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为构建南极冰藻Chlamydomonassp.ICE-L的cDNA文库,提取对数生长期南极冰藻ICE-L的总RNA,以此为模板,通过PowerScript逆转录酶逆转录合成第一链cDNA;再以第一链cDNA产物为模板,用LD-PCR合成第二链cDNA。该cDNA产物经分级分离转入大肠杆菌中,即获得南极冰藻ICE-L的cDNA原始文库,其滴度为1.6×106cfu/mL。扩增后的cDNA文库的滴度为1.0×1010cfu/mL。用PCR方法测得文库的重组率大于97%,插入cDNA的长度为0.5~1.8 kb,0.9 kb以上插入片段占50%以上。取适量扩增文库稀释并铺平板,挑取72个独立菌落,对其中22个独立菌落所插入的cDNA进行测序,克隆到了一个具有5′和3′非编码区的40S ribosomalprotein S5全长基因序列,GenBank收录号为AM167929。 相似文献
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In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6(PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity(258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃ for 30 min. Al163 activity was stimulated by Cd~(2+), Ca~(2+), Fe~(3+), and Mn~(2+), but inhibited by Cu~(2+), Si~(2+), Fe~(2+), and Ni~(2+). Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, poly M, and poly G, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides. 相似文献
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以金黄色葡萄球菌为指示菌,采用琼脂扩散法从国家海洋局海洋生物活性物质重点实验室广西钦州湾红树林微生物资源库中筛选获得一株具有明显抑菌效果的菌株5-8,对该菌株进行了分子鉴定与系统发育分析。结果表明,此菌为欧文氏菌属(Erwinia)。通过单因子试验对其发酵条件进行了优化,优化后发酵条件为:最佳氮源为蛋白胨,最佳碳源为葡萄糖,发酵培养基初始pH值为8.0,发酵温度为25℃,盐度为1.5,摇瓶装液量30%,接种量为1%(V种子液∶V培养液=1∶100)。 相似文献
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利用过氧化氢耐受性实验对北极海洋沉积物中具有高抗氧化活性的菌株进行初筛,通过其对DPPH(1,1-二苯基-2-三硝基苯肼)自由基、羟自由基和超氧阴离子清除能力的测定进一步验证其抗氧化能力;并对其进行了分子鉴定与系统发育分析。根据过氧化氢耐受性实验,从145株供测菌株中筛选出25株对过氧化氢具有较强耐受性的抗氧化活性菌株,占总供测菌株的17.2%;其对DPPH自由基、羟自由基和超氧阴离子均具有较强的清除能力,可分别高达84.15%,88.61%和45.77%。基于16SrRNA基因的分子鉴定与系统发育分析表明,筛选出的25株活性菌株多样性较高,分别属于细菌域的5个纲,15个属,20个种;其中以γ-Proteobactria纲的菌株数量最多。北极海域的海洋沉积物中存在着丰富的具抗氧化活性的菌株资源,将为了解极地微生物的抗氧胁迫机制及极地来源的新型生物活性物质的研发打下一定的基础。 相似文献
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Phylogenetic analysis based on 16S rDNA of 8 strains of cultivable bacteria isolated from Arctic sea-ice was studied.The results showed that strain BJ1 belonged to genus Planococcus,which was a genus of low mole percent G+C gram-positive bacteria;strain BJ6 belonged to genus Burkholderia of β-proteobacteria and the rest 6 strain all belonged to γ-proteobacteria,of which strain BJ8 was a species of Pseudoalteromonas,strain BJ2-BJ5 and BJ7 were members of genus Psychrobacter.Phylogenetic analysis also indicated that bacteria of genus Psychrobacter of the isolates formed a relatively independent phylogenetic cluster in comparison with other bacteria belonged to genus Psychrobacter. 相似文献
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为了探讨微生物多糖剂量效应在水产动物对不良环境刺激反应中的作用,本研究利用从南极冰海水中分离得到的南极细菌Pseudoalteromonas sp.3-3-1-2产生的胞外多糖,采用注射不同浓度的胞外多糖溶液的方式处理刺参,定期记录其生长状况并测定体腔液免疫相关指标的变化。结果表明,南极细菌胞外多糖处理后的刺参保护率升高,即较高浓度(4mg/mL)的细菌多糖可以有效降低刺参的死亡率。细菌多糖可以降低体腔液中MDA含量,虽然体腔液ACP、CAT和SOD活性在处理前期有所受到抑制,但是试验后期细菌多糖处理明显增强了上述免疫酶活性,以保护细胞免受氧自由基诱导的氧化损伤。处理过程中刺参体腔液的溶菌酶活性降低,但是细菌多糖的处理减弱了溶菌酶活性下降的幅度。此外,与2mg/mL处理组相比,4mg/mL处理组对刺参体腔液免疫酶的促进作用稍弱,但无显著差异。研究结果表明,极地细菌胞外多糖可对刺参产生一定的免疫保护作用,从而提高刺参逆境下的生长活力。 相似文献
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从南极第24次科学考察采集的样品中分离出产低温脂肪酶菌株Psychrobacter sp.G,通过PlackettBurman法及响应面法对其产脂肪酶条件进行优化,得出最适条件为:蛋白胨0.75％、酵母粉0.2％、NaCl 2％、MgSO40.1％、(NH4)2SO4 0.1％、K2HPO40.2％、Tween-80 1.5％、培养温度15℃、培养时间72 h、装液量150 mL/500 mL、振荡转速150 r/min.在此条件下发酵的脂肪酶活力为16.5 U/mL. 相似文献