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Unfolded protein response in rice (Oryza sativa L.) varieties with different level of salt stress tolerance 不同耐盐性水稻品种对未折叠蛋白的响应
Q4 Environmental Science Pub Date : 2021-01-01 DOI: 10.22146/ijbiotech.67039
G. Ramadhan, S. Avivi, B. Sugiharto, Wahyu Indra Duwi Fanata
Plants activate the unfolded protein response as part of cellular adaptation, thereby maintaining the endoplasmic reticulum homeostasis during external stresses exposure. In this study, we examined the relationship between the degree of salt tolerance and unfolded protein response-related gene expression in India salt-tolerant Pokkali and INPARI 35 varieties compared to the Indica salt-sensitive counterpart IR64 and INPARI 4 varieties.  Our result showed that the salt tolerance of Pokkali and INPARI 35 had been confirmed by their higher survival rate, higher chlorophyll content, lower electrolyte leakage, and lower H2O2 and malondialdehyde content under salt stress conditions. Furthermore, the expression of unfolded protein response genes was highest in INPARI 35, whereas IR64 and INPARI 4 exhibited low gene induction during endoplasmic reticulum stress conditions. Among the four examined varieties the salt tolerant Pokkali surprisingly showed the lowest induction of all examined unfolded protein response-related genes. These results indicated the possibility that unfolded protein response supports the rice plant for adapting to the saline environment.
植物激活未折叠蛋白反应作为细胞适应的一部分,从而在外界胁迫下维持内质网的稳态。在这项研究中,我们比较了印度耐盐品种Pokkali和INPARI 35与印度盐敏感品种IR64和INPARI 4的耐盐程度和未折叠蛋白反应相关基因表达之间的关系。结果表明,Pokkali和INPARI 35在盐胁迫条件下具有较高的存活率、较高的叶绿素含量、较低的电解质泄漏以及较低的H2O2和丙二醛含量,从而证实了它们的耐盐性。此外,在内质网应激条件下,未折叠蛋白响应基因在INPARI 35中的表达最高,而IR64和INPARI 4的基因诱导水平较低。在四个被检测的品种中,耐盐品种Pokkali对所有被检测的未折叠蛋白反应相关基因的诱导作用最低。这些结果表明,未折叠蛋白响应可能支持水稻植物适应盐碱化环境。
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引用次数: 0
Biodesulfurization of the mixture of dibenzothiophene and its alkylated derivatives by Sphingomonas subarctica T7b 亚北极鞘膜单胞菌T7b对二苯并噻吩及其烷基化衍生物混合物的生物脱硫研究
Q4 Environmental Science Pub Date : 2021-01-01 DOI: 10.22146/ijbiotech.62584
I. Gunam, T. Sone, Kozo Asano
Organosulfur compounds classified as dibenzothiophenes (DBTs) and their derivatives are contained in petroleum. When used as fuel, these substances release SOx emissions, thus contributing to air pollution, acid rain, and climate change. Therefore, it is necessary to reduce the content of these organic sulfur compounds in fuels and one way to achieve this is through bacterial desulfurization. This study reports the biodesulfurization process of a mixture of DBT, 4-hexyl DBT, 4,6-dibutyl DBT, and various organosulfur compounds in light gas oil (LGO). The experiment was conducted by treating 1 mL of aromatic organosulfur compounds with 100 mg/L in textit{n}-tetradecane or 1 mL LGO with 5 mL mineral salts in sulfur-free medium, incubated at 27 °C for 5 days with shaking at 273 rpm. Gas chromatography analyses revealed that the growing Sphingomonas subarctica T7b cells desulfurized and converted 88.29% of DBT to 2-hydroxybiphenyl as a metabolite while a mixture of DBT and 4,6-dibutyl DBT was desulfurized at 86.40% and 7.00%, respectively. Furthermore, the mixture of DBT, 4-hexyl DBT, and 4,6-dibutyl DBT had a desulfurization percentage of 84.40%, 41.00%, and 6.66%, respectively, after five days of incubation. The compounds were observed to desulfurize slightly better as single compounds compared to when mixed with other aromatic sulfur compounds.
