芽苗菜中蜡样芽孢杆菌的污染研究

为了控制芽苗菜工厂化生产中蜡样芽孢杆菌的源头污染,实验采用人工污染芽苗菜种子的方式,对芽苗菜生长、收获、储藏等环节进行蜡样芽孢杆菌变化的全程控制;采用电子扫描显微镜对蜡样芽孢杆菌的附着、内化进行观察;采用基质解析电离飞行时间质谱(MALDI-TOF MS)对蜡样芽孢杆菌进行定性鉴定;利用Baranyi Roberts模...     

硝态氮垂直运移过程中的影响因素研究

用模拟土柱的方法对黄淮海平原主要土壤类型中硝态氮垂直运移的影响因素进行了研究。结果表明:在水分饱和条件下,硝态氮的垂直运移过程主要受土壤粘粒含量的影响,随着粘粒含量的增加硝态氮出流的时间推迟,硝态氮的穿透曲线的峰值变低、峰面分布变宽;在非饱和条件下硝态氮的穿透曲线变得不规则,有明显的拖尾现象,硝态氮运移的时间增长,穿透曲线变得平缓;示踪剂的流速对硝态氮运移有较大的影响,流速大的穿透曲线峰值高于流速小的穿透曲线峰值,且出流的时间和达到平衡的时间提前;不同价态阳离子由于带的电荷量不同,对硝态氮运移略有影响,表现为与硝态氮结合的高价阳离子产生的穿透曲线的峰值略高于低价阳离子;不同浓度的硝态氮示踪剂对硝态氮运移也有影响,在重力势和溶质势的共同作用下,浓度越高出流速度越快,穿透曲线峰值也高,完成出流的时间也迅速。     

Influence of Desiccation on the Transport of <Emphasis Type="Italic">Escherichia coli</Emphasis> through Saturated Sand Packs

Bacterial cells that enter the groundwater system commonly experience desiccation stresses (i.e., bacterial cells are directly exposed to air) when traveling through the unsaturated layer of soil. Little is known about the effects of desiccation on the transport of bacterial cells in the groundwater system. In this research, we investigated the transport of desiccated and non-desiccated Escherichia coli K12 (ATCC 10798) cells through saturated sand packs using laboratory column transport experiments. Cell desiccation was performed at 25°C under relative humidity (RH) levels of 22%, 53%, 75%, and 97%, respectively, and the desiccation duration was 22 h. Our results showed that desiccation reduced the viability of E. coli cells under all RH levels and increased the transport of E. coli cells under ≥75% RH levels. The increase in the transport of the desiccated E. coli cells was not related to changes in cell size or cell zeta potential. Desiccation under high (i.e., ≥75%) RH levels, however, led to lower cell hydrophobicity, which was found to be positively correlated with cell transport.     

蜡状芽胞杆菌群三大类全基因组序列的同源性分析

全基因组比对一般采用全基因组范围内的序列比对,本文首次采用单基因在全基因组范围内进行基因排列比对,选取了蜡状芽胞杆菌群中的44个必须基因,在该群中3大类共5个(Bt.97-27,Bc.14579,Bc.10987,Bc.E33L和Ba.Ames Ancestor)全基因组进行横向比对,所有基因在各个基因组中的排列顺序完全一致,少有交叉,进一步证明:在蜡状芽胞杆菌群中,特别是在蜡状芽胞杆菌、苏云金芽胞杆菌和炭疽芽胞杆菌之间,存在相当大的基因相似性,这与前人的研究结果一致。这种相似性有利于将来用于在该群中所有菌株的全基因组测序装配中。     

蜡质芽孢杆菌超氧化物歧化酶(SOD)的纯化研究

蜡质芽孢杆菌经培养后,离心收集菌体,将菌体破碎,离心,经（ＮＨ４）２ＳＯ４分级沉淀,Ｓｅｐｈａｄｅｘ Ｇ－７５凝胶过滤和ＤＥＡＥ－５２柱层析所获得的超氧化物歧化酶进行了ＳＤＳ－ＰＡＧＥ电泳为一条带,亚基分子量为１７７８３Ｄ,表明制备得到了电泳纯品。将该样品经氰化钾,双氧水,氯仿－乙醇抑制实验和特征吸收光谱分析,鉴定出此酶为Ｆｅ－ＳＯＤ,比活为：１７５３．８ｕ。     

