枯草杆菌SOD高产菌株的诱变选育及产酶条件研究

本实验采用低能氮离子注入技术对枯草芽孢杆菌(Bacillus subtilis)进行辐照诱变处理,选育出一株SOD高产菌株(编号为BsB8)。其生物量略高于出发菌株、SOD产量达3439．3U／g湿菌体,为实验出发菌的1．88倍。该菌株最佳产酶条件为：起始pH为8．2,装液量为150ml／500ml,接种量为1．5％;添加Mn^2 的培养基可显著提高SOD酶活。     

高生物量富铁酵母菌的选育及其发酵条件的研究

对402株不同种属的酵母菌株进行初筛、复筛,筛选到一株生物量较高的二倍体菌株ZY-46(Saccharomyces cerevisiae)和一株铁富集量较高的二倍体菌株ZY-173(Saccharomyces kluyveri)。然后以它们为出发菌,分别进行单倍体分离、硫酸二乙酯(DES)诱变,并通过原生质体融合,得到一株高生物量富铁酵母融合菌株ZYF-15。在优化的发酵条件下,该融合菌株生物量可达11．2g／L,细胞铁含量达24．5mg／g干细胞,细胞总铁含量分别比原始亲株ZY-46和ZY-173提高了2．6倍和1．9倍。     

高生物量富硒酵母的选育及培养条件初步优化

通过筛选、单倍体分离、诱变和原生质体融合,从融合子中选育了一株高生物量富硒酵母菌株（编号为ZFF-28）,其细胞硒总含量分别是原始亲株ZY-67和ZY-198的2.8倍和2.0倍。通过单因素实验和正交试验设计,确定了优化培养条件：6%糖浓度的蔗糖糖蜜,添加0.5% (NH4)2SO4、0.1% H3PO4、60μg/mL Se,pH60～6.5,装液量50mL/250mL三角瓶,接种量10%,培养时间25h。在优化培养条件下,菌株ZFF_28的生物量可达8.2g/L,细胞中硒的含量达2050μg/g,硒总含量达到了16810μg/L,是培养条件优化前的1.3倍。细胞硒含量的91%为有机硒。     

番茄灰霉病病原菌分离鉴定及拮抗菌筛选

灰葡萄孢菌(Botrytis cinerea)引起的番茄灰霉病是番茄生产上的重要病害。从番茄果实表面成功分离了1株真菌BC2016-2,经过鉴定确定其为灰霉病的病原菌——灰葡萄孢菌;以灰葡萄孢菌BC2016-2为指示菌,筛选到了3株具有明显抑制作用的菌株,分别为解淀粉芽孢杆菌(Bacillus amyloliquefacien)CM3、巨大芽孢杆菌(Bacillus megaterium)Y-30和解淀粉芽孢杆菌Y-48。盆栽实验结果显示,菌株CM3、Y-30和Y-48对灰葡萄孢菌BC2016-2引起的番茄灰霉病的防治效果分别为65.58%、54.1%和72.13%。     

SOD高产菌株乳酸菌的选育及其产酶条件的研究

采用常规筛选方法从 200多株不同种属的乳酸菌中筛选出一株超氧化物歧化酶(SOD)产量较高的菌株 (Sn 898)作为实验出发菌株 ,经紫外线 (UV) ,硫酸二乙酯(DES) ,亚硝基胍 (NTG)复合诱变 ,选育出一株 (Lactobacillusplantarum-578简写L .plan-578)SOD产量高达6400u/g湿菌体的高产菌株。该突变株的SOD产量较出发菌株提高了3.5倍 ,并研究了影响SOD产生的最适温度 ,起始pH值 ,通气量 ,培养时间等因素。在优化     

