深海沉积物微生物元基因组文库来源的新的酯酶基因的克隆、表达及酶学性质

【目的】从深海沉积物微生物元基因组文库中克隆新的酯酶基因,并进行酶学性质研究。【方法】利用含有三丁酸甘油酯的酯酶选择性筛选培养基,从深海沉积物微生物元基因组文库中筛选得到酯酶阳性Fosmid克隆。对筛选得到的fosmid FL10进行部分酶切构建亚克隆文库,筛选得到酯酶阳性亚克隆pFLS10。PCR扩增目的片段后与pET28a连接构建酯酶基因原核表达质粒,转化大肠杆菌(Escherichia coli)BL21。纯化表达产物并对其进行活性测定及酶学性质研究。【结果】序列分析显示该pFLS10亚克隆质粒含有一段924bp的ORF(Open Reading Frame),与一海洋元基因组文库中筛选出的酯酶ADA70030序列一致性为71%。该酶为一新的低温酯酶,对C4底物(对硝基苯丁酸酯)水解能力最强。该酶最适作用温度为20℃,最适作用pH为7.5,20℃时较为稳定,pH8-10的范围内有良好的pH稳定性,K+、Mg2+对该酶具有一定的激活作用,Mn2+等对其具有不同程度的抑制作用。【结论】应用元基因组技术筛选到了新的酯酶基因fls10并进行了克隆表达,该酶在低温及碱性条件下较为稳定且活力较高,对于工业化生产具有一定的应用潜力。关键词:深海沉积物;元基因组文库;低温酯酶;酶学特征     

生物素对地衣芽孢杆菌生长和乳酸形成的影响

生物素对地衣芽孢杆菌生长和乳酸形成的影响胡尚勤（重庆师范学院生物系重庆６３００４７）地衣芽孢杆菌（Ｂａｃｉｌｕｓｌｉｃｇｅｎｉｆｏｒｍｉｓ）是我国９０年代首次从正常待产妇阴道拭子分离出来的一个无毒新菌株。现在国内已经用它来生产“整肠生”活菌剂。它是一...     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

山东地区犬冠状病毒的分子流行病学调查及其基因型多样性分析

【目的】了解分析山东地区健康犬及腹泻犬中犬冠状病毒(CCoV)的分子流行病学及其基因型。【方法】采集2017年以来山东省宠物市场、流浪犬中心、犬舍、各地宠物医院等的健康和腹泻犬只的肛拭子样品,采用PCR方法检测CCoV及其CCoV-Ⅰ和CCoV-Ⅱ(CCoV-Ⅱa和CCoV-Ⅱb)基因分型情况,并对扩增出的M、S全基因进行测序分析。【结果】199份样品中,共检出CCoV阳性样品79份,健康犬中的检出率为40.2%(33/82),腹泻犬中的检出率为39.3%(46/117)。健康犬中CCoV-Ⅰ型与CCoV-Ⅱ型阳性检出率分别为78.8%(26/33)和51.5%(17/33);腹泻犬中CCoV-Ⅰ型与CCoV-Ⅱ型阳性检出率分别为39.13%(18/46)和80.4%(37/46)。基因型阳性比率分析表明,健康犬中单独感染CCoV-I型的比率最高,为48.5%(16/33);腹泻犬中单独感染CCoV-Ⅱa比率最高,为47.8%(22/46)。测序分析获得48株M基因序列,遗传进化分析表明,12株CCoV-Ⅰ型毒株中除1株外,其余均从健康犬中分离。36株CCoV-Ⅱ型毒株中除7株来自健康犬外,其余均为腹泻犬中获得。获得的3株S全基因均来自腹泻犬且均属于CCoV-Ⅱa亚型。【结论】山东地区犬群中CCoV检出率较高,说明CCoV广泛流行,健康犬和腹泻犬中均存在多重感染情况,健康犬中主要流行CCoV毒株为CCoV-Ⅰ型,腹泻犬中主要流行CCoV-Ⅱa型。     

农杆菌介导的单子叶植物早熟禾的转化

利用种子和胚分别在两种培养基K3和K5诱导产生了早熟禾(Poa pratensis L.)一个品种Mado的胚性愈伤组织.K3培养基含有10.0μmol/L的二氯苯氧乙酸(2,4-D)、0.5μmol/L的苄氨基嘌呤(BAP).K5培养基是K3另加0.5μmol/L的硫酸铜.光照条件为20～30 μmol.m-2.s、16 h光照、8 h黑暗.温度保持在24℃.用携有bar基因和gus基因的pDM805质粒转化的农杆菌AGL1对胚性愈伤组织进行转化.共得到4个转基因株系.影响转基因效率的主要因素有愈伤组织的胚性、光照条件、共转化时间、抗生素浓度、选择压力.本研究建立了单子叶早熟禾农杆菌介导的转基因方案.     

