七星瓢虫保幼激素环氧水解酶基因克隆及表达分析

保幼激素(juvenile hormone, JH)可以调控昆虫滞育,保幼激素环氧水解酶(juvenile hormone epoxide hydrolase, JHEH)是调节保幼激素代谢的关键酶之一。为探索JHEH在七星瓢虫Coccinella septempunctata L.滞育中的调控作用,利用RT-PCR和RACE技术克隆获得七星瓢虫JHEH全长基因,命名为Csjheh(GenBank登录号:MH932586),该基因cDNA全长2 077 bp,开放阅读框(ORF)1 380 bp,编码459个氨基酸,预测蛋白质分子量为51.39 kD,理论等电点(pI)为8.79。疏水性分析结果显示该基因具有典型环氧水解酶的N末端疏水结构。氨基酸序列比对结果表明,Csjheh与中欧山松大小蠹、赤拟谷盗、丽蝇蛹集金小蜂、内华达古白蚁保幼激素环氧水解酶同源性达到64.24%。利用实时荧光定量PCR技术研究其时空表达模式,结果表明Csjheh基因在七星瓢虫成虫初羽化阶段表达量较高,滞育诱导条件下表达量呈先下降后上升的趋势,滞育60 d时与初羽化阶段接近。本研究结果对揭示JHEH参与JH的调控作用,进而调控昆虫滞育提供了理论参考。     

蔗糖水解酶注释基因XC0805与野油菜黄单胞菌8004菌株的蔗糖利用及致病相关

 野油菜黄单胞菌(Xanthomonas campestris pv.campestris,Xcc)是造成十字花科植物减产的重要病原菌,其通过水孔或伤口进入植物体内并在维管束中快速繁殖。光合产物(蔗糖)的运输主要依靠植物维管组织,同时蔗糖也是维管组织重要的能量来源。本研究利用生物信息学和遗传学等方法对Xcc中参与蔗糖利用的基因进行了分析,并研究了这些基因与其致病力的关系。Xcc 8004菌株中注释的参与蔗糖分解蛋白的编码基因有XC1002、XC1642、 XC1645和XC0805。 XC1002和XC1642的编码产物属于糖苷水解酶GH97蛋白超家族,而XC1645和XC0805的编码产物则属于GH13家族。突变分析发现除XC0805外,其他3个都不影响Xcc的蔗糖利用能力。接种实验表明,XC0805参与Xcc的侵染或在植物体内的生长,其他3个不参与致病过程。另外,Xcc 8004中预测参与蔗糖转运的基因(XC0806和XC0807)突变并不影响细菌的蔗糖利用和致病力,表明其可能存在其他的蔗糖转运通路。上述结果说明XC0805可能是Xcc 8004主要的蔗糖水解酶基因,且在致病中起重要作用。     

Cloning and Expression of Bile Salt Hydrolase Gene from Lactobacillus plantarum M1-UVS29

We cloned and expressed bile salt hydrolase gene of Lactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from Gen Bank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector p NZ8148 and yielding vector p NZ8148-BSH. p NZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol · min-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.     

圆叶决明添加量对红壤可溶性氮及酶活性的影响

为了探明圆叶决明(Chamaecrista rotundifolia)降解后红壤可溶性氮及氮水解酶活性的变化规律,本研究采用室内好气恒湿培养法,研究占红壤质量0.5%(T1),1%(T2)和2%(T3)的圆叶决明添加至红壤中,培养7~88d内红壤硝态氮(NO^-₃-N)、铵态氮(NH⁺₄-N)和可溶性有机氮(Soluble organic nitrogen,SON)及脲酶、蛋白酶和天冬酰胺酶的变化。结果表明：添加圆叶决明后,红壤NO^-₃-N和NH⁺₄-N含量在培养前期降低,培养中期增加;而整个培养期SON含量及脲酶、蛋白酶和天冬酰胺酶活性均增加,且圆叶决明添加量越大,效果越显著。NO^-₃-N和SON含量及脲酶、蛋白酶和天冬酰胺酶活性可用2次或3次函数方程拟合;而NH⁺₄-N含量可用线性函数拟合。氮水解酶与NO^-₃-N负相关,与NH⁺₄-N和SON正相关,且相关性从大到小的顺序为蛋白酶>天冬酰胺酶>脲酶。综上,添加圆叶决明提高了红壤供氮水平和红壤氮转化能力。     

Enzyme activity and microbial biomass in a field soil amended with municipal refuse