蜡样芽孢杆菌ATCC14579毒力基因plcR缺失株的构建及其一般特性

蜡样芽孢杆菌是土壤中的优势菌,具有作为益生菌的潜在能力,同时它也是条件致病菌,能引起食物中毒等。蜡样芽孢杆菌的多种毒力因子受到多效性调控子（pleiotropic regulator,plcR）的调控,在其条件致病性作用中起着重要作用。真养产碱杆菌JMP34（Alcaligenes eutrophus）质粒上的2,4-二氯苯氧乙酸（2,4-dichlorophenoxyacetic acid,2,4-D）单加氧酶（tfdA）基因可以降解2,4-D。本研究利用同源重组技术使tfdA基因置换掉蜡样芽孢杆菌的plcR基因,构建了蜡样芽孢杆菌ATCC14579突变株B.cereus△plcRΩtf-dA,并对其毒性、一般生理生化特性进行分析。研究结果表明,突变株B.cereus△plcRΩtfdA的毒性显著减弱;生理生化实验结果显示突变株与野生株并没有明显区别,且突变株并没有表现出tfdA酶活性。所有的结果表明plcR控制着蜡样芽孢杆菌ATCC14579的致病性,同时剔除plcR并不破坏其酶系统。本研究为今后构建蜡样芽孢杆菌工程菌提供了新的思路和依据。     

蜡状芽孢杆菌HY-1降解甲基对硫磷和毒死蜱的影响因素研究

采用富集驯化培养和紫外分光光度计定量的方法,从农药生产企业的废水处理系统中分离筛选出1株能够降解甲基对硫磷和毒死蜱的蜡状芽孢杆菌（Bacilluscereus）HY-1,并系统研究了影响其降解甲基对硫磷和毒死蜱的主要因素。研究表明,菌株HY-1能够利用甲基对硫磷和毒死蜱为唯一磷源降解农药。HY-1降解甲基对硫磷的适宜条件为：培养温度30～35℃,pH为6～8,甲基对硫磷初始浓度为10～50mg·L^-1,接种量20%（体积比,菌体密度：稀释到菌悬母液（OD600=3.0）的0.8～1倍）,添加葡萄糖不能促进菌株对甲基对硫磷的降解。HY-1降解毒死蜱的适宜条件为：葡萄糖浓度6g·L^-1,培养温度30～35℃,pH为7.0,毒死蜱初始浓度80～200mg·L^-1,接种量20%（体积比,菌体密度：稀释到菌悬母液（OD600=3.0）的0.8～1倍）。结果表明,HY-1菌株降解甲基对硫磷和毒死蜱的适宜条件相类似,只是降解所需的最适葡萄糖浓度和底物浓度不同。     

Enumeration and confirmation of Bacillus cereus in foods: collaborative study

A collaborative study was conducted in 15 laboratories to evaluate 2 different techniques for enumerating Bacillus cereus in foods. A direct plating technique using mannitol-egg yolk-poly-myxin agar and a most probable number (MPN) technique using trypticase-soy-polymyxin broth were compared for the enumeration of high and low populations of B. cereus in mashed potatoes. The collaborative results showed that the overall mean recovery obtained with the low population level was essentially the same by both techniques. However, the overall mean recovery was significantly higher by the direct plating technique at the high population level. A statistical evaluation of the data also showed that the direct plating technique had better repeatability and reproducibility than did the MPN technique at both the high and low population levels. These results suggest that the MPN technique is suitable for examining foods containing low populations of B. cereus, but that the direct plating technique is preferable for foods that contain a high population of this organism. The confirmatory technique used in the proposed method is reliable for presumptive identification of isolates as B. cereus. The method has been adopted as official first action.     

