Isolation and characterization of a novel tannase from a metagenomic library     

Yao J  Fan XJ  Lu Y  Liu YH 《Journal of agricultural and food chemistry》2011,59(8):3812-3818

A novel gene (designated as tan410) encoding tannase was isolated from a cotton field metagenomic library by functional screening. Sequence analysis revealed that tan410 encoded a protein of 521 amino acids. SDS-PAGE and gel filtration chromatography analysis of purified tannase suggested that Tan410 was a monomeric enzyme with a molecular mass of 55 kDa. The optimum temperature and pH of Tan410 were 30 °C and 6.4. The activity was enhanced by addition of Ca(2+), Mg(2+) and Cd(2+). In addition, Tan410 was stable in the presence of 4 M NaCl. Chlorogenic acid, rosmarinic acid, ethyl ferulate, tannic acid, epicatechin gallate and epigallocathchin gallate were efficiently hydrolyzed by recombinant tannase. All of these excellent properties make Tan410 an interesting enzyme for biotechnological application.  相似文献   

Isolation and identification of the DNA aptamer target to acetamiprid   总被引：1，自引：0，他引：1  

He J  Liu Y  Fan M  Liu X 《Journal of agricultural and food chemistry》2011,59(5):1582-1586

As an alternative to antibodies, aptamers have a great potential as analytical tools for pesticide detection. In this work, aptamers targeting acetamiprid were selected by a specific systematic evolution of ligands by exponential enrichment (SELEX) strategy, where a single-stranded DNA (ssDNA) library was immobilized and target modification was eliminated. After 18 rounds of repeated selection, the ssDNA pool was enriched and then 14 sequences were selected and carefully identified. At last, an acetamiprid-specific aptamer with the apparent dissociation constant (K(d)) estimated to be 4.98 μM was successfully obtained. Further work is ongoing to develop an aptamer-based detection method for field determination of this pesticides in agricultural products and environmental samples.  相似文献   

Habitat and food choice of the critically endangered cao vit gibbon (Nomascus nasutus) in China: Implications for conservation     

Peng-Fei Fan  Han-Lan Fei  Matthew B. Scott  Wen Zhang  Chang-Yong Ma 《Biological conservation》2011,(9):2247-2254

Knowledge of habitat and feeding ecology is essential for developing an effective conservation management plan for threatened primates. Despite having been rediscovered nearly a decade ago, very little is known of the critically endangered cao vit gibbon (Nomascus nasutus) or its habitat. We analysed forest cover, made direct observations and conducted interviews of local elders to better understand forest changes and to determine the forest characters and species important to the survival of the gibbon. Interviews indicated human-induced forest resource exploitation focused on species-specific and larger trees most easily accessed. Gibbon forest habitat comprised four primary forest types. The mean canopy height over the whole site was 10.52 m. Gibbons consumed 81, or nearly half of the tree and liana species recorded in the site; however, only 19 species provide 77.8% of the diet. Six of the 19 food species were logged for different reasons throughout the history of the site. We conclude that effective conservation management of primates with highly limited distributions, focused dietary needs, and in degraded ecosystems will require active forest restoration, such as planting important food species in degraded sites.  相似文献   

精准施肥决策模型与数据库系统   总被引：2，自引：0，他引：2  

马晓蕾  范广博  李永玉  徐杨  彭彦昆  汤修映 《农业机械学报》2011,42(5):193-197

以华北地区小麦及玉米为研究对象,以北京市昌平区崔村镇大辛峰村种植区为例,研究了精准施肥决策系统中施肥参数,并建立了相应的数学模型.将算法模型与数据库相结合,采用线性回归的方法,计算施肥时土壤对氮、磷、钾的需求量,利用二维插值的方法,建立了实验区中已知肥力样点与区域内未知肥力样点的相关关系,从而建立了土壤肥力查询数据库.通过以上方式,结合土壤的养分含量、作物种类及目标产量等相关参数来确定实验区域的施肥量.结果表明,该系统可实现施肥量的精确控制.  相似文献   

柴油机掺烧桐油制备生物柴油的动力性和经济性     

张天顺  张汝坤  玄伟东  范焱虎 《农机化研究》2011,33(3)

