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21.
目的:通过在原核表达系统中表达蓖麻的可溶性脂肪酸去饱和酶基因和线虫的fat-1脂肪酸去饱和酶基因,为脂肪酸去饱和酶序列结构与功能的研究奠定基础。方法:将蓖麻RCD△9脂肪酸去饱和酶和线虫fat-1脂肪酸去饱和酶基因亚克隆到大肠杆菌BL21表达载体pET32a+中,获得重组表达载体pET32a+-R9,pET32a+- F1,并通过SDS-PAGE和Western Blotting鉴定蛋白的表达情况。结果:经PCR和测序鉴定,证实两个重组质粒含有目的基因片段;SDS-PAGE和Western Blotting证实两种蛋白在大肠杆菌中获得表达,但表达量具有明显的不同;Anthepro软件对蛋白跨膜结构的分析,验证蓖麻△9脂肪酸去饱和酶和线虫fat-1脂肪酸去饱和酶在结构上的不同。结论:蓖麻的RCD脂肪酸去饱和酶和线虫的fat-1脂肪酸去饱和酶都得到了表达,但线虫fat-1脂肪酸去饱和酶表达量偏低;这可能与fat-1脂肪酸去饱和酶是一类跨膜蛋白的性质直接相关。因此,对于线虫fat-1脂肪酸去饱和酶的基于蛋白纯化的结构分析有待进一步的研究。 相似文献
22.
Jafar A. Mammadov Wei Chen Ruihua Ren Reetal Pai Wesley Marchione Feyruz Yalçin Hanneke Witsenboer Thomas W. Greene Steven A. Thompson Siva P. Kumpatla 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2010,121(3):577-588
The duplicated and the highly repetitive nature of the maize genome has historically impeded the development of true single
nucleotide polymorphism (SNP) markers in this crop. Recent advances in genome complexity reduction methods coupled with sequencing-by-synthesis
technologies permit the implementation of efficient genome-wide SNP discovery in maize. In this study, we have applied Complexity
Reduction of Polymorphic Sequences technology (Keygene N.V., Wageningen, The Netherlands) for the identification of informative
SNPs between two genetically distinct maize inbred lines of North and South American origins. This approach resulted in the
discovery of 1,123 putative SNPs representing low and single copy loci. In silico and experimental (Illumina GoldenGate (GG)
assay) validation of putative SNPs resulted in mapping of 604 markers, out of which 188 SNPs represented 43 haplotype blocks
distributed across all ten chromosomes. We have determined and clearly stated a specific combination of stringent criteria
(>0.3 minor allele frequency, >0.8 GenTrainScore and >0.5 Chi_test100 score) necessary for the identification of highly polymorphic
and genetically stable SNP markers. Due to these criteria, we identified a subset of 120 high-quality SNP markers to leverage
in GG assay-based marker-assisted selection projects. A total of 32 high-quality SNPs represented 21 haplotypes out of 43
identified in this study. The information on the selection criteria of highly polymorphic SNPs in a complex genome such as
maize and the public availability of these SNP assays will be of great value for the maize molecular genetics and breeding
community. 相似文献
23.
Glucose oxidase production was optimized using an isolated strain of Aspergillus niger and an economical nutrient source, corn steep liquor (CSL). The culture produced 580 +/- 30 units/ml of the enzyme using 70 g/l sucrose as the carbon source. Using CSL as the sole nutrient source enzyme synthesis was increased to 640 +/- 36 units/ml. None of the nitrogen sources (nitrates of calcium, sodium, ammonium, potassium and yeast extract, malt extract, and peptone) was beneficial to the enzyme synthesis. Aeration and agitation enhanced enzyme synthesis to 850 +/- 45 units/ml. Glucose oxidase has numerous applications in food industry and clinical fields. 相似文献
24.
Biochemical and genetic characterization of dehydrobiotin resistant mutants of Escherichia coli 总被引:2,自引:0,他引:2
C. H. Pai 《Molecular & general genetics : MGG》1974,134(4):345-357
Summary Dehydrobiotin (DHB) resistant mutants were isolated from strains of Escherichia coli K-12 and were classified into two groups; dhbA and dhbB.In dhbB mutants the structural genes for enzymes of the biotin pathway are expressed constitutively at a high rate. The dhbB gene is co-transducible with argE at a frequency of about 50% by P1 transduction and maps on the chromosome between arg EC BH and rif. The dhbB
+ gene is trans-dominant over the mutant allele indicating that the dhbB
+ gene controls the production of a diffusible substance such as a repressor molecule.The dhbA mutants show biotin biosynthetic activity comparable to the wild type and are as sensitive to repression by biotin as the parent strain. The mutants appear to be deficient in DHB transport as suggested by the findings that the ability of the mutants to take up biotin is reduced significantly and that DHB, a competitive inhibitor of biotin uptake, is much less inhibitory to biotin uptake in the mutants than in the wild type. 相似文献
25.