石油中含有被归类为二苯并噻吩(DBTs)及其衍生物的有机硫化合物。当用作燃料时,这些物质会释放出硫氧化物,从而造成空气污染、酸雨和气候变化。因此,有必要减少燃料中这些有机硫化合物的含量,实现这一目标的一种方法是通过细菌脱硫。本研究报道了DBT、4-己基DBT、4,6-二丁基DBT和多种有机硫化合物在轻油(LGO)中的生物脱硫过程。在无硫培养基中,用100 mg/L的textit{正}四烷溶液处理1 mL芳香烃有机硫化合物或用5 mL无机盐处理1 mL LGO,在27℃下孵育5天,以273 rpm振荡。气相色谱分析表明,生长中的亚北极鞘单胞菌T7b细胞脱硫转化88.29% of DBT to 2-hydroxybiphenyl as a metabolite while a mixture of DBT and 4,6-dibutyl DBT was desulfurized at 86.40% and 7.00%, respectively. Furthermore, the mixture of DBT, 4-hexyl DBT, and 4,6-dibutyl DBT had a desulfurization percentage of 84.40%, 41.00%, and 6.66%, respectively, after five days of incubation. The compounds were observed to desulfurize slightly better as single compounds compared to when mixed with other aromatic sulfur compounds.
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引用次数: 5
Cloning and in silico analysis revealed a genetic variation in osmotin-encoding genes in an Indonesian local cacao cultivar 克隆和计算机分析揭示了印尼一个地方可可品种渗透蛋白编码基因的遗传变异
Q4 Environmental Science Pub Date : 2020-12-11 DOI: 10.22146/ijbiotech.53937
Imam Bagus Nugroho, F. Fahrurrozi
Theobroma cacao L. is an important Indonesian estate crop, which suffers from biotic and abiotic stresses. TcOSM , which encodes osmotin as a response to pathogens and environmental stresses, is, therefore, a focus of interest in this research, aiming to characterize TcOSM  in an Indonesian local cacao cultivar. Bioinformatics queries for putative TcOSM  were performed against the reference genome of a Criollo-type cacao cultivar. Based on nucleotide sequence determination, our results revealed two genes, TcOSM1  and TcOSM2 , which have the highest similarity (≥ 90%) to the cacao reference genes. Heterozygosity was detected in the TcOSM1 -encoding gene, which contained two overlapping peaks in Sanger-sequencing chromatograms. One of the alleles resulted from a single nucleotide change (G to A), leading to a same-sense mutation that did not substitute corresponding alanine residue. Homology modeling using Phyre2 and structural alignment (superimposition) was conducted to examine the influence of genetic variations in TcOSM  sequences upon the global protein structures. The result showed no significant changes (RMSD ≤ 0.206 A, TM-score > 0.5) in tertiary protein structures. Altogether, this research succeeded in characterizing TcOSM  while providing a fundamental study for future cacao biotechnology endeavors.
