十里香茶离体培养及再生体系研究

为了保护珍贵的十里香茶树资源,掌握利于十里香茶的最优组培条件,本研究对十里香茶组培过程中外植体类型、消毒时间、培养基添加物及培养条件进行了筛选。结果表明,采用轻微木质化的带腋芽茎段经升汞消毒15 min,接种后暗培养5 d,MS+0.2 mg·L^-1 NAA培养基中添加3 mg·L^-1 PVP+2000 mg·L^-1 Vc或者6 mg·L^-1 PVP+3000 mg·L^-1 AC,可在低污染率的前提下将褐化率降为10%左右,一定程度上解决了茶树组培中褐化率高的难题;采用1/8 MS+1 mg·L^-1 IBA培养基有利于外植体生根且增殖迅速。本研究建立了十里香茶的无菌苗离体培养及再生体系,可通过组培手段对其进行大量繁殖。     

Colonization and histopathology of susceptible and resistant carnation cultivars infected with Fusarium oxysporum f. sp. dianthi

Stems of the susceptible Early Sam and resistant Novada carnations were inoculated with a conidial suspension ofFusarium oxysporum f. sp.dianthi. Stem segments of either cultivar were sampled regularly and used for determination of fungal growth and for microscopical investigation.Early Sam showed typicalFusarium wilt symptoms and its stems were colonized intensively. The observed vascular browning appeared to be caused by discolouration of primary walls of infected vessels and surrounding cells. Vessels were rarely occluded with gel. Cell wall degradation led to the formation of stem cavities. Hyperplasia of xylem parenchyma was not seen.In Novada, fungal colonization remained low throughout the experiment. Macroscopic symptoms were absent except for longitudinal bursts in the stem, which appeared to be caused by hyperplasia of xylem parenchyma bordering infection. Vascular gelation occurred in the infected tissues, causing some vascular browning also. Xylem vessel regeneration was observed in the hyperplastic layer. Cavities were not formed, and wall discolouration was rare. Vascular gelation is considered part of theFusarium wilt resistance mechanism. It is followed by xylem vessel regeneration, which expresses a general plant response to vascular dysfunction rather than being part of the resistance mechanism.Although of different origin, vascular browning as such occurs in both susceptible and resistant interactions. In breeding for resistance, care should hence be taken with the current use of browning as an indication of disease.Samenvatting Anjers van de vatbare cultivar Early Sam en de resistente cultivar Novada werden geïnoculeerd met een conidiënsuspensie vanFusarium oxysporum f. sp.dianthi. Van beide cultivars werden regelmatig stengeldelen geoogst om deze microscopisch te onderzoeken en om de schimmelgroei te bepalen.Early Sam vertoonde de voor deze verwelkingsziekte kenmerkende symptomen en werd intensief gekoloniseerd. Aan het vaatweefsel waargenomen bruinkleuring bleek veroorzaakt te worden door verkleuring van de primaire wanden van geïnfecteerde vaten en de hen omringende cellen. Zelden trad er in de vaten gomvorming op. Celwandafbraak veroorzaakte de vorming van holten in de stengel. Hyperplasie van het houtparenchym werd niet waargenomen.In Novada bleef de schimmelgroei gedurende het hele experiment beperkt. Macroscopisch waren er enkel lengtescheuren in de stengel te zien, die veroorzaakt bleken te worden door hyperplasie van aan de infectie grenzend houtparenchym. In het geïnfecteerde vaatweefsel optredende gomvorming veroorzaakte ook enige bruinkleuring. In het hyperplastische weefsel werd regeneratie van houtvaten waargenomen. In de stengel werden geen holten gevormd, en verkleuring van de celwanden kwam weinig voor. De vorming van gommen in de houtvaten maakt waarschijnlijk deel uit van het resistentiemechanisme. De daarop volgende houtvatregeneratie is eerder een algemene reactie van de plant op vaatverstopping dan een deel van het resistentiemechanisme.Vaatverbruining, zij het van verschillende oorsprong, komt voor in zowel vatbare als resistente interacties. Om die reden moet men in de resistentieveredeling bij de anjer voorzichtig zijn met het gebruik van bruinkleuring als ziekteïndicatie.     