高产Y-氨基丁酸酵母菌株的亚硝基胍诱变选育

目的：探讨亚硝基胍诱变选育高产Y-氨基丁酸酵母菌株的方法。方法：使用亚硝基胍对酵母菌株进行诱变;采用含溴甲酚绿的YEPD培养基筛选突变菌,采用薄层层析法和比色法鉴定变异菌株发酵液中的Y-氨基丁酸及其含量;对突变菌株连续继代培养4代,测定各代发酵液中Y-氨基丁酸的含量,鉴定诱变菌株的遗传稳定性：结果：亚硝基胍诱变酵母的最佳浓度为1．0g．L^-1,最佳诱变时间为15min;获得了5株突变菌株,菌落呈绿色;薄层层析法鉴定突变菌株都能产Y-氨基丁酸;诱变菌发酵液中的Y-氨基丁酸含量各异,但高于对照,且增长幅度很大;对突变菌株后代遗传稳定性进行了鉴定,结果表明突变菌株4遗传性较稳定。结论：采用1．0g．L^-1的亚硝基胍溶液处理酵母菌15min,经筛选鉴定,获得了一株遗传稳定的高产Y-氨基丁酸的酵母菌株。     

产类胡萝卜素酵母菌原生质体的制备、再生与诱变

确定了1株产类胡萝卜素红酵母Y-35的原生质体最佳制备条件和再生条件,以及在此基础上的诱变育种,通过实验,初步确定Y-35菌株原生质体形成和再生的适宜条件为;菌龄16h,蜗牛酶浓度1%,30℃处理60min。红酵母Y-35原生原体经紫外线诱变后得到18株诱变菌株,分别测定其生物量,类胡萝卜素含量及产量,获得2株类胡萝卜素产量明显提高的变异菌株RY-10和RY-19。其产量分别比出发菌株提高49%和54%。     

一株硅酸盐细菌的鉴定及其紫外线诱变育种

从油用牡丹的根际土壤中分离到一株硅酸盐细菌,将其编号为CLM8。采用火焰光度法对该菌株在30℃、160 r/min条件下发酵培养7d的发酵上清液中水溶性钾素含量进行测定,最高含量达到0.9μg/mL,故将CLM8定性为硅酸盐菌株。综合CLM8的显微观察特征、生化特征和16S rRNA序列的克隆分析结果,确定菌株CLM8为成团泛菌属中的一个种,暂命名为Pantoea sp.CLM8。为提高野生菌株CLM8的解钾活性,通过对其紫外线诱变选育,筛选得到活性较高的突变株CLM8-B,其在30℃、160 r/min条件下发酵培养过程中,发酵上清液中水溶性钾素含量最高达到1.7μg/mL,是出发菌株CLM8的1.9倍。CLM8可作为油用牡丹及其它作物生物钾肥开发的备用菌株。     

不同类型菌根菌对烟草幼苗生理代谢的影响

在自然土壤中以VA菌根菌接种烟草幼苗,测得叶片中N、P、K、叶绿素含量,硝酸还原酶活性及蛋白质含量均高于对照;与对照相比,接种的幼苗中丙二醛（MDA）、过氧化氢（H2O2）含量减少,超氧物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）活性增加,同时以外生菌根菌两个菌株分别接种,与所测指标变化趋势相同,而且其中的Calvatia lilacina菌株超过VA菌根菌接种的效果。     

啤酒酵母工业菌株单倍体的诱导、分离和鉴定

【目的】探索适宜的方法进行啤酒酵母工业菌株单倍体的诱导、分离和鉴定,为啤酒酵母改良和遗传学研究提供便利。【方法】首先,选择产孢效果最好的培养基进行产孢诱导,诱导产生的孢子在YPD培养基上形成菌落后,用流式细胞技术检测其DNA含量,进而判断其倍性;单倍体菌株的交配型通过MAT-PCR和杂交实验确定。【结果】啤酒酵母工业菌G-03通过产孢诱导和孢子分离、富集后得到26株菌,最终通过流式细胞技术确定了其中4株为单倍体,MATa和MATα型各2株。通过扫描电镜法观察4株单倍体菌株及出发菌G-03的细胞形态,发现单倍体菌株的形态和出发菌有较大区别,单倍体菌株长期培养没有假丝生长的现象发生。【结论】啤酒酵母工业菌单倍体育种较为困难,严格的单倍体筛选、鉴定尤其具有挑战性。     