Directed evolution relies on both random and site-directed mutagenesis of individual genes and regulatory elements to create variants with altered activity profiles for engineering applications. Central to these experiments is the construction of large libraries of related variants. However, a number of technical hurdles continue to limit routine construction of random mutagenesis libraries in Escherichia coli, in particular, inefficiencies during digestion and ligation steps. Here, we report a restriction enzyme-free approach to library generation using megaprimers termed MegAnneal. Target DNA is first exponentially amplified using error-prone polymerase chain reaction (PCR) and then linearly amplified with a single 3′ primer to generate long, randomly mutated, single-stranded megaprimers. These are annealed to single-stranded dUTP-containing template plasmid and extended with T7 polymerase to create a complementary strand, and the resulting termini are ligated with T4 DNA ligase. Using this approach, we are able to reliably generate libraries of approximately 107 colony-forming units (cfu)/μg DNA/transformation in a single day. We have created MegAnneal libraries based on three different single-chain antibodies and identified variants with enhanced expression and ligand-binding affinity. The key advantages of this approach include facile amplification, restriction enzyme-free library generation, and a significantly reduced risk of mutations outside the targeted region and wild-type contamination as compared with current methods. 相似文献
26.
Ping-Jung Yi Cheng-Kang Pai Je-Ruei Liu 《World journal of microbiology & biotechnology》2011,27(5):1035-1043
The worldwide contamination of cereals, oilseeds, and other crops by mycotoxin-producing moulds is a significant problem.
Mycotoxins have adverse effects on humans and animals that result in illnesses and economic losses. Reduction or elimination
of mycotoxin contamination in food and feed is an important issue. This study aimed to screen soil bacteria for degradation
of zearalenone (ZEN). A pure culture of strain CK1 isolated from soil samples showed most capable of degradation of ZEN. Using
physiological, biochemical, and 16S rRNA gene sequence analysis methods, CK1 was identified as Bacillus licheniformis. Addition of 2 ppm of ZEN in Luria–Bertani (LB) medium, B. licheniformis CK1 decreased 95.8% of ZEN after 36 h of incubation. In ZEN-contaminated corn meal medium, B. licheniformis CK1 decreased more than 98% of ZEN after 36 h of incubation. In addition, B. licheniformis CK1 was non-hemolytic, non-enterotoxin producing, and displayed high levels of extracellular xylanase, cellulase, and protease
activities. These findings suggest that B. licheniformis CK1 could be used to reduce the concentrations of ZEN and improve the digestibility of nutrients in feedstuffs simultaneously. 相似文献
27.
28.
Tsuyoshi Shimo Kenichi Matsumoto Kiyofumi Takabatake Eriko Aoyama Yuichiro Takebe Soichiro Ibaragi Tatsuo Okui Naito Kurio Hiroyuki Takada Kyoichi Obata Pai Pang Masahiro Iwamoto Hitoshi Nagatsuka Akira Sasaki 《PloS one》2016,11(3)
Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b+ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment. 相似文献
29.
Use of a chemiluminescence detector for quantitation of nitric oxide produced in assays of denitrifying enzymes 总被引:1,自引:0,他引:1
We have developed a closed-flow system that continuously sweeps away gases evolved in enzyme assay mixtures into a commercially available oxides of nitrogen (NOx) analyzer for the quantitation of any nitric oxide present in these gases. The system enabled us to study both the stoichiometry and the kinetics of NO production by a copper-containing nitrite reductase (EC 1.7.99.3) purified from Achromobacter cycloclastes IAM 1013. In addition to its much greater sensitivity in comparison with standard gas chromatographic (GC) techniques, the method offers the advantage that NO, a very reactive free radical, is immediately swept away and quantitated, obviating the necessity for periodic manipulations and disturbances of the reaction mixture characteristic of other GC quantitations. The characteristics of the system are discussed and its utility in studies of the kinetics and stoichiometry of NO production from nitrite is confirmed by comparison with results obtained using manometric and GC techniques. 相似文献
30.
Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans (ATCC 21783) was immobilised on a silica-based support: purified seasand. Although adsorption of 98% was achieved, considerable
desorption was encountered. This problem was minimised by crosslinking the adsorbed enzyme with glutaraldehyde. The immobilised
enzyme after crosslinking could be used repeatedly for cyclodextrin (CD) production in a batch process. The activity retention
was 80% at the end of the eighth cycle. The immobilised enzyme showed a shift in the pH optimum towards the alkaline side
and also an improvement in the pH stability compared to the free enzyme. It catalysed the formation of β-CD as a major product.
A significant amount of α-CD production was also observed on prolonged incubation.
Electronic Publication 相似文献