棉铃虫核型多角体病毒ORF27基因的表达和细胞内定位

棉铃虫核型多角体病毒（HaSNPV）ORF27编码区全长768bp，编码255个氨基酸残基组成的多肽，预计分子量29.5kD。PCR扩增获得该基因，克隆到融合表达载体pGEX-4t-2中，表达蛋白分子量55.5kD，表达量约占细菌总蛋白的9.2%。割胶透析法纯化融合蛋白，免疫家兔制备多克隆抗体。再把该基因插入到转移载体pFastBacHTb，转化大肠杆菌DH10Bac感受态细胞，构建重组病毒；提取病毒基因组DNA，在Lipofectin介导下转染昆虫细胞Tn-5B1-4。表达蛋白分子量为32kD，表达量约占细胞总蛋白的2.9%。将ORF27在昆虫细胞中表达产物固定，免疫电镜法确定表达蛋白主要存在于细胞的细胞质中。ORF27基因表达及在细胞内定位为其进一步研究提供了基础。     

TOH间隙法测定不同类型公羔体成分沉积的研究

本研究采用氚水(TOH)间隙法活体测定不同类型公羔体成分沉积。结果表明,9—11月龄羔羊体成分的变化,在不同品种羔羊之间存在差异:高营养水平时,体蛋白和体脂肪的沉积量依次为长毛半血羊>东北细毛羊>中国美利奴;低营养水平时,惟有东北细毛羊获得较高的体蛋白和体脂肪沉积量,其他羔羊则沉积甚少。     

粉纹夜蛾颗粒体病毒重组增效蛋白的生物学特性

在大肠杆菌(Escherichiacoli)中表达的粉纹夜蛾颗粒体病毒(Trichoplusianigranulovirus)重组增效蛋白P96能显著提高多种微生物杀虫剂对多种害虫的毒力,具广谱增效活性。基于SDS-PAGE和一系列生物测定进一步研究P96的生物学特性,结果表明:P96在大肠杆菌中表达时形成包涵体较为稳定,但4!存放3个月后有部分解离;P96包涵体可由Na2CO3打开或由棉铃虫(Helicoverpaarmigera)5龄幼虫离体中肠液有效溶解;取食1.9×105￣1.9×106OBs(occlusion-bodies)/mLP96后,棉铃虫幼虫围食膜上的肠粘蛋白基本降解完全;随P96浓度增加,其增效活性愈强,至6.76×105OBs/mL时趋于饱和;金属螯合剂乙二胺四乙酸对P96的增效活性有强烈抑制作用。对P96上述生物学特性的了解,为P96具生物活性提供了生物测定之外的证据支持,并为其合理生产应用提供了依据。     

慢病毒介导shRNA干扰颗粒体蛋白前体(GRN)促进猪前体脂肪细胞分化

为研究颗粒体蛋白前体(granulin,GRN)在脂肪细胞发育过程中的作用,本实验构建了GRN短发夹RNA(short hairpin RNA,shRNA)慢病毒干扰载体,包装并感染猪前体脂肪细胞,采用油红O染色、油红O提取比色法检测猪前体脂肪细胞分化情况,采用Real-time PCR法检测成脂关键基因mRNA的表达变化情况。结果显示,GRN慢病毒干扰载体病毒滴度在5×107TU/mL以上,病毒感染前体脂肪细胞后显著降低了GRN的表达,shRNA2干扰效率最高,达到76%,沉默GRN后能够促进猪前体脂肪细胞分化;脂肪细胞分化标志基因过氧化物酶体增殖物激活受体γ(PPARγ)、固醇调节原件结合蛋白(SREBP-1c)、脂肪细胞型脂肪酸结合蛋白(ap2)和脂蛋白酯酶(LPL)基因mRNA表达量均升高。结果表明,降低GRN基因表达促进了猪前体脂肪细胞分化,揭示GRN在猪前体脂肪细胞分化过程中可能起到抑制作用。     

不同品种花生油脂体粒径电位和蛋白质组成的分析

为了探索不同品种花生油脂体的物理和化学性质差异,以5种（豫花23,豫花27,豫花9719,豫花9830和豫花9502）油脂含量不同的花生品种为原料,采用水剂法提取油脂体,并对提取后油脂体的粒径、ζ电位、氨基酸组成、蛋白质分子量分布进行分析比较。结果表明：提取后,5种花生油脂体粒径间存在一定差异,以豫花9719的粒径较大;花生油脂体均含有油脂体蛋白和贮藏蛋白,但不同品种间存在蛋白质种类的差异;5种花生油脂体在pH值为3.0时ζ电位为正值,在pH值为7.4和pH值为9.0时ζ电位为负值,盐浓度的增加会降低油脂体ζ电位的绝对值;5种花生油脂体的蛋白质均为极性带负电氨基酸质量分数均大于非极性带正电或不带电氨基酸,但氨基酸总量各不相同,以豫花27较低。该研究可为花生油脂体的品质特性研究和应用产品开发提供参考。     