Purification and Characterization of Yeast β-Glucosidases

Constitutive beta-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) precipitated at the same concentration of ammonium sulfate. The partially purified enzymes had similar activation energies, molecular weights, affinities for certain natural and synthetic beta-glucosides, and optimal pH values for substrate hydrolysis, and they were stable over approximately the same pH range. The enzymes, however, could be clearly distinguished by other criteria. Affinities of the synthetic, sulfur-containing beta-glucosides for Y-18 enzyme were many times greater than for Y-19 enzyme. The latter enzyme was more resistant to heat. The two enzymes eluted from diethylaminoethyl cellulose at different concentrations of sodium chloride. In precipitin tests, homologous enzyme-antisera systems were highly specific. The beta-glucosidase synthesized by a hybrid, S. fragilis x S. dobzhanskii (Y-42), was unique. Characterization of this enzyme produced values which were intermediate to those for the enzymes from the parental yeast strains. Heat-inactivation slopes and Lineweaver-Burk plots for the Y-42 enzyme were anomalous. It is suggested that hydrolytic activity in Y-42 preparations is due to a spectrum of hybrid enzyme molecules composed of varying amounts of two distinct polypeptides. It is further suggested that these polypeptides may be identical to those synthesized by the parental Y-18 and Y-19 yeast strains.     

Isolation and Characterization of Bile Acid 7-Dehydroxylating Bacteria from Human Feces

Methods for isolation of fecal 7α-dehydroxylating bacteria are presented. A total of 219 strains were isolated from feces of healthy humans, and their ability to 7-dehydroxylate cholic, chenodeoxycholic, and ursodeoxycholic acids were examined. Of all the isolates, 14 strains were found to be capable of eliminating the hydroxy group at C-7α and/or C-7β. All the isolates were strictly anaerobic, Gram-positive rods. Thirteen isolates were non-sporeforming bacteria showing certain saccharolytic properties with the production of acid and gas from dextrose, and were catalase-positive but indole-, lecithinase-, urease- and oxidase-negative. Based on the data available at present, it was concluded that they could be regarded as members of the genus Eubacterium. One strain, however was identified as Clostridium sordellii. The isolated strains capable of 7α-dehydroxylating cholic acid and chenodeoxycholic acid were also able to oxidize the hydroxy group at C-7α. Nine strains (10, 12, 36S, M-2, M-17, M-18, Y-98, Y-1112, and Y-1113) of the 7α-dehydroxylating bacteria were confirmed to have 7β-dehydroxylation ability, but five strains (O-51, O-52, O-71, O-72, and Y-67) could not transform ursodeoxycholic acid to lithocholic acid.     

高效降解棉酚菌株的分离鉴定及诱变选育

作为一种蛋白资源, 棉籽粕因其含有毒素——游离棉酚限制了其在饲料工业中的应用。为获得能高效降解棉籽粕中棉酚的菌株, 以醋酸棉酚为唯一碳源培养基从16份样品中筛选出一株高效降解棉酚菌株(Y-2), 经生理生化及18S rDNA鉴定为Pichia guilliermondii, 此菌种为非致病酵母, 且首次报道用于棉酚的降解, 在工业生产中具有潜在的应用前景。通过紫外诱变获得一株棉酚降解率更高的突变株YUV-51。通过对突变株YUV-51发酵温度、时间及接种量的初步优化, 获得其固态发酵优化条件: 30 °C培养48 h, 接种量0.025 g湿菌体/g棉籽粕, 初始水分含量50%。为避免游离棉酚在前处理中大量降解棉籽粕, 不进行湿热处理, 经接种发酵后脱毒率可达到58%。这使微生物脱毒在实际生产中应用成为可能。     