Transport of microorganisms through soil

Microorganisms migrating into and through soil from sources on the land surface may cause a serious threat to both ground and surface waters. It has been estimated that microorganisms can migrate significant distances in the field. Results from various studies suggested that preferential flow through macropores, worm holes, cracks, and fractures is the main reason for such observations. However, a quantitative representation of this phenomenon has not been provided. Microorganisms migrate through soil by advection and dispersion, while being subjected to effects of filtration, adsorption, desorption, growth, decay, sedimentation and chemotaxis. Both laboratory and field investigations have contributed important information on bacterial movement in soils. Qualitative comparisons are generally transferable from laboratory to field situations. Quantitative agreement is much more difficult to establish. Available mathematical modelling of microbial transport is limited in practical application because of the simplifying assumptions used in its development.     

蜡状芽孢杆菌抗重金属性能及对镉的累积

所研究的蜡状芽孢杆菌RC可以在镉浓度为200mg·L-1的固体培养基平板上生长良好,表明菌株具有强抗镉的能力。该菌株在液体培养基中Cd2＋、Cr3＋、Pb2＋浓度均为75mg·L-1和Mn2＋浓度为100mg·L-1培养时,菌株生长正常。在重金属Cr3＋、Pb2＋、Mn2＋存在时,采用红外光谱与原子吸收光谱分析菌株对Cd2＋的积累,结果表明,培养基中其他重金属离子的加入,会影响菌株对Cd2＋的积累率;当Cr3＋存在时,Cr3＋可以增加细胞壁上有效官能团活性,明显提高RC菌体对Cd2＋的积累率,而其他重金属组合对Cd2＋吸附积累能力影响不大;RC细胞壁上活性基团-OH、-NH-、-COOH、-PO34-和-M-O（O-M-O）活跃参与Cr3＋、Pb2＋、Mn2＋和Cd2＋多种重金属离子的络合作用。通过高温和十二烷基硫酸钠处理菌株进行质粒消除试验,未发现该菌株的抗镉性质与抗性质粒的存在相关。     

蜡样芽孢杆菌及其生物被膜对有机酸及乙醇的抗性比较

为比较蜡样芽孢杆菌及其生物被膜对环境压力的抗性,该文主要研究了4种有机酸（乙酸、柠檬酸、乳酸和苹果酸）和乙醇对蜡样芽孢杆菌及其生物被膜存活率的影响。结果表明：生物被膜态蜡样芽孢杆菌对乙酸的抗性高于浮游态菌。扫描电镜结果显示,经乙酸处理后,浮游态蜡样芽孢杆菌的细胞表面严重受损,而生物被膜态菌的表面形态未发生明显变化。在柠檬酸、乳酸、苹果酸和乙醇存在的条件下,生物被膜态蜡样芽孢杆菌比浮游态菌表现出更强的压力抗性,特别是在高浓度（有机酸16%～20%,乙醇50%～60%）的情况下,该现象尤为显著。因此,在食品工业中控制被膜态蜡样芽孢杆菌对预防和阻止食品腐败非常重要。该研究结果为实际生产中有效控制蜡样芽孢杆菌及其生物被膜的形成提供参考。     

蜡样芽孢杆菌M22锰超氧化物歧化酶基因在毕赤酵母中的表达

实验成功地构建了毕赤酵母（Pichia pastoris）表达质粒pPICZA—Mn—sod,质粒线性化后通过电激法导入毕赤酵母GSl15，抗生素zeocin抗性梯度筛选得到高拷贝重组菌株。PCR鉴定及Mut^＋表型分析表明，目标基因已经重组到宿主基因组染色体上；0．5％甲醇诱导表达后，SDS—PAGE结果显示，表达蛋白的相对分子量约为23kD，活性电泳出现明显活性条带；酶活性测定显示，重组菌株SOD活性比对照提高5倍左右；氯仿-乙醇（3：5／V：V）和KCN（5mmol／L）抑制反应进一步证明，所表达的SOD为锰超氧化物歧化酶C。来源于蜡样芽孢杆菌（Bacillus cereus）M22的Mn—sod基因在毕赤酵母中得到正确表达。为研究该酶的生理功能提供了必要的物质条件。     