苏打盐碱化土壤pH与团聚体中球囊霉素相关土壤蛋白含量的关系

球囊霉素相关土壤蛋白(Glomalin-related soil protein,GRSP)在土壤团聚体形成中起重要作用,与土壤团聚体稳定性正相关。土壤盐碱化破坏土壤结构,降低土壤中GRSP含量,但影响多大尺寸团聚体GRSP含量并不清楚。本文采集45个松嫩盐碱化草地土壤样品,通过干筛法分离出直径0.25、0.25~1和1~2 mm 3种不同粒级团聚体,采用Bradford法测定土壤中GRSP含量,并测定土壤盐碱化指标,经Pearson相关分析和前向选择变量多元线性回归分析,结果显示:土壤pH显著影响各粒级团聚体总球囊霉素相关土壤蛋白(T-GRSP)含量和难提取球囊霉素相关土壤蛋白(DE-GRSP)含量,二者存在显著负相关关系,特别是0.25~1 mm粒级团聚体DE-GRSP含量与土壤pH存在极显著负相关关系,可解释22.3%的DE-GRSP含量变化。土壤pH、电导率、碱解氮和有效磷对各粒级团聚体易提取球囊霉素相关土壤蛋白(EE-GRSP)影响不显著。结果表明土壤苏打盐碱化影响0.25~1 mm团粒结构中较稳定的DE-GRSP含量,可能对土壤团聚体胶联和土壤碳存储产生负影响。     

黄芪组方浓缩物和半胱胺对肥育猪生长性能和体蛋白动态代谢的影响

采用稳定同位素(15N-Gly)示踪技术和氮平衡试验,比较研究了外源代谢调节物(黄芪组方浓缩物、半胱胺)对PIC肥育猪生长性能、体蛋白动态代谢和氮沉积的影响。选用32头体重59.82±1.32kg的PIC商品猪,随机分成4个处理组,CON组、NCAE组、CAE组和CS组。每处理设4个重复,每重复2头猪。试验结果表明:(1)黄芪组方浓缩物和半胱胺均明显提高肥育猪的生长性能,其中基础日粮+半胱胺70mg/kg组(CS组)显著提高肥育猪日采食量(P<0.05),基础日粮+黄芪组方浓缩物250mg/kg组(CAE组)和基础日粮+非稳定化处理黄芪组方浓缩物250mg/kg组(NCAE组)显著提高饲料利用率(P<0.05),从而使肥育猪日增重均显著提高(P<0.05)。(2)黄芪组方浓缩物和半胱胺均显著增加肥育猪体蛋白沉积速率、显著提高氨基酸利用效率和生物学效价(P<0.05);黄芪组方浓缩物显著提高可消化氨基酸用于合成体蛋白质的速率(P<0.05),降低内源尿氮排泄速率。(3)黄芪组方浓缩物和半胱胺调控体蛋白沉积的强度和途径不同:半胱胺主要通过降低体蛋白降解速率26.71%,增加体蛋白沉积速率63.53%(P<0.05);黄芪组方浓缩物通过降低体蛋白降解速率和提高体蛋白合成速率,共同增加体蛋白沉积,与对照组相比,NCAE和CAE组体蛋白质降解速率分别降低了24.84%和13.66%,体蛋白合成速率分别提高了22.86%和19.18%。(4)黄芪组方浓缩物和半胱胺均可改善氮代谢,体现在CS组显著提高氮的净利用率和生物学效价(P<0.05),CAE组极显著改善沉积氮、氮的净利用率和生物学效价(P<0.01),NCAE组显著提高氮的净利用率和生物学效价(P<0.05)。     