在ZX195型直喷式柴油机上进行了桐油制备的生物柴油和石化柴油不同配比的混合燃料试验研究.试验结果表明:在不对柴油机做任何调整的情况下,燃用B10和B20的桐油生物柴油对发动机动力性和经济性影响不大;燃用B50和B100的生物柴油时,动力性和经济性下降明显,其功率下降11%,油耗增加35%左右;综合考虑发动机的动力性和经济性,燃用B20较为合适.  相似文献   

Linking plant identity and interspecific competition to soil nitrogen cycling through ammonia oxidizer communities     

Fenliang Fan  Fusuo Zhang 《Soil biology & biochemistry》2011,43(1):46-54

Both plants and microbes influence soil nutrient cycling. However, the links between plants, microbes and nutrient cycling are poorly understood. In this study, we investigated how plant identity and interspecific competition influence soil nitrogen cycling and attempted to link plant identity and interspecific competition to community structures of bacterial and archaeal ammonia oxidizers based on terminal restriction fragment length polymorphism analysis (T-RFLP) of bacterial and archaeal ammonia monooxygenase (amoA) genes. Faba bean and maize monocultures and a faba bean/maize mixture were planted with two nitrogen levels (0 and 100 mg N kg⁻¹ soil as urea). Soil mineral nitrogen, ammonia oxidizer function (potential nitrification activity, PNA) and community structures were measured 28 and 54 days after plant emergence. Faba bean and maize substantially differed in their influences on mineral nitrogen concentrations and PNA in rhizosphere soils. Soil mineral nitrogen and PNA in the rhizosphere soils of the faba bean/maize mixture were closer to those of the maize monoculture than to those of the faba bean monoculture. T-RFLP with restriction enzymes BsaJI and Hpy8I distinguished variations in bacterial and archaeal ammonia oxidizers community structure, respectively, and detected both between-cluster and within-cluster variations in bacterial ammonia oxidizers. T-RFLP data showed that nitrogen addition favored part of a Nitrosospira cluster 3b sequence type and suppressed part of a cluster Nitrosospira 3a sequence type of bacterial ammonia oxidizers, while it had no influence on the archaeal ammonia oxidizer community structure. Although multivariate analysis showed that the function and community structure of bacterial ammonia oxidizers were significantly correlated, plant species and interspecific competition did not significantly change the community structure of bacterial and archaeal ammonia oxidizers. These results indicate that plant species and interspecific competition regulate soil nitrogen cycling via a mechanism of other than alteration in the community structure of ammonia oxidizers as investigated by DNA based methods.  相似文献   

拟南芥Alpha-dioxygenase2(AtDOX2)在毕赤酵母中的表达、纯化及活性鉴定   总被引：1，自引：0，他引：1  

魏少华  杨宇衡  安瑞  单丽伟  范三红 《农业生物技术学报》2011,19(1)

为了获得具有生物活性的拟南芥(Arabidopsis thaliana)alpha-dioxygenase2(AtDOX2),将其对应基因AtDOX2编码区克隆到酵母表达载体pPIC9k中,获得重组表达载体pPIC9k-AtDOX2,将线性化的重组载体电击转化入毕赤酵母(Pichia pastoris)表达菌株GS115,经G418筛选、PCR鉴定和甲醇诱导时间优化,获得重组AtDOX2的高效表达菌株GS115/pPIC9k-AtDOX2。SDS-PAGE分析结果显示,0.5%甲醇诱导96h重组蛋白表达量最高,其表达量占胞外总蛋白的15%。重组AtDOX2的表观分子量约为70kD,经Ni-NTA柱亲和层析可获得纯度大于80%的重组蛋白。2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)法测定结果表明重组蛋白具有过氧化物酶活性,且其活性受Ca2+和Mg2+激活,受EDTA、咪唑和Mn2+抑制;2,4-二硝基苯肼(2,4-DNP)法测定结果显示,重组AtDOX2具有双加氧酶活性,Ca2+对其双加氧酶活性也有激活作用。结果说明利用酵母表达系统获得...  相似文献   

烟草丛顶病毒(TBTV)ORF3和ORF4的原核表达、抗血清制备及应用     

龙亚芹  王万东  左瑞娟  李凡  尹朝先  陈海如 《农业生物技术学报》2011,19(2)