横斑锦蛇的现状

结合近几年来的野外调查,对横斑锦蛇的现状进行了报道。横斑锦蛇目前仅见于四川省西部的汶川、宝兴、石棉、泸定和雅安5个县、市,其中石棉和宝兴两县为其新分布区。横斑锦蛇栖息于海拔1650－2500m的湿润山地落叶阔叶林及灌草丛中,数量稀少,亟待加强保护和研究。 此外,还对唯一的幼蛇标本进行了描述。     

深黄被孢霉△^6—脂肪酸脱氢酶基因在转基因烟草中的表达

γ—亚麻酸(GLA)是人体和动物饮食中具有营养作用的重要的多烯不饱和脂肪酸,在大多数油料作物种子中不含有GLA,而只含有其前体物亚油酸,只有少数油料植物种子中含有GLA,如夜来香(Oenothera spp),琉璃苣(Borago officinalis)等。△^6—脂肪酸脱氢酶可将亚油酸转化为γ—亚麻酸,为了能够在传统的油料作物种子中产生GLA,我们将从深黄被孢霉中克隆的△^6—脂肪酸脱氢酶基因,与植物表达载体pGA643连接,构建了重组质粒pGAM—ICL6,将其通过农杆菌介导法,导入模式植物烟草中。经PCR和Southern杂交分析表明该基因已导入并整合到烟草的基因组中,Northern杂交结果表明该基因在转基因烟草的mRNA水平上获得表达。对转基因植株进行脂肪酸分析,结果显示,GLA和十八碳四烯酸(OTA)分别占总脂肪酸含量的19．7％和3．5％。     

Single nucleotide polymorphisms in <Emphasis Type="Italic">TaER</Emphasis> genes and their association with carbon isotope discrimination in wheat genotypes under drought

Candidate gene association studies implicate the detection of contributing single nucleotide polymorphism (SNP) for the target traits and have been recommended as a promising technique to anatomize the complex characters in plants. The ERECTA gene in plants controls different physiological functions. In this study, we identified SNPs in 1.1 kb partial sequences of TaER-1 and TaER-2 of wheat (Triticum aestivum L.). Thirty-nine SNPs were identified in the coding regions of TaER-1 gene in 33 wheat genotypes, of which 20 SNPs caused non-synonymous mutations while 19 SNPs produced synonymous mutations; 31 SNPs were located in the coding regions of TaER-2 gene in 26 genotypes, of which 18 SNPs caused non-synonymous mutations and 13 SNPs caused synonymous mutations. In addition, 32 SNPs in TaER-1 and 9 SNPs in TaER-2 were also identified in the non-coding regions. Moreover, the significant genetic associations of SNPs of TaER-1 and TaER-2 genes with carbon isotope discrimination, stomatal conductance, photosynthetic rate, transpiration rate, intrinsic water use efficiency (iWUE), leaf length, leaf width, stomatal density, epidermal cell density, and stomatal index were noted in wheat genotypes. This study confirms the importance of TaER-1 and TaER-2 genes which could improve iWUE of wheat by regulating leaf gas exchange and leaf structural traits. These identified SNPs may play a critical role in molecular breeding by means of marker-assisted selection.     

葡萄糖苷转移酶生产菌种的筛选

用甲基葡萄糖及麦芽糖为底物,对５０多株黑曲霉α－葡萄糖苷酶活性作了测定,结果表明各菌株对这二类底物之水解能力并不一致,甲基葡萄糖水解活性高的,其麦芽糖水解能力未必也高。通过ＴＬＣ测定这些菌株生成异麦芽低聚糖之能力,结果表明甲基葡萄糖和麦芽糖为底物的水解能力并不能正常反映转苷反应的能力。酶活力高的,生成异麦芽低聚糖的能力未必比活性低的为强,造成上述结果的原因,或许是胞外酶中存在着仅有水解作用的α－葡