中南地区典型地带性土壤团聚体抗张强度的变化及影响因素

团聚体的力稳性是决定土壤抗侵蚀能力的关键因素。为探究地带性土壤团聚体抗张强度的变化规律及其影响因素,自北向南选取我国中南部地区6种典型地带性土壤(褐土、黄褐土、棕红壤、红壤、赤红壤和砖红壤)的不同粒径(1~2,2~3,3~5,5~10mm)团聚体作为研究对象,通过测定团聚体的抗张强度(TS),探究其与土壤基本理化性质的关系,揭示该区域团聚体抗张强度的变化特点和稳定机制。结果表明:(1)供试土壤皆为黏性土壤,自北向南,随着水热条件的增加,土壤的pH值逐渐降低,高岭石含量和游离氧化物(Fe_d、Al_d)呈现明显的增加趋势;有机质含量随土壤深度的增加而降低。(2)同种土壤团聚体的抗张强度随着粒径的增大而逐步减小,从北至南,相同粒径团聚体的抗张强度整体呈现减小的趋势。(3)TS与pH、粉粒含量、蛭石含量呈极显著正相关(r0.63,p0.01),TS与黏粒含量、1.4nm过渡矿物含量、高岭石含量、游离氧化铁、铝(Fe_d、Al_d)呈显著负相关(r-0.53,p0.05)。(4)逐步回归分析表明,Fe_d和CEC可以较好的预测和评价3~5mm团聚体的抗张强度(R~2=0.80,p0.01)。总体而言,黏土矿物类型及其含量是影响地带性土壤团聚体力稳性的重要因素。研究结果可为该区域土壤侵蚀预测提供一定的理论依据。     

辐照螺旋藻粉的杀菌效果对营养活性成分的影响

研究了60Coγ射线辐照螺旋藻粉的杀菌效果及对其主要营养活性成分、矿物质、维生素、氨基酸含量和超氧化物歧化酶(SOD)、过氧化物酶(POD)活性的影响。结果表明:经6.0 kGy辐照处理后,螺旋藻粉样品中微生物指标均达到国家标准。当剂量为4.0~8.0 kGy时,对螺旋藻中矿物质(除Se)和氨基酸含量无显著影响;螺旋藻中多糖含量随剂量增加有所增加,经6.0和8.0 kGy辐照后多糖分别增加了9%和27.2%;SOD、POD活性在辐照后有所降低,藻胆蛋白、藻蓝蛋白和维生素含量则明显下降;剂量达6 kGy以上时,藻胆蛋白和藻蓝蛋白含量显著低于对照;4.0、6.0、8.0 kGy剂量辐照后VC分别减少了40.6%、62.8%和14.1%,VA分别减少了36.3%、17.6%和17.0%,VE分别减少了25.8%、61.7%和52.6%。综合试验结果,提出螺旋藻粉辐照杀菌的适宜剂量范围为4.0~6.0 kGy。     

大麦离体诱变效应的研究

以二稜皮大麦品种舟麦2号和秀四的幼胚及其愈伤组织为离体诱变材料,研究了γ射线不同剂量辐照后的离体培养反应、生理损伤及诱变效应。结果表明,用γ射线辐照离体大麦幼胚及其愈伤组织的适宜剂量是10—20Gy,MR_2代的突变频率可比常规诱变技术(300Gy γ射线辐照干种子)分别提高33.9％—82.1％和32.7％—229.6％;比离体培养技术分别提高84.0％—150.3％和82.4％—352.9％,而且仍可维持相对较好的培养反应。用30Gy辐照幼胚的诱变效果则可提高9—13倍,但严重抑制了离体培养物的再生能力,并导致了MR_1代严重的生理损伤。     

玉米叶绿体铁氧还蛋白1的原核表达及部分性质分析

叶绿体铁氧还蛋白(Fd)通过活性中心的铁硫簇传递还原力,在各种氧化还原途径中起重要作用.本研究中,氨基酸序列比对显示玉米中5种Fd的叶绿体导肽同源性很低,而去除导肽的成熟蛋白氨基酸序列具有很高的同源性.采用RT-PCR技术从玉米幼叶总RNA中克隆了编码成熟Fd1的基因.并分别插入pQE 80和p28SUMO表达载体,转...     

小麦花药蛋白质组双向电泳技术体系的优化

以小麦单核期花药为材料,比较了两种不同蛋白质提取方法TCA/丙酮法和酚提取法及不同的蛋白质裂解液组成对双向电泳的影响,并在蛋白质上样量和SDS-PAGE胶浓度等方面也进行了探索与优化。结果表明,采用TCA/丙酮提取法比用酚提取法提取的蛋白质所得的2-DE胶图谱上蛋白质点数增加,图谱背景也比较清晰;样品溶解于含有硫脲和TBP的蛋白质裂解液Ⅱ中,可显著提高蛋白质的溶解性,在2-DE图谱上可分辨出554个蛋白质点,比用蛋白质裂解液Ⅰ提取的蛋白多39个点。以TCA-丙酮法提取小麦花药组织中的蛋白,用蛋白质裂解液Ⅱ[7mol/L尿素、2mol/L硫脲、4%CHAPS、2%TBP、65mmol/L DTT、0.2%载体两性电解质(其中0.1%pH 3~10,0.1%pH4~6)]溶解蛋白,以pH4~7线性17cm的IPG胶条进行双向凝胶电泳,在上样量为800μg,13%SDS-PAGE胶浓度下,蛋白质得到了更好的分离,2-DE图谱上可分辨出631个蛋白点,图谱质量最佳。优化后的双向电泳技术体系,适合于小麦花药全蛋白质的双向电泳分析。     

