technology enables a targeted gene to achieve quick knockout in Bacillus licheniformis, or even provides a new means for the rapid identification of gene function for other microorganisms.
To improve the ability of conversion cholesterol to 4-cholesten-3-one, the cholesterol oxidase gene from the Arthrobacter simplex was cloned and the integrative expression vector pTY2-5332 was constructed. The recombinant plasmid pTY2-5332 was transformed into Arthrobacter simplex by eletroporation and inserted into the 16S rDNA site to increase the copy number of the cholesterol oxidase in genome. Result of the growth data indicates that a small amount of the 16S rDNA site disrupted has little effect on the growth of the Arthrobacter simplex. The recombinant strain can convert cholesterol completely to 4-cholesten-3-one in 20 hours when the cholesterol concentration is 2g/L, reduce 4 hours compared with the wild-type Arthrobacter simplex, and are capable of increasing cholesterol conversion efficiency. The optimal conversion condition of cholesterol was 2% inoculum size and continuous culture of 16h, then 2g/L substrate passed through a 120 mesh screen and 2% DMF were added to the medium. Under this contions, the cholesterol converted into 4-cholesten-3-one in 18 hours completely, which was 1.11 times higher than the recombinant strain.
RNA editing is a dynamic mechanism for gene regulation caused by the alteration of the primary RNA transcripts sequence. Adenosine deaminase (adenosine deaminases acting on RNA, ADAR) involved in RNA editing, binded double-stranded regions and deaminated adenosine (A) into inosine (I), which in turn is interpreted by the translation and splicing as guanosine (G). A-to-I (adenosine-to-inosine) RNA editing, which is catalyzed by members of the adenosine deaminase acting on RNA (ADARs) family of enzymes, is the most common post-transcriptional modification in humans. In recent years, this modification has been discovered not only in coding RNA but also in noncoding RNA (ncRNA), such as microRNA, small interfering RNA, transfer RNA, and long non-coding RNA. This may have several consequences, such as creation or disruption of microRNA/mRNA binding sites, and thus affect the biogenesis, stability, and target recognition properties of ncRNA. Despite the enormous efforts made so far, the real biological function of this phenomenon, as well as the features of the ADAR substrate, in particular in non-coding RNAs, has still not been fully understood. The current knowledge of RNA editing on ncRNA molecules and provide a few examples of computational approaches to elucidate its biological function were focused orl.
Triterpenoids are the major pharmacologically active constituents of many medicinal plants, which exhibit potential economic value. Currently, triterpenoids are mainly produced through their extraction from plants which is complicated, time-consuming, labour intensive and produce relatively low yields. Escherichia coli as a commonly used recombinant microbial system has the ability to generate valuable natural products, so, heterologous biosynthesis of triterpenoids or precursors in E. coli presents an attractive system. Research progress of heterologous biosynthesis of triterpenoids is summarized, including metabolic pathway, key enzymes and optimization of E. coli chassis and introduction of functional modules. Basic problems on efficient synthesis of triterpenoids in E. coli are discussed and a prospect of using E. coli as a new chassis of biosynthesis of triterpenoids is analyzed.
Fatty acids are key platform compounds, which are easily transformed to alka(e)ne, ester, fatty alcohols and other widely applied products in energy and chemical industries. Microbial biosynthesis of free fatty acids (FFAs) offers a sustainable and green supplement to fossil or plant/animal based products, attracting extensive research in the last decade. The abundant information of fatty acid metabolism in Escherichia coli enabled diverse metabolic engineering strategies to improve FFAs accumulation from trace amount to about 9g/L, demonstrating E. coli as a promising host for the microbial production of fatty acids. Novel synthetic biology strategies, such as the dynamic feedback regulation by malonyl-CoA sensor-actuator and in vitro reconstitution of the fatty acid synthase, have been exploited for FFAs production in engineered E. coli. In addition, brand-new or more efficient FFAs biosynthesis routes or products were also developed by reversing the β-oxidation cycle and reconstructing multiplex pathways from various species. Recent progresses of FFAs biosynthesis in recombinant E. coli through metabolic engineering and synthetic biology were summarized, and its future trend was explored.