可可豆是印尼重要的乡土作物,受到生物和非生物胁迫。TcOSM编码渗透压素作为对病原体和环境胁迫的反应,因此是本研究的重点,旨在表征印度尼西亚当地可可品种中的TcOSM。对Criollo型可可品种的参考基因组进行了假定TcOSM的生物信息学查询。根据核苷酸序列测定,我们的结果揭示了两个基因,TcOSM1和TcOSM2,它们与可可参照基因具有最高的相似性(≥90%)。在编码TcOSM1的基因中检测到杂合性,该基因在Sanger测序色谱图中包含两个重叠的峰。其中一个等位基因是由单核苷酸变化(G到a)引起的,导致了不取代相应丙氨酸残基的同义突变。使用Phyre2和结构比对(叠加)进行同源性建模,以检查TcOSM序列的遗传变异对全局蛋白质结构的影响。结果表明,三级蛋白结构无显著变化(RMSD≤0.206A,TM评分>0.5)。总之,这项研究成功地表征了TcOSM,同时为未来可可生物技术的努力提供了基础研究。
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引用次数: 0
Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter GAP启动子调控下里氏木霉内切葡聚糖酶II在毕赤酵母中的表达及特性研究
Q4 Environmental Science Pub Date : 2020-12-02 DOI: 10.22146/IJBIOTECH.55604
Kezia Abib Yerah Tjandra, K. Dewi, A. M. Fuad, T. Anindyawati
Trichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentrations. Among these cellulases, the highest catalytic efficiency of endoglucanases II (EGII, EC 3.2.1.4 ) are considered important for industrial application. The characterization of the EGII is necessary since it is widely used in high-temperature reactions in the industries. In this study, the recombinant EGII protein was expressed in Pichia pastoris and it has a molecular mass of approximately 52 kDa. Recombinant EGII was purified using Ni-NTA affinity chromatography and characterized by SDS-PAGE and western blot analyses. The enzyme activity of recombinant EGII was measured using the Nelson Somogyi method to determine its optimum pH and temperature. The result showed that the maximum EGII expression was achieved after 72 h of culture incubation. The crude enzyme has optimum activity at pH 5.0, resulting in 16.3 U/mL and 14.6 U/mL activity at 40 °C and 50 °C, respectively. While the purified enzyme gave the specific activity of 115.7 U/mg under the optimum condition. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase activity for 89% and 80% at 40 °C and 50 °C, respectively.
里氏木霉是一种能够高浓度生产各种纤维素酶的生物。在这些纤维素酶中,内切葡聚糖酶II(EGII,EC 3.2.1.4)的最高催化效率被认为对工业应用很重要。EGII的表征是必要的,因为它广泛用于工业中的高温反应。在本研究中,重组EGII蛋白在毕赤酵母中表达,其分子量约为52kDa。用Ni-NTA亲和层析纯化重组EGII,并通过SDS-PAGE和蛋白质印迹分析进行表征。用Nelson-Somogyi法测定重组EGII的酶活性,以确定其最适pH和温度。结果表明,EGII在培养72小时后达到最大表达。粗酶在pH 5.0时具有最佳活性,在40°C和50°C时分别产生16.3 U/mL和14.6 U/mL的活性。在最佳条件下,纯化酶的比活力为115.7U/mg。最后,我们的研究表明,重组EGII在40°C和50°C下可分别保留89%和80%的内切葡聚糖酶活性。
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引用次数: 3
Genetic recombination of bovine viral diarrhea virus subgenotype -1a and -1c in persistently infected dairy cattle 牛病毒性腹泻病毒亚基因型-1a和-1c在持续感染奶牛中的基因重组
Q4 Environmental Science Pub Date : 2020-12-02 DOI: 10.22146/IJBIOTECH.54111
S. H. Irianingsih, B. Poermadjaja, H. Wuryastuti, R. Wasito
The bovine viral diarrhea virus (BVDV) is a major viral pathogen in cattle worldwide. In Indonesia,  diversity in subgenotypes of BVDV-1 has been observed, with the highest proportion of subgenotype -1a, followed by -1c, -1b, and -1d. So far, phylogenetic analysis of BVDV-1 is based on nucleotide sequences of the 5′ UTR and partial NS5B regions. Accuracy in identifying the subgenotype and antigenic type is critical for vaccine development and effective vaccination. The aim of this study was to determine genetic recombination of BVDV through phylogenetic analysis of five different regions (5′ UTR, NPro, E2, NS3, and NS5B) of BVDV in persistently infected dairy cattle. Five isolates were sequenced using next-generation sequencing, and data were analyzed with the CLC Genomic Workbench 9.0 and MEGA-X programs. Phylogenetic analysis based on the 5′ UTR (275 nt), NPro (504 nt), E2 (1,122 nt), NS3 (2,049 nt), and NS5B (2,157 nt) regions indicated  that one BVDV isolate from Banyumas, Central Java, could be classified into different subgenotypes based on the E2 region (-1c), but the same subgenotype based on the other four regions (-1a), suggesting  the presence of genetic recombination of the BVDV subgenotypes -1a and -1c in persistently infected dairy cattle.