Effects of wood char and activated carbon on the hydrolysis of cellobiose by β-glucosidase from Aspergillus niger

Biochar amendments to soils have been suggested as a strategy to sequester carbon and therefore mitigate global climate change. The enrichment of soils with charred materials also increases their fertility. This fertilising effect of biochar may be caused by various mechanisms; an acceleration of nutrient cycling has been suggested as one such mechanism. The rate-limiting step in nutrient cycling is thought to be the extracellular enzymatic attack on biological macromolecules. In this study, therefore, the effects of chestnut wood char (specific surface area 2.0 m² g⁻¹) and of activated carbon (specific surface area approximately 900 m² g⁻¹) on an extracellular enzymatic reaction involved in the degradation of cellulose (i.e., hydrolysis of cellobiose by β-glucosidase from Aspergillus niger) were investigated. Cellobiose was not adsorbed by chestnut wood char, whereas activated carbon absorbed more than 97% of it. Both charred materials adsorbed more than 99% of β-glucosidase. For chestnut wood char, adsorption of the enzyme caused a decrease of approximately 30% in the reaction rate, whereas for activated carbon, the nearly complete absorption of both substrate and enzyme entirely inhibited the reaction. These results show that β-glucosidase from A. niger retains most of its activity when adsorbed to chestnut wood char and that the reaction it catalyses in nature is only slightly affected by this charred material. On the other hand, a material characterised by a high specific surface area and high porosity, such as activated carbon, can make even a highly soluble substrate unavailable for soil enzymes and therefore completely inhibit the reaction. Thus, charred materials may affect nutrient cycling mainly by regulating the availability of substrates: the degradation of highly soluble substrates may be accelerated by materials with low specific surface area, which maintain an active and protected enzyme pool, whereas materials with high specific surface and high porosity may slow down the degradation by making substrates unavailable.     

一株曲霉的一种中温糖化酶的纯化及其部分酶学特性

本研究从广西花坪自然保护区采集的土壤中筛选获得了一株产糖化酶丝状真菌菌株57-45,通过形态学观察和真菌内转录间隔区（internal transcribed spacer,ITS）序列比对分析,将其初步鉴定为曲霉属（Aspergillus sp.）的一个种。纯化真菌57-45所产的一种胞外糖化酶经过硫酸铵分级沉淀、疏水层析和阴离子交换层析三步蛋白质纯化步骤,得到在SDS-PAGE上约60kD的单一蛋白质条带,薄层层析分析表明该纯化的蛋白质水解可溶性淀粉的产物只有葡萄糖,证明该纯化的蛋白质为糖化酶。纯化的糖化酶Km值为1.9mg/mL,Vmax为4148μmol/（min·mg）,最适作用pH5.5,最适作用温度50℃,在同步糖化发酵中有应用的潜力。金属离子Fe3＋、Zn2＋、Cu2＋对酶活有较强的抑制作用,EDTA对酶活有较强的促进作用。本文结果将为进一步研究曲霉糖化酶的酶学特性提供新的材料。     

快速脱水抑制葡萄干制过程中膜脂过氧化及褐变

为探索脱水速度对无核白葡萄干制过程中膜脂过氧化作用及褐变的影响,以新疆无核白葡萄为试验材料,经过葡萄促干剂处理后,采用快速脱水和缓慢脱水2种处理,质量每减轻10%进行取样,测定脱水过程中果实干基含水率、干燥速率、褐变度、细胞膜透性、丙二醛(malonaldehyde,MDA)含量、脂氧合酶(lipoxygenase,LOX)、超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、过氧化物酶(peroxidase,POD)、多酚氧化酶(polyphenol oxidase,PPO)活性以及总酚含量的变化。结果表明:与缓慢脱水相比,快速脱水处理显著(P0.05)降低了无核白葡萄褐变度的上升,减少MDA生成量以及膜透性的增加,抑制LOX活性的升高,保持较高的活性氧清除酶SOD、CAT及POD活性及较高的总酚含量,且使PPO活性保持在一个较低的水平。因此认为,快速脱水可以有效地抑制无核白葡萄的膜脂过氧化作用对细胞膜的破坏,保持细胞的完整性,且PPO活性较低,从而减少无核白葡萄脱水褐变的发生。研究结果为快速脱水在无核白葡萄干制中的应用提供参考。     