不同寡营养培养条件下南海水体细菌群落结构及其对碳源的利用特征

【背景】绝大多数海洋微生物不可培养,为挖掘海洋生态系统中可培养的微生物资源,研究者尝试寡营养培养等方法。【目的】比较不同寡营养培养条件下南海水体细菌数量、群落结构及其对碳源的利用特征差异。【方法】采用原2216E培养液(Y)、稀释10倍(Y-10)和稀释50倍(Y-50)的2216E培养液培养南海海水样品,用荧光定量PCR法和16S rRNA基因检测细菌数量和菌群结构;利用平板计数法计数异养细菌的数量,纯化鉴定可培养细菌;采用Biolog EcoPlateTM微板法分析不同培养基中细菌群落对碳源的利用特征。【结果】Y组细菌总数高于Y-10组和Y-50组,差异不显著(P0.05),但异养细菌数量显著高于Y-10组和Y-50组(P0.05)。16S rRNA基因测序结果表明,不同稀释倍数下的细菌群落结构差异明显,Y组检测出10门193属,优势类群为Proteobacteria(56.44%)和Bacteroides (37.27%);Y-10组检测出15门220属,优势类群为Proteobacteria (40.30%)、Bacteroides(36.91%)和Firmicutes (17.30%);Y-50组检测出14门226属,优势类群为Proteobacteria (45.19%)、Bacteroides(25.29%)、Planctomycetes (13.58%)和Firmicutes(11.21%)。通过平板培养,Y组和Y-10组均分离到6属14株优势菌,Y-50组分离到7属13株优势菌,其中,Bacillus为其共有的优势菌属,稀释10倍培养液筛得的Microbacterium(1株)、Vibro(1株)、Idiomarina(1株)、Halobacillus(1株)共4株优势菌和稀释50倍培养液筛得的Alcanivorax(1株)、Sulfitobacter(1株)、Alteromonas(1株)、Pseudomonas (1株)、Exiguobacterium (2株)、Vibro (3株)共9株优势菌不同于原培养液。通过寡营养培养,可培养细菌群落的代谢活性和McIntosh指数显著增加(P0.05),其对聚合物、羧酸、氨基酸、糖类的利用率也显著提高(P0.05)。【结论】通过寡营养培养能增加细菌群落的丰富度和多样性,提高可培养细菌的代谢活性和对碳源尤其是聚合物、羧酸、氨基酸和糖类的利用率,分离纯化可获得原培养基未筛选得到的细菌。因此,在南海远洋海域可培养细菌样品的采集及复苏时,可通过寡营养培养法获得更丰富的南海可培养微生物资源。     

二倍体和四倍体杂交兰幼苗对低温胁迫的生理响应差异分析

为探究不同倍性杂交兰（Cymbidium hybrid）对低温胁迫的生理响应差异,对低温胁迫下（昼温10℃、夜温4℃）二倍体杂交兰以及四倍体杂交兰株系T1和T2幼苗叶片中脯氨酸、可溶性糖、可溶性蛋白质、MDA和叶绿素含量,O-·2产生速率以及SOD、POD和CAT活性的动态变化进行了测定和分析;在此基础上,比较了二倍体和四倍体杂交兰的耐低温特性差异。结果显示：随着胁迫时间的延长,二倍体杂交兰和四倍体杂交兰株系T1和T2幼苗叶片中脯氨酸、可溶性糖、可溶性蛋白质和MDA含量,O-·2产生速率以及POD和CAT活性总体呈逐渐增加的趋势;叶绿素含量呈逐渐下降的趋势;SOD活性呈先上升后下降的趋势,并分别在胁迫后第9天和第6天达到峰值。在常温（25℃）下恢复生长3d后,二倍体杂交兰和四倍体杂交兰株系T1和T2幼苗叶片中脯氨酸、可溶性糖、可溶性蛋白质和MDA含量,O-·2产生速率以及SOD、POD和CAT活性均降低,而叶绿素含量升高。总体上,二倍体杂交兰叶片中脯氨酸、可溶性糖和可溶性蛋白质含量以及SOD、POD和CAT活性的增幅明显小于四倍体杂交兰,MDA含量和O-·2产生速率的增幅则大于四倍体杂交兰,而叶绿素含量的降幅则明显大于四倍体杂交兰。综合分析结果表明：在低温胁迫下,四倍体杂交兰叶片中渗透调节物质含量和保护酶活性均较高,其对低温的耐性强于二倍体杂交兰。     