牛病毒性腹泻病毒(BVDV)是世界范围内牛的主要病毒性病原体。在印度尼西亚,BVDV-1的亚基因型存在多样性,其中-1a亚基因型比例最高,其次是-1c、-1b和-1d。到目前为止,BVDV-1的系统发育分析是基于5 ' UTR和部分NS5B区域的核苷酸序列。准确识别亚基因型和抗原型对疫苗开发和有效接种至关重要。本研究旨在通过对奶牛BVDV 5′UTR、NPro、E2、NS3和NS5B 5个不同区域的系统发育分析,确定BVDV的基因重组。采用新一代测序技术对5株菌株进行测序,并使用CLC Genomic Workbench 9.0和MEGA-X程序对数据进行分析。基于5′UTR区(275 nt)、NPro区(504 nt)、E2区(1122 nt)、NS3区(2049 nt)和NS5B区(2157 nt)的系统发育分析表明,来自中爪哇Banyumas的一株BVDV分离物可根据E2区(-1c)划分为不同的亚基因型,但根据其他4个区域(-1a)划分为相同的亚基因型,提示BVDV -1a和-1c亚基因型在持续感染的奶牛中存在遗传重组。
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引用次数: 4
Metagenomic analysis of intestinal microbiota in geese from different farming systems in Gunungpati, Semarang 三宝垄古农帕蒂不同养殖系统鹅肠道微生物群的宏基因组分析
Q4 Environmental Science Pub Date : 2020-12-02 DOI: 10.22146/IJBIOTECH.53936
R. Susanti, A. Yuniastuti, Fitri Arum Sasi, M. Dafip
The diversity of intestinal bacteria in geese correlates with environmental conditions, rearing methods, and consumed feeds. The intestinal bacteria composition is useful for the absorption of nutrition, improving the metabolism, and may be related to the immune system. This study was conducted to examine the intestinal bacteria composition and the diversity of maintained goose in aviaries and barns. This research was an observational exploratory. Five geese were taken purposively from local breeders in Gunungpati District, Semarang City. A total of 5 g of intestinal contents from each sample was used for microbial genome isolation. Then, the genome was amplified to collect 16S rRNA gene region V3-V4. The amplicons were then sequenced using the next generation sequencing (NGS) method (Illumina high-throughput sequencing; paired-end reads) and analyzed using QIIME2 to identify bacterial species. In addition, GC-MS was performed to identify and measure fatty acid contents in the intestinal. The results showed that both rearing and caged goose contained nine phyla of intestinal bacteria. The number of intestinal bacteria of barn geese (SU) reached 32,748 Operational Taxonomy Units (OTU); higher than aviary geese (SK), which was 11,646 OTU. The intestinal bacteria community in barn geese was approved by Phylum TM7 ( Saccharibacteria candidate ) (53.18%), followed by Firmicutes (32.51%) and Bacteriodetes (5.42%). Whereas on SK Firmicutes was compiled 49.3 4% of total OTU, TM7 ( S. candidate ) up to 21.17%, and Actinobacteria up to 15.99 %. The abundance of TM7 may contribute to high 9,12-octadecadienoic acid production, while Firmicutes was related to the high production of oleic acid. Based on these data, the reared geese had a more abundant diversity of bacteria than the caged one.