Isolation of extracellular protein from greenhouse soil

Although extracellular proteins may play an important role in the soil environment, these proteins are difficult to isolate because they are immediately degraded by soil microbes, or become associated with clay mineral and humic substances. We developed a method of isolating extracellular proteins from greenhouse soils. Phosphate buffer (pH 6.0) was used to extract protein from soil. A phosphate buffer with higher pH was not recommended because it extracted a large amount of non-proteinaceous organic matter as well as protein and, as a result, the extracted protein was difficult to separate by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After removing cells by filtration, proteins dissolved in the soil extract were recovered by precipitation with 5% trichloroacetic acid (TCA) and isolated by SDS-PAGE. Proteins were detected in 10 of 32 soil samples derived from different greenhouses and the protein bands ranged in apparent molecular mass from 35 to 68 kDa, suggesting that some of soils derived from greenhouse culture contained significant amounts of a specific protein soluble in 67 mM phosphate buffer (pH 6.0). N-terminal amino acid sequence of one of the isolated proteins was found to be a homologue of thermostable cellulase produced by the genus Humicola, a thermophilic fungus.     

Fluorescence spectroscopy of aqueous pine litter extracts: effects of humification and aluminium complexation

Fluorescence excitation, emission, and synchronous-scan spectra were obtained for aqueous extracts of needle and litter layer (O-horizon) samples from ponderosa pine collected at a plantation site. The spectral lineshapes differed markedly between the needle and litter samples, and showed an increasing overall intensity with increasing extent of humification (increasing depth in the litter layer). At a dissolved organic C (DOC) concentration of 100 g m^?3, these effects were accompanied by a general shift in spectral density from lower to higher wavelength such that, in the excitation spectra, there was increasing prominence of a peak at 390 nm. When the DOC concentration was decreased from 100 to 25 g m^?3, the overall spectral intensity decreased and the peak at 390 nm in the excitation spectra of the litter samples gave way to a rising peak at 340 nm. Changes in pH from 4 to 5, characteristic of the undiluted litter extracts, produced little effect on the spectra. The addition of A1 at 40 mmol m^?3 generally produced enhancement of the fluorescence intensity in all three kinds of spectra for the needle and upper litter-layer extracts at 100 g C m^?3 and pH 4. Spectral density below 320 nm in the excitation and synchronous-scan spectra of the needle solution (possibly attributable to gentisic acid on the basis of model experiments) was, however, diminished by Al addition. For the lower litter-layer extracts, A1 addition decreased the fluorescence intensity in excitation and emission spectra, whereas it increased the intensity in synchronous-scan spectra. These trends indicated that the water-soluble fluorophores in the litter layer differed significantly from those in the fresh needles, and changed with the extent of humification. The 390-nm peak in the excitation spectrum, particularly its behaviour in the presence of added Al, may be useful as a spectral signature of products formed by litter humification processes.     

Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.     

中国传统细菌型豆豉溶栓酶的提取纯化技术研究(简报)

为深入开发利用中国传统发酵细菌型豆豉溶栓酶,对豆豉溶栓酶分离纯化工艺进行了研究探讨,确认了最佳的纯化条件:首先用饱和度75%的硫酸铵沉淀豆豉溶栓酶;然后利用DEAE-Sepharose FF (Diethylaminoethyl Sepharose Fast Flow,二乙基氨基琼脂糖快速凝胶)阴离子交换柱进行层析,优化的层析条件为用10 mmol/L Tris(Trishydroxymethylaminomen,三羟甲基氨基甲烷)-HCI (pH 8.7)作为上样缓冲液,采用线性梯度洗脱,洗脱液为10mmol/L Tris-HCI(pH 8.7)和0.6mol/LNaCl,洗脱流速为1 mL/min;最后经过Sephadex G-75(葡聚糖凝胶G-75)凝胶过滤,得到纯化倍数为11.29倍的豆豉溶栓酶.利用SDS-PAGE (Sodium dodecyl sulphate-polylamide gel electroresis,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳) 显示经纯化后的酶无杂蛋白,初步鉴定其分子量接近31 ku.     