Comparison of the catalytic and immunological properties of beta-glucosidases from three strains of Saccharomyces lactis

beta-Glucosidase from Saccharomyces lactis strains Y-123 (B(h)), Y-14 (B(m)), and Y-1057A (B(1)) was partially purified. The pH optima, Michaelis constants, and activation energies were determined for the hydrolysis of p-nitro-phenyl-beta-d-glucoside by each of the enzymes. Differences among these constants were not enough to account for the low specific activity of beta-glucosidase in strains Y-14 and Y-1057A. Enzyme-inhibitor constants were measured for a series of alkyl and aryl glucosides. In general, the three enzymes are arylglucosidases. Tris(hydroxymethyl)-aminomethane inhibited all three enzymes in an uncompetitive fashion. The inhibition was antagonized by Mg(++). An antiserum was prepared to the highly purified (200-fold) beta-glucosidase from strain Y-123. The nature and degree of cross-reaction between the three beta-glucosidases was investigated by double diffusion in agar and neutralization tests. Spur formation in the immunodiffusion tests and similar equivalence points in the neutralization tests indicated a strong degree of cross-reaction between the three enzymes. The ratio of enzyme activity to antigenicity was used to compare the relative molecular activity of beta-glucosidase in the three strains. Each strain produced the same amount of beta-glucosidase per milligram of cell protein. The results are consistent either with a lower turnover number for the beta-glucosidase in strains Y-14 and Y-1057A or with the production of beta-glucosidase with a "normal" turnover number and enough cross-reacting material to effectively reduce the specific activity to the observed levels.     

Efficacy of yeasts in the management of root-knot nematode Meloidogyne incognita,in Flame Seedless grape vines and the consequent effect on the productivity of the vines

Twenty-two yeast strains were screened for their efficacy in suppression of root-knot nematode Meloidogyne incognita on Flame Seedless grapevines. The most effective seven strains were chosen to study their effect on nematode population in vitro and in vivo as well as their impact on crop productivity and fruit quality. Data showed that all treatments significantly reduced the number of juveniles in vitro after 24 and 48 h. The highest percentage of nematode mortality was achieved by the application of Pichia gluilliermondii Moh10, Pachytrichospora transvaalensis Y-1240, Candida albicans Moh Y-5 and Geotichum terrestre Y 2162. Similarly, under greenhouse conditions, all treatments significantly reduced the number of juveniles and disease index compared to the untreated plants during both incubation periods (30 and 50 days). Under field conditions, the tested yeast strains as well as Nemacour (nematocide) resulted in a significant reduction (P<0.05) of populations of Meloidogyne incognita compared to the control in two successive seasons tested. The beneficial impact of application of yeast strains was reflected in crop productivity on Flame Seedless grapevines as well as fruit quality. Percentages of vegetative buds, flower buds, shoot length and leaf number were significantly enhanced as a result of application of the different yeast strains and Nemacour. Bunch weight, berry weight and berry numbers were significantly increased by Pachytrichospora transvaalensis Y-1240, Geotichum terrestre Y-2162 and Candida albicans Moh Y-5. Nemacour and other yeast strains did not show any significant differences compared to the infected vines. Percentage of total soluble solids (TSS%) increased by applying all yeast strains, whereas Nemacour treatment gave the highest value of both TSS percent and percentage of acidity and increased anthocyanins in the skin of berries.     