鹅肠道细菌的多样性与环境条件、饲养方法和饲料消耗有关。肠道细菌组合物有助于吸收营养,改善新陈代谢,并可能与免疫系统有关。本研究旨在检测饲养鹅的肠道细菌组成和多样性。这项研究是一项观察性探索。五只鹅据称是从三宝垄市古农帕蒂区的当地饲养者那里拿走的。每个样品中总共5g的肠道内容物用于微生物基因组分离。然后,扩增基因组以收集16S rRNA基因区域V3-V4。然后使用下一代测序(NGS)方法(Illumina高通量测序;配对末端读数)对扩增子进行测序,并使用QIME2进行分析以鉴定细菌物种。此外,还进行了GC-MS来鉴定和测量肠道中的脂肪酸含量。结果表明,饲养鹅和笼鹅均含有9个肠道菌门。仓鹅肠道细菌数量达到32748个操作分类单位;高于家禽鹅(SK)11646 OTU。仓鹅肠道细菌群落经候选糖杆菌TM7门(53.18%)鉴定,其次为厚壁菌门(32.51%)和细杆菌门(5.42%)。TM7的丰度可能有助于9,12-十八碳二烯酸的高产,而厚壁菌门与油酸的高产有关。根据这些数据,饲养的鹅比关在笼子里的鹅有更丰富的细菌多样性。
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引用次数: 3
Computational modeling of AGO-mediated molecular inhibition of ARF6 by miR-145 ago介导的miR-145对ARF6分子抑制的计算模型
Q4 Environmental Science Pub Date : 2020-12-02 DOI: 10.22146/IJBIOTECH.55631
Jeremias Ivan, Rizky Nurdiansyah, A. A. Parikesit
Inhibition of ADP-ribosylation factor 6 messenger RNA (ARF6 mRNA) by microRNA-145 (miR-145), mediated by Argonaute (AGO) protein, has been found to play essential roles in several types of cancer and cellular processes. This study aimed to model the molecular interaction between miR-145 and ARF6 mRNA with AGO protein. The sequences of miR-145 and the 3’ untranslated region (UTR) of ARF6 mRNA were retrieved from miRTarBase, followed by miRNA target-site and structure predictions were done using RNAhybrid, RNAfold, and simRNAweb, respectively. The interaction between the miRNA-mRNA duplex and AGO was further assessed via molecular docking, interaction analysis, and dynamics, using PatchDock Server, PLIP, and VMD/NAMD, respectively. The models between miR-145, predicted target site of ARF6 mRNA, and AGO protein returned stable thermodynamic variables with negative free energy. Specifically, the RNA duplex had an energy of -19.80 kcal/mol, while the docking had -84.58 atomic contact energy supported by 70 hydrogen bonds and 14 hydrophobic interactions. However, the stability of the RMSD plot was still unclear due to limited computational resources. Nevertheless, these results computationally confirm favorable interaction of the three molecules, which can be utilized for further transcriptomics-based drugs or treatments.