为制备烟草丛顶病毒(TBTV)ORF3和ORF4多克隆抗体,采用RT-PCR方法克隆了TBTV保山龙陵分离物(TBTV-BSLLi)的ORF3和ORF4,亚克隆至pMD18-T载体中,经测序正确后,将该基因插入原核表达载体pET28a(+)中,通过热击转化大肠杆菌(Escherichia coli)BL21(plysS),以IPTG诱导6×His融合蛋白的表达,并经Ni2+亲和柱层析纯化,成功地克隆了TBTV的ORF3和ORF4,建立了其原核表达和表达产物的纯化体系,获得了高纯度的ORF3、ORF4蛋白.用纯化的蛋白免疫大白兔,获得高效价的特异性抗血清.DAS-ELISA检测结果表明,制备的抗血清可用于田间烟草(Nicotiana tabacumL.)样品及传播介体的检测.本研究为用血清学方法检测该病毒提供了基础条件.  相似文献   

一株降解生木薯淀粉的曲霉菌株的筛选、鉴定及其淀粉降解特性     

宋贤冲  田会会  陈国利  许剑  樊宪伟  李有志 《广西农业生物科学》2011,30(5):596-602

本研究利用以生木薯淀粉为唯一碳源的筛选培养基,从腐烂木薯渣中分离筛选出一株可以降解生木薯淀粉的真菌菌株RSDF-7。根据RSDF-7形态和18SrDNA与28SrDNA之间的内转录间隔区（internal transcribed spacer,ITS）序列分析的结果,初步认定该菌株为曲霉属。菌株RSDF-7的粗酶液对多种不同的生淀粉底物均有水解效果;在以大米和玉米淀粉为底物时,其生淀粉分解活力比较高,分别为42%和40%。菌株RSDF-7的粗酶液具有良好的低pH稳定性,对生木薯淀粉的最适作用温度为50℃,最适作用pH为4.5。在30min的吸附后,RSDF-7的粗酶液对生木薯淀粉的吸附力高达60%。使用HPLC对粗酶液的酶解产物进行检测,结果发现酶解产物中仅存在葡萄糖,表明菌株RSDF-7所产的生淀粉降解酶主要为糖化酶。扫描电镜观察结果发现,经RSDF-7粗酶液酶解后的生木薯粉颗粒破裂,形成空洞,说明RSDF-7粗酶液对生淀粉有较强的水解作用。可以预见,经纯化后的曲霉菌株RSDF-7生淀粉酶将来可以用于基于酶解的木薯淀粉转化。  相似文献   

胡杨果糖-1,6-二磷酸醛羧酶基因PeALD的克隆及耐盐性分析     

张帆  常李伟  丁明全  侯佩臣  沈昕  陈少良 《广西农业生物科学》2011,30(3):257-267

胡杨（Populus euphratica Oliv）是优良的抗逆树种,有很强的耐盐碱性。前期胡杨转录组研究结果显示盐胁迫可诱导果糖-1,6-二磷酸醛羧酶基因（PeALD）转录上调,提示该基因可能对胡杨耐盐性方面有某种贡献。为分析PeALD基因在胡杨耐盐性中的作用,本研究以胡杨为材料,利用RT-PCR方法克隆了胡杨果糖-1,6-二磷酸醛羧酶基因的全长cDNA,通过基因转化法尝试在烟草中过量表达该基因,并对转基因株系的耐盐性进行初步分析。研究结果显示,克隆的cDNA编码果糖-1,6-二磷酸醛羧酶,ORF为1177bp,是由247个氨基酸编码的疏水性蛋白,理论等电点为6.84,分子量为26.79kD。PCR与RT-PCR检测结果表明,外源的PeALD基因通过转化已经整合到烟草的染色体中,并在4个株系中得到较强的表达。转化株系表型筛选实验表明,在200mmol/L NaCl的MS培养基中培养15d后,野生型烟草植株无明显高生长,叶片全部黄化并萎缩;而转基因烟草高生长基本没有受到抑制,植株生长良好。说明在烟草中过表达PeALD使转化植株的耐盐性显著提高。光合数据结果显示,ALD基因能够降低盐胁迫对烟草叶片净光合速率的影响,可能是PeALD过表达加速了卡尔文循环的运转,提高了CO2的固定速率,进而促进了光合活性,通过光合作用促进碳的积累,利于呼吸作用和氧化磷酸化产生ATP,为维持跨膜质子浓度梯度提供能量,从而有可能促进质膜上的Na＋/H＋逆向转运,利于细胞质膜保持拒盐性。这些结果初步验证了PeALD基因的耐盐性功能,为进一步深入研究该基因在耐盐机制中的作用奠定了良好基础。  相似文献