Antimicrobial synergistic effect of linolenic acid and monoglyceride against Bacillus cereus and Staphylococcus aureus

The antimicrobial effect of linolenic acid with or without monoglyceride (glycerol laurate or glycerol myristate) against six food-borne microorganisms was determined in broth medium. Minimum inhibitory concentration of linolenic acid on Bacillus cereus and Staphylococcus aureus was 20 and 50 ppm, respectively. The growth of B. cereus treated with linolenic acid at 10 ppm with 10 ppm monoglyceride was more inhibitory than that of linolenic acid alone, and the viable cell population was reduced 2-4 log cycles compared to that of the control. When linolenic acid was added at that level, the adenosine triphosphate (ATP) concentration of extracellular fluid was drastically increased compared with that of the control, and the combined effect with monoglyceride was higher than that with linolenic acid alone. However, the intracellular ATP concentration decreased compared with that of the control. From these results, we concluded that linolenic acid has a strong antimicrobial activity against B. cereus and S. aureus, and that linolenic acid combined with monoglyceride showed stronger antimicrobial activity than using linolenic acid alone.     

一株产耐高温蛋白酶蜡样芽胞杆菌的分子鉴定和酶学性质研究

为筛选具有工业应用价值的产蛋白酶新菌种,本研究采用酪素培养基进行新菌株筛选,利用生理生化试验和16S rDNA测序进行菌种鉴定,并结合生化方法测定其蛋白酶的酶活。结果表明,本研究筛选的一株产蛋白酶菌株HSU-2 (GenBank登录号:MN315516)为蜡样芽胞杆菌(Bacillus cereus)。该菌种产蛋白酶的最适pH值和温度分别是7.0和60℃,为一种耐高温的中性蛋白酶。该蛋白酶可耐受5.0%过氧化氢、5.0%十二烷基磺酸钠和5.0%去污剂Triton X-100作用;且5.0%去污剂吐温80可显著提高该蛋白酶的酶活力。此外,Mg²⁺、Zn²⁺、K⁺、Ni²⁺和Cu²⁺ 5种金属离子可显著降低该蛋白酶酶活力,其中Zn²⁺抑制作用最强;而Ca²⁺可提高该蛋白酶酶活力。本研究为产蛋白酶菌株HSU-2的应用开发和大规模生产奠定了理论基础。     

黄泛风沙区耕地土壤风蚀影响因子的通径分析

[目的]研究黄泛风沙区影响耕地土壤风蚀量的主要因子及其相互作用关系,为土壤风蚀的防治及改善耕作措施提供依据。[方法]通过影响因子的的野外定位观测,及对风蚀数据进行收集整理,在逐步回归分析的前提下,对风蚀量主要影响因子进行通径分析。[结果]风速累计时间、作物盖度、粗糙度是影响耕地土壤风蚀的主要因素;风蚀量的直接影响因素作用大小的排序为风速累计时间粗糙度作物盖度;风蚀量间接影响因素作用大小的排序为粗糙度作物盖度风速累计时间;风蚀量决定系数排序为d_(风速累计时间·风速累计时间)d_(风速累计时间·作物盖度)d_(风速累计时间·粗糙度)d_(作物盖度·粗糙度)d_(作物盖度·作物盖度)d_(粗糙度·粗糙度);风速累计时间与作物盖度主要通过其本身直接影响风蚀量,粗糙度主要通过间接作用减少风蚀量。[结论]可适当地增加作物种植密度,提高粗糙度、作物盖度,减少风蚀危害。     

The electro-ultrafiltration method for controlling the effect of Bacillus cereus on phosphorus mobilization in a calcareous soil

Summary The effects of phosphate-solubilizing bacteria on the phosphorus mobilization and P utake by Lolium multiflorum from a calcareous soil were investigated using a greenhouse pot experiment. The soil was inoculated with Bacillus cereus and the pots were either planted with Lolium multiflorum or left fallow. Treatments were designed to study the effect of inoculation of phosphate-solubilizing bacteria on phosphorus dynamics throughout the experiment. The dynamics were studied by the electro-ultrafiltration method, the results of which were used to interpret phosphorus mobilization in the soil. Phosphate uptake by L. multiflorum from the inoculated soil showed a 40% increase over the control soil, and an increase in total dry matter of 50%.     