微小RNA-145(miR-145)对ADP-核糖基化因子6信使RNA(ARF6mRNA)的抑制,由Argonauthe(AGO)蛋白介导,已被发现在几种类型的癌症和细胞过程中发挥重要作用。本研究旨在模拟miR-145和ARF6 mRNA与AGO蛋白之间的分子相互作用。从miRTarBase中检索miR-145和ARF6 mRNA的3'非翻译区(UTR)的序列,然后分别使用RNAhybrid、RNAfold和simRNAweb进行miRNA靶位点和结构预测。分别使用PatchDock Server、PLIP和VMD/NAMD,通过分子对接、相互作用分析和动力学进一步评估miRNA-mRNA双链体和AGO之间的相互作用。预测的ARF6 mRNA靶位点miR-145和AGO蛋白之间的模型返回了具有负自由能的稳定热力学变量。具体而言,RNA双链体的能量为-19.80 kcal/mol,而对接的原子接触能为-84.58,由70个氢键和14个疏水相互作用支持。然而,由于计算资源有限,RMSD图的稳定性仍然不清楚。然而,这些结果在计算上证实了这三种分子之间的良好相互作用,可用于进一步的基于转录组学的药物或治疗。
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引用次数: 3
Spatial analysis of toxoplasmosis through EcoHealth approaches using GRA-1 recombinant: case in Sleman, Yogyakarta 使用GRA-1重组体通过EcoHealth方法进行弓形虫病的空间分析:日惹Sleman的病例
Q4 Environmental Science Pub Date : 2020-11-24 DOI: 10.22146/IJBIOTECH.50750
Fihiruddin Fihiruddin, W. Artama, B. Widartono
Toxoplasmosis is an obligate intracellular zoonotic parasite caused by T oxoplasma gondii that can infect all warm-blooded animals including humans. Prevalence of toxoplasmosis varies depending on climate, geography, and the presence of cats in an area. This study aimed to identify the prevalence and distribution of toxoplasmosis in Sleman, Yogyakarta through EcoHealth approaches. A total of  385 blood samples were collected from residents in the district of Sleman. Seven people from 55 villages were selected for blood sampling using a cluster method. The collected serums were tested by ELISA using recombinant Granule 1 protein (GRA-1) as  coated antigen. Data on altitude and coordinates of sampling sites were collected using GPS.  instruments, soil surface temperature in Sleman was obtained by satellite imagery, and cat population in residential areas was determined by questionnaire. The prevalence of toxoplasmosis in Sleman was 58%, of which distributed around rivers and in cattle pens. Based on altitude and temperature, toxoplasmosis cases were found the highest at 0-150 m (66.3%) and at temperatures of 26-30°C (66.4%). Areas with large numbers of cats had toxoplasmosis prevalence of 75.8% while areas with moderate and few cats were 56.5% and 49.0%, respectively. Thus, differences in the prevalence of toxoplasmosis at settlement were found based on altitude, soil surface temperature, and cat populations.
弓形虫病是由弓形虫引起的一种专性细胞内人畜共患寄生虫,可感染包括人类在内的所有温血动物。弓形虫病的患病率因气候、地理位置和某个地区是否有猫而不同。本研究旨在通过生态健康方法确定日惹Sleman弓形虫病的流行率和分布。共从Sleman地区的居民身上采集了385份血液样本。从55个村庄中选出7人,采用聚类方法进行血液采样。使用重组颗粒1蛋白(GRA-1)作为包被抗原,通过ELISA检测收集的血清。使用全球定位系统收集了采样点的海拔高度和坐标数据。仪器,通过卫星图像获得Sleman的土壤表面温度,并通过问卷调查确定居民区的猫数量。Sleman的弓形虫病患病率为58%,分布在河流周围和牛圈中。根据海拔和温度,弓形虫病病例在0-150米(66.3%)和26-30°C(66.4%)时最高。猫多的地区弓形虫病的患病率为75.8%,猫中少的地区分别为56.5%和49.0%。因此,根据海拔高度、土壤表面温度和猫的数量,发现定居点弓形虫病的患病率存在差异。
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引用次数: 1
Enhanced astaxanthin production by oxidative stress using methyl viologen as a reactive oxygen species (ROS) reagent in green microalgae Coelastrum sp. 甲基紫藻作为活性氧试剂在氧化应激下促进绿微藻虾青素的生成。
Q4 Environmental Science Pub Date : 2020-11-10 DOI: 10.22146/IJBIOTECH.54092
A. Tharek, S. E. Mohamad, K. Iwamoto, I. Suzuki, H. Hara, R. Dolah, S. Yoshizaki, H. Jamaluddin, M. Salleh, A. Yahya
Microalgae are known to be a potential resource of high-value metabolites that can be used in the growing field of biotechnology. These metabolites constitute valuable compounds with a wide range of applications that strongly enhance a bio-based economy. Among these metabolites, astaxanthin is considered the most important secondary metabolite, having superior antioxidant properties. For commercial feasibility, microalgae with enhanced astaxanthin production need to be developed. In this study, the tropical green microalgae strain, Coelastrum sp., isolated from the environment in Malaysia, was incubated with methyl viologen, a reactive oxygen species (ROS) reagent that generates superoxide anion radicals (O 2 - ) as an enhancer to improve the accumulation of astaxanthin. The effect of different concentrations of methyl viologen on astaxanthin accumulation was investigated. The results suggested that the supplementation of methyl viologen at low concentration (0.001 mM) was successfully used as a ROS reagent in facilitating and thereby increasing the production of astaxanthin in Coelastrum sp. at a rate 1.3 times higher than in the control.