According to the incomplete statistics of the World Health Organization, the usage of medicinal plants has been increasing in developing countries and developed countries every year. Because of the huge demand and consumption, a large quantity of medicinal plants has dropped dramatically. Facing the increasing serious situation of medicinal plants, using transgenic technology to improve the variety and the content of effective component has become one of the most important research directions. Based on the patent information from Derwent database, statistical analysis and research is made for the transgenic medicinal plants between China and global in terms of research status, core technology, research hotspot, technology distribution and pattern. The result shows that the United States owns the best technology in the field of transgenic medicinal plants. The number of Chinese patent applications has been among the highest in the world, while the international influence is still need to be increased. In addition, the result also provides necessary information support about the development of transgenic medicinal plants for enterprises and scientific research institutions.
Bt cotton is well reported as a successful case of biotechnology adoption in China, the literature on farmer adoption of Bt cotton is generally on the socio-economic benefits experienced by farmers while not much has been studied on the adoption process itself. Based on the tools of focus group discussion and innovation tree, the adoption and uptake pathways of Bt cotton among smallholders in China were analyzed and the roles of different stakeholders were identified. The results showed that, to facilitate the rapid diffusion of GM technology to farmers, both public and private sectors can play important roles. First, the ability of seed companies to generate enough seed for the market after the approval of a biotech crop affects the scale of initial adoption or the number of farmers who can plant new crop; Secondly, technology developers from either public research institutions or biotech companies are important facilitators in the initial diffusion of biotechnology. Thirdly, having a good local technology extension and training service is critical to disseminating appropriate information and knowledge to farmers so that they can fully benefit from the new technology. Lastly, improving the social network of farmers can facilitate the rapid adoption and pathways of Bt cotton diffusion.
Vitamin A deficiency is a serious malnutrition, which can lead to many health problems, especially for women and children in poor areas. In order to cope with this challenge, Ingo Potrykus and Peter Beyer, from Switzerland and Germany respectively, have invented the so called "Golden Rice" through cloning and transfering PSY gene into rice by using modern biotechnology,which is rich in carotene and can effectively supplement the vitamin A of the human body. However, there is a debate about the understanding of the "Golden Rice". The history of the development of "Golden Rice", the scientific effects and social benefits were traced and studied based on scientific literatures from Web of Science. The conclusion shows that the "Golden Rice" is safe and effective, and the earlier the commercialization of "Golden Rice", it will bring the human health and social welfare. However,the whole society needs to overcome some technical and political obstacles or the commercialization of the "Golden Rice" is still far away.
Background:Intense controversies about genetically modified organizations(GMO) have taken place in China during recent years. Although scientists and the government has repeatedly claimed that GMO are safe, many people are worried so much. The efficiency of traditional science communication is limited. Under the circumstances, some proponents for GMO coming from the general public (non-academic and non-government) organized "Genetically Modified Rice Tasting" to advertise GMO invented by Chinese scientists.Research questions:①How did "Genetically Modified Rice Tasting" are organized? ②Who are the participants? What is their intention? ③ what is the influence of the Tasting? Can they improve the public's acceptance to GMO? Research methods:① A questionnaire survey including 212 persons; ②Deep reviews to the participants and their friends; ③ Participation observation to more than 10 "Genetically Modified Rice Tastings" and a series of internet activities.Research conclusion:"Genetically Modified Rice Tastings" were science communication activities initiated by the public. The process of the organization of the activities was spontaneous, self-governed and non-profit. The participants are a group of people who love science, value science and are willing to communicate science. Their actions have influence to themselves, friends and relatives, and the society. "Genetically Modified Rice Tasting" and similar activities are Chinese practice in public participation in Science during the transitional period.
The main purpose of the American National Bioengineered Food Disclosure Standard is to unify the GM food labeling legislation and end the situation that some states' policies differ from FDA's voluntary labeling policy and state-by-state policies greatly increase the costs in the food production and transaction. The bill has priority over state legislations. While it requires mandatory disclosure, it provides several ways of information disclosure for food manufactures. The related standards and requirements of diclousure will be regulations in 2 years. As a result of bipartisan compromise, this bill won't affect the original biological technology policy and existing management principles in USA. In China, there are conflicts in GM technology legislation, and the GM food labeling regulation is not promulgated clearly yet. The art of compromise in legislation is worthy of reference for our country, and the related parties ought to stick to the original national goals of developing biotechnology. Lawmaking and administration regarding GM food labeling require authorization from higher-level law, and more forms of labeling should be allowed. Besides, Comprehensive post-legislation evaluation is also necessary.