生防菌对青枯雷尔氏菌的致弱特性

青枯雷尔氏菌(Ralstonia solanacearum)的致病性与弱化指数存在着特定的相关性,弱化指数＜0.60时,菌株回接番茄组织培养苗的发病率为100%,定义为强致病力菌株;弱化指数＞0.80,回接发病率为0,定义为无致病力菌株,弱化指数介于0.60和0.80之间,回接发病率5.3%～100%,菌株的致病性为不确定性,定义为过度性菌株.010703-01菌株进行培养致弱、物理致弱、化学致弱和生物致弱的实验结果表明培养时间延长至120 h未发现致弱作用继代培养11代以前未出现明显致弱;物理因子超声波处理无致弱作用;化学因子农用链霉素处理有抑制作用,但无致弱作用;生物因子15种细菌的致弱作用可用聚类分析将之分为三类,第一类具致弱特性,包括青枯病生防菌ANTI-8098A(Bacillus cereus)和球形芽孢杆菌(B.sphaercus),第二类具抑制特性,而无致弱作用,包括Bt-9408(B.thuringiensis)等3种芽孢杆菌;第三类抑制作用低和无致弱作用,包括阴沟肠杆菌(Enterobactercloacae)和未知学名的10株芽孢杆菌(Bacillus spp.)等.研究结果表明,生防菌对青枯雷尔氏菌的致弱作用与因其它因素产生的致弱作用有着本质区别.     

Bt cry I A(c)杀虫基因向棉花内生细菌Bacillus cereus染色体中整合的研究

将来自Bacillusthuringiensis subsp.kurstaki的BtcryIA(c)杀虫基因通过综合质粒载体pBC601,整合到棉花(Gossypium hirsutum)优势内生细菌Bacillusceieus(Bc9002)的染色体上,得到的工程菌对棉铃虫(Helicoverpa armigera Hb.)有杀虫活性.此综合质粒含有能在营养期表达Btcry IA(c)基因的强启动子、cryIA纠杀虫基因、四环素抗性标记基因tet'、8.0kb的EcoRI-NcoIB.ceieus染色体片段.将综合质粒通过电击导入B.cereus(Bc9002)中,综合质粒因含B.cereus染色体片段,可与B. cereus染色体发生同源重组,从而将BtcryIA(c)基因整合到B.cereus的染色体上.通过对转化子的DNA酶切分析、PCR扩增、SDS-PAGE凝胶电泳检测、ELISA检测、电镜观察、毒力测定,结果表明Btcry IA(c)基因已经整合到内生细菌Bc9002的染色体上,并可高效表达.     

水分对沙生先锋植物生长的影响

结合荒漠地节水灌溉试验，对具有典型代表性的3种沙生先锋植物进行了土壤含水量，植株相对含水量以及生物量的测定，进一步掌握了沙生植物极其抗旱的特性，总结了水分对沙生植物生长的影响。     

锰超氧化物歧化酶（Mn-SOD）基因的分子改造及在毕赤酵母中的表达

根据毕赤酵母（Pichia pastoris）的密码子偏好性,以不改变氨基酸序列为原则,对源于蜡样芽孢杆菌M22（Bacillus cerues M22）的Mn-SOD基因进行分子改造,设计、合成新的基因序列Mn-SOD-2。构建酵母表达载体pPICZαA/Mn-SOD-2,并整合至毕赤酵母GS115染色体。结果表明,所构建的酵母工程菌株YM103,经0.5%甲醇诱导表达后,Native-PAGE检测证实有清晰活性条带;SDS-PAGE检测证实重组蛋白的分子量为24kD,与预期大小一致。酶活分析表明,外源蛋白的活性较改造前增加了2.2倍,且表达稳定性良好。