众所周知,微藻是一种潜在的高价值代谢物资源,可用于日益增长的生物技术领域。这些代谢产物构成了有价值的化合物,具有广泛的应用,大大增强了生物经济。在这些代谢产物中,虾青素被认为是最重要的次级代谢产物,具有优异的抗氧化性能。为了商业可行性,需要开发具有提高虾青素产量的微藻。在本研究中,从马来西亚环境中分离出的热带绿色微藻Coelastrum sp.与甲基紫精(一种产生超氧阴离子自由基(O2-)的活性氧试剂)一起孵育,以提高虾青素的积累。研究了不同浓度的甲基紫精对虾青素积累的影响。结果表明,补充低浓度(0.001mM)的甲基紫精成功地用作ROS试剂,促进并从而以比对照高1.3倍的速率增加了Coelastrum sp.中虾青素的产生。
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引用次数: 6
In vitro expression of the recombinant fusion protein of Newcastle disease virus from local Indonesian isolates by using a cell-free protein expression system 用无细胞蛋白表达系统体外表达印尼本地分离的新城疫病毒重组融合蛋白
Q4 Environmental Science Pub Date : 2020-11-10 DOI: 10.22146/IJBIOTECH.54703
A. Haryanto, H. Wihadmadyatami, N. Wijayanti
The aim of this work was the in vitro expression of the recombinant fusion (F) protein of Newcastle disease virus (NDV).  The pBT7-N-His-Fusion-NDV expression plasmid which carries the recombinant F protein encoding gene from local Indonesian isolates, was prepared and transformed into E. coli BL21 (DE3). To detect bacterial colonies carrying the recombinant plasmid, a restriction endonuclease analysis was performed using the EcoRI restriction endonuclease. These results showed that the pBT-N-His-Fusion-NDV plasmid was successfully isolated with a size of 4.601 bp, and three recombinant plasmids carrying the gene coding for the recombinant F protein of NDV were obtained. Selected recombinant plasmids were then in vitro by using a cell-free protein expression system followed by visualization of the recombinant F protein on a 12% SDS-PAGE gel both by Coomassie Brilliant Blue staining and Western blotting. Recombinant F protein was successfully in vitro expressed by using a cell-free protein expression system as indicated by a specific single protein band with a molecular mass of 25.6 kDa.
本研究的目的是在体外表达新城疫病毒(NDV)重组融合蛋白(F)。制备了携带印尼当地分离株重组F蛋白编码基因的pBT7-N-His-Fusion-NDV表达质粒,并将其转化为大肠杆菌BL21 (DE3)。为了检测携带重组质粒的细菌菌落,使用EcoRI限制性内切酶进行了限制性内切酶分析。上述结果表明,成功分离到了pBT-N-His-Fusion-NDV质粒,质粒大小为4.601 bp,获得了3个携带NDV重组F蛋白编码基因的重组质粒。选择重组质粒,用无细胞蛋白表达系统进行体外表达,然后用12% SDS-PAGE凝胶对重组F蛋白进行考马斯亮蓝染色和Western blotting。重组F蛋白通过无细胞蛋白表达系统在体外成功表达,其特异的单蛋白带分子量为25.6 kDa。
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引用次数: 1
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Indonesian Journal of Biotechnology
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