Summary Changes in enzyme activity levels, in biomass-C content, and in the rate of fluorescein diacetate hydrolysis were measured in a loamy soil to which solid municipal refuse had been applied as compost over a 3-year period at two different rates. Addition of the compost caused significant increases in the activity of all enzymes tested. The increases were much higher at 90 t ha^-1 year^-1 than at 30 t ha^-1 year^-1. Significant increases were also observed in the biomass-C content and in the rate of fluorescein diacetate hydrolysis. Significant correlations among changes in biomass-C content and the rate of fluorescein diacetate hydrolysis and the changes in all enzymes tested were found.Two activity indices were calculated; a biological index of fertility and an enzyme activity number. No correlations were found between the biological index of fertility and the changes in the various enzyme activities. However, significant correlations were found either between enzyme activity number and most of the changes in enzyme activity, or between the enzyme activity number index and the biomass-C content (r=0.850). The use of a new activity index, the hydrolysis coefficient, is proposed. This coefficient was significantly correlated with biomass-C content (r=0.925) and with the enzyme activity number index (r=0.780).     

产胆盐水解酶植物乳杆菌降胆固醇作用的研究

采用茚三酮显色法测定了5株具有潜在益生特性的植物乳杆菌的胆盐水解酶(BSH)活性,采用邻苯二甲醛法测定了发酵上清液、菌体洗涤液和菌体破碎液中胆固醇含量,并对二者进行了相关性分析。结果表明,5株菌的BSH比活力为0.0149~0.0299U/mg总蛋白,去除胆固醇能力为37.64~47.88μg/mL。5株乳杆菌BSH活性与菌体洗涤液中胆固醇含量呈显著正相关,推断是菌株BSH水解结合胆盐生成游离胆酸,后者与胆固醇共沉淀,从而达到降胆固醇的效果。上述研究结果为利用益生菌生产具有降胆固醇功效的产品提供了理论依据。     

热稳定性对甲基对硫磷水解酶在毕赤酵母中分泌的影响

甲基对硫磷水解酶可生物降解有机磷农药,由于其特殊的应用价值,越来越受到人们的关注。来源于苍白杆菌的甲基对硫磷水解酶MPH-Och在毕赤酵母系统中不能有效地分泌表达。将热稳定性提高的突变体MPH-S274Q在毕赤酵母中表达,发现其毕赤酵母重组菌株经摇瓶诱导培养120 h后,培养上清的酶活力达到0.7 U/mL,蛋白分泌量是野生型的14倍,SDS-PAGE电泳图中培养上清有明显的蛋白质条带,进一步证明了分泌效率提高,该结果表明提高蛋白质的热稳定性可以作为提高外源蛋白在毕赤酵母中的分泌效率的一条有效途径。     

胆盐水解酶基因结构与功能研究现状

胆盐水解酶是微生物生长、繁殖过程中产生的一种胞内酶,因其可能与降低血胆固醇、预防心血管疾病有关而受到广泛关注。本文从胆盐水解酶的特性出发,综述了胆盐水解酶的生理功能、酶学活性、微生物菌群的来源及特征,以及胆盐水解酶的氨基酸结构等方面的研究进展,以期为进一步深入研究胆盐水解酶的作用机理及相关制品的开发利用提供参考。     

青岛泊子盐场放线菌多样性及其功能酶的筛选

采用平板涂布法利用3种培养基从青岛即墨市田横镇泊子盐场盐池底泥样中共分离纯化出15株放线菌,对分离菌株进行16S rRNA基因测序分析。发现这15株放线菌分属于链霉菌属(Streptomyces)、拟诺卡氏菌属(Nocardiopsis)和1个新属,其中Streptomyces属为优势菌属;菌株CXB832与Nocardiopsis arabia DSM 45083T和Haloactinospora albus DSM 45015T最接近,同源性分别为95.4%和94.9%,根据其表型和分子特征可以初步判定该菌株为放线菌新属。培养基及培养温度对盐池环境中放线菌的分离效果均有影响,利用淀粉酪素培养基在37℃分离的放线菌种类和数量较多。在15株放线菌中,分别有12株、3株、2株和1株放线菌产淀粉酶、酯酶、蛋白酶和纤维素酶。     

纤维素酶及其基因结构特征与功能的关系

纤维素是一种具有重要发展潜力的生物型能源.研究纤维素酶及其基因的结构和功能,阐明基因表达和降解机制是利用这一巨大资源的基础之一.本文综述了纤维素酶及其基因结构和功能研究的最新进展,并对其发展前景进行了分析.