Characterization of a 19 kDa alpha-zein of high purity

A highly pure alpha-zein was extracted from corn flour using ethanol (95%). Subsequently, ion-exchange chromatography was performed, using SP-Sepharose that yielded a highly homogeneous protein. This protein migrated as a single band in 20% SDS-PAGE and in pH gradient gels, showing an isoelectric point of 6.8. Mass spectrometry (MALDI-TOF-MS) showed a single peak with a molecular mass of 24 535 Da. It was identified as Z19, when comparing the sequence obtained in an automatic Edman sequencer with the Swissprot database using BLAST. The molar extinction coefficient, determined by dry weight in 70% methanol, was 12 415.49 M(-1) cm(-1) at 280 nm. Light scattering showed its presence in a monodispersed state of 44-66 kDa aggregates in methanol (70%). Circular dichroism spectra allowed the estimation of an alpha-helix content that was lower than the one found for a mixture of two alpha-zeins but with a higher content of beta sheets.     

Purification and characterization of polyphenol oxidase from garland chrysanthemum (Chrysanthemum coronarium L.)

Polyphenol oxidase (PPO) of garland chrysanthemum (Chrysanthemum coronarium L.) was purified approximately 32-fold with a recovery rate of 16% by ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme appeared as a single band on PAGE and SDS-PAGE. The molecular weight of the enzyme was estimated to be about 47000 and 45000 by gel filtration and SDS-PAGE, respectively. The purified enzyme quickly oxidized chlorogenic acid and (-)-epicatechin. The K(m) value (Michaelis constant) of the enzyme was 2.0 mM for chlorogenic acid (pH 4.0, 30 degrees C) and 10.0 mM for (-)-epicatechin (pH 8.0, 40 degrees C). The optimum pH was 4.0 for chlorogenic acid oxidase (ChO) and 8.0 for (-)-epicatechin oxidase (EpO). In the pH range from 5 to 11, their activities were quite stable at 5 degrees C for 22 h. The optimum temperatures of ChO and EpO activities were 30 and 40 degrees C, respectively. Both activities were stable at up to 50 degrees C after heat treatment for 30 min. The purified enzyme was strongly inhibited by l-ascorbic acid and l-cysteine at 1 mM.     

β-琼脂糖酶ⅠDagA的原核表达和活性鉴定

采用PCR技术从假别单胞菌（Pseudoalteromonas atlantica）19262基因组DNA中获得β-琼脂糖酶Ⅰ基因（dagA）及去除信号肽的编码序列dagA(▽),分别与载体pET21a连接后转入大肠杆菌（Escherichia coli）ER2566中,共表达分子伴侣DsbC及FkpA,筛选出以包涵体为主要表达形式的高效表达体系：ER2566-pET21a-dagA(▽)-DsbC菌株。包涵体蛋白达到菌体总蛋白的60%左右。包涵体用8 mol/L尿素溶解、镍离子亲和层析纯化和梯度稀释复性。SDS-PAGE检测表明,复性后的DagA蛋白相对分子质量约为30.8 kD,且具有水解琼脂糖的生物活性。酶学特性分析表明,在pH 4.8~6.8范围内,DagA蛋白活性保持60%以上,最适pH 5.8;在温度37~60 ℃均有活性,最适温度为55 ℃。     

Resveratrol and a novel tyrosinase in Carignan grape juice

Polyphenol levels in wines are affected by the wine-making process. Resveratrol is one polyphenol which has been the subject of a commendable amount of recent research. In this work, we found that resveratrol is immediately degraded by tyrosinase. A novel tyrosinase was purified from Carignan grapes. The purification process included salting out and separation on a cation-exchange column, followed by gel filtration. Tyrosinase was purified in a homogeneous form by SDS-PAGE and was characterized: its specific activity toward 3-(3,4-dihydroxyphenyl)-L-alanine (DOPA) increased by a factor of 24 with an overall recovery of 3% of initial activity. The apparent molecular mass of the purified tyrosinase was 40 kDa as determined by SDS-PAGE, and 42 kDa as determined by gel filtration. Its activity was optimal at pH 6 and at 25 degrees C. The enzyme exhibited high activity toward phenylenediamine, epicatechin, pyrogallol, DOPA, and resveratrol. Tyrosinase activity was inhibited by KCN, thiourea, and SO(2). Resveratrol levels were stable following the removal of proteins from the juice, suggesting that early spraying of grapes with SO(2) is an important factor affecting the final amount of resveratrol in wine.