Purification of β-Glucosidase from Saccharomyces lactis Strains Y-14 and Y-1057A

Two strains of Saccharomyces lactis (Y-14 and Y-1057A), medium (B(m)) and low (B(1)) constitutive producers of beta-glucosidase, were grown in enriched medium. beta-Glucosidase was extracted by autolysis and purified by ammonium sulfate precipitation, gel filtration, and calcium phosphate gel adsorption-elution. The kinetics of release, purification, and stability of beta-glucosidase from strains Y-14 and Y-1057A were compared with the enzyme from strain Y-123. The ability of glycerol, sorbitol, and mannitol to stabilize the beta-glucosidases is presented. A lower molecular weight, labile form of the Y-14 enzyme is demonstrated. Differences in the initial specific activites of beta-glucosidase among the three strains are discussed.     

Physical and genetic characterization of linear DNA plasmids from the heterothallic yeastSaccharomycopsis crataegensis

Five strains of the heterothallic yeastSaccharomycopsis crataegensis have been previously shown to contain DNA and/or RNA plasmidlike molecules (Shepherd et al. 1987). Three DNA plasmids, designated pScrl-1,-2 and -3, were found in strain NRRL Y-5902, while two were identified in each of NRRL strains Y-5903 and Y-5904. DNA plasmids were not identified inS. crataegensis strains Y-5910 or YB-192. FourS. crataegensis strains (Y-5903, Y-5904, Y-5910 and YB-192) were also shown to possess double-stranded RNA (dsRNA) molecules not found in strain Y-5902 (Shepherd et al. 1987). Hybridization studies now demonstrate the DNA plasmids in Y-5903 and Y-5904 to be highly homologous to their respective size counterparts (pScrl-1 and pScrl-2) in Y-5902 and to show some homology to pScrl-3. Restriction endonuclease mapping studies confirm the linear nature of each plasmid and establish identical restriction maps for a 1.4 kilobase (kb) region in pScrl-2 and -3. This 1.4 kb region accounts for the hybridization homology of pScrl-2 and pScrl-3 noted by Shepherd et al. (1987) and for homology of the plasmids of Y-5903 and Y-5904 to pScrl-3 of Y-5902. The pScrl plasmids show no homology to the dsRNA molecules ofS. crataegensis, the 2 M circular DNA ofStaccharomyces cerevisiae, the killer plasmids ofKluyveromyces lactis, or the linear DNA plasmids ofPichia inositovora.In crosses between linear DNA plasmid-containing and dsRNA-containing strains, only progeny containing the pScrl plasmids were recovered. Poor spore viability and a lack of complete tetrad recovery limited the extent of the analysis, but the findings suggest a cytoplasmic mode of inheritance for these linear DNAs.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

ON THE ORIGIN OF INCIPIENT REPRODUCTIVE ISOLATION: THE CASE OF DROSOPHILA ALBOMICANS AND D. NASUTA

The nasuta subgroup of Drosophila consists of 12 known species classified within the immigrans group. D. nasuta and D. albomicans are two sibling species widely distributed throughout the Indo-Pacific tropics, which, although morphologically indistinguishable, have different meta-phase-chromosome configurations: chromosomes X and 3 are attached in D. albomicans, so that about 60% of its genes are sex-linked. Our experiments show that, at least in the laboratory, there is no sexual, mechanical, or gametic isolation between the two species. There is, however, hybrid “breakdown” expressed in three ways: 1) reduction in the number of F₂ hybrids produced per culture; 2) reduction in the fertility of F₂ (males) and F₃ (males and females) hybrid progenies; and 3) abnormal sex ratios in the progenies of crosses between strains of certain localities. In experimental populations, the karyotypes of both species are still present in substantial frequencies after 20 generations, although the frequencies of the two karyotypes vary depending on the geographic origin of the strains. Our results support the hypothesis that, in allopatry, the evolution of postzygotic isolation precedes that of prezygotic isolation. The mtDNA is polymorphic in both D. nasuta and D. albomicans and fairly similar between them. Assuming typical rates of mtDNA evolution, the two species would have diverged from each other about 500,000 years ago, whereas the African and Indian populations of D. nasuta (considered to be different subspecies by some authors) might have diverged some 350,000 years ago.