产低温淀粉酶菌株的筛选、鉴定及酶学性质初步研究

对低温淀粉酶资源的开发是功能酶研究的新热点,本研究从东帕米尔高原江布拉克冰川冻土中分离获得多株高产低温淀粉酶菌株,最高产酶菌株D5-2经16S rDNA鉴定为假单胞菌属(Pseudomonas sp.)细菌.对D5-2所产淀粉酶性质进行初步分析,结果表明,其最适作用温度和pH值分别为25 ℃和6.5,酶的热稳定性较差,40 ℃处理1h后酶活急剧下降,至70℃酶活力仅存23％;在pH 5.5～8.0条件下,酶活力相对稳定;Mn2+和Mg2+对淀粉酶有激活作用,而乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)、Fe2+和Na+则抑制酶活.结果提示,菌株D5-2分泌的淀粉酶符合低温酶特性,应用空间较大,值得深入探究开发.     

普鲁兰酶氮端氨基酸的截短对其酶学性质的影响

淀粉是丰富的生物质资源,已被广泛地应用到食品、酿酒、医药等各个相关的领域.普鲁兰酶(pullulanase,pulA,EC 3.2.1.41)作为淀粉脱支酶,可以水解淀粉中的α-1,6糖苷键,最大限度地降解淀粉原料,提高淀粉利用率.因此,寻找一条国产化的道路开发我国自己的普鲁兰酶,具有长远的意义.课题组前期从淀粉厂附近的土壤中筛到一株普鲁兰酶的高产菌株变栖克雷伯氏菌(Klebiella variicola)strain 7,克隆获得pulA基因(GenBank登录号:KJ146839.1)后在大肠杆菌(Escherichia coli) BL21 (DE3)中异源表达.为提高该蛋白的表达量和分泌效率,同时改善普鲁兰酶的性质,本研究利用基因工程的手段构建了普鲁兰酶去信号肽突变体M1和氮端31个氨基酸的截短突变体M2,结果显示M1和M2最适温度均为45 ℃,二者的最适pH分别是6.0和5.6;M2的半衰期是37 min,是M1的6.17倍;M2的比酶活是582.204U/mg,是M1的1.6倍.动力学分析显示,以普鲁兰糖为底物时,M2的Vmax、Kcat、Km和Kcat/Km分别为0.001 3μmoL/(mL·s)-1、191.80-1 s.-1、0.30 mg/mL、693.30,与M1相比,M2的底物亲和能力增加,同时催化效率是M1的2倍.本研究通过普鲁兰酶氮端氨基酸的截短,为提高酶的比酶活和半衰期提供了新的方法和思路.     

抑制荸荠中多酚氧化酶活性的研究

通过单因素及多因素试验,探讨抑制荸荠中多酚氧化酶活性的效果。L_9(3~3)正交试验结果表明:柠檬酸预煮液的质量分数0.5%,预煮温度100℃,预煮时间5 min为最优预煮条件。     

鲜切莲藕中褐变因素分析的研究进展

鲜切莲藕的褐变问题很大程度上限制了产品的生产和销售,分析其褐变因素对解决贮运保鲜问题具有重要意义。本文针对文献资料介绍的导致鲜切莲藕褐变的各因素(酚类物质、多酚氧化酶及环境条件)进行总结和分析,阐明各因素在保鲜贮藏中的变化规律,以期为莲藕加工提供理论借鉴和技术指导。