毛壳菌产抗真菌活性物质菌株筛选及种间原生质体融合研究

毛壳菌属很多种类具有重要生防价值，其生防机理包括对植物病原真菌的重寄生作用、诱导植物产生抗病性、产生抗真菌活性的次生代谢产物等。迄今，学界对毛壳菌的研究主要集中在毛壳菌的生防机理，毛壳菌活性次生代谢产物的分离等方面。本研究致力于产抗生素的毛壳菌的种间原生质体融合，从产抗生素毛壳菌菌株的筛选开始，进而对产抗生素的角毛壳菌进行诱变选育，最终用产不同抗生素的角毛壳菌与球毛壳菌进行种间原生质体融合。主要有以下五方面研究结果。 1、毛壳菌抗真菌活性物质产生菌株的筛选：不同毛壳菌菌株发酵液采用琼脂扩散法对植物病原真菌进行抑菌活性试验，结果显示，菌株CH08和CH23的发酵液对芒果炭疽、苹果炭疽和马铃薯晚疫菌具有抑制作用。菌株CH16和CH17的发酵液对芒果炭疽菌、苹果炭疽菌有抑制作用。菌株CH21发酵液对辣椒炭疽菌和西瓜枯萎菌有抑制作用。经形态学研究，菌株CH08、CH16、CH17和CH23鉴定为球毛壳菌，菌株CH21鉴定为角毛壳菌。对角毛壳菌与球毛壳菌菌株发酵液抑菌谱比较，发现角毛壳菌与球毛壳菌发酵液具有明显不同的抑菌谱，表明角毛壳菌与球毛壳菌产生不同的抗真菌活性物质。 2、角毛壳菌（CH21）和球毛壳菌（CH08）原生质体制备和再生条件研究：考察了菌龄、酶浓度、稳渗剂及其浓度、酶解温度、酶解时间及再生培养基对原生质体制备和再生的影响。用菌龄为生长54 h的角毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1.5 h，原生质体释放量2.02×107个/g；以PDA为再生培养基，0.7 mol/L的蔗糖再生稳渗剂，再生率可达51.45%。用菌龄为生长48 h的球毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1 h，原生质体释放量达1.57×108个/g；以PDA为再生培养基，0.7 mol/L的蔗糖为再生稳渗剂，再生率可达41.48%。 3、角毛壳菌（CH21）原生质体紫外诱变选育：以CH21为出发菌株，制备原生质体进行紫外诱变，诱变条件为：15 w紫外灯，距离30 cm，照射90 s，致死率80%~85%。建立了诱变菌株初筛的双层平板筛选模型。经平板初筛和摇瓶复筛，获得一株突变菌株CH21-I-402，其发酵液抑菌活性较出发菌株提高18.3%。 4、抗性标记菌株的获得：菌株CH21-I-402和CH08抗生素药敏试验表明， CH21-I-402菌株对潮霉素有抗性、对G418（Geneticin）敏感，菌株CH08对潮霉素和G418都敏感。根癌农杆菌EHA105介导的新霉素磷酸转移酶基因转化球毛壳菌，经PCR检测，新霉素磷酸转移酶基因成功转化进菌株CH08-GR70，CH08-GR120。转化子对G418抗性提高3～4倍，对潮霉素仍然比较敏感。 5、以G418和潮霉素抗性为筛选标记的原生质体融合与融合菌株AFLP分析：制备角毛壳菌CH21-I-402和球毛壳菌CH08-GR70原生质体，以35％的PEG6000为助融剂进行原生质体融合，以65 μg/ml的潮霉素和60 μg/ml G418为抗性筛选标记，获得46个再生菌株。再生菌株连续传代5代后，再生菌株表现出多种形态类型。利用AFLP技术对再生菌株及亲本菌株基因组DNA分析表明，再生菌株PF1、PF26为融合菌株。抑菌活性测试表明，融合菌株PF26发酵液对芒果炭疽菌和苹果轮纹菌有强的抑制作用，且抑菌活性比亲本球毛壳菌明显提高。 Chaetomium spp. have great potentials as biocontrol agents against a range of plant pathogens on the basis of its mycoparasitism, induced plant disease resistance, production of antifungal metabolites, and so on. Previous researches on C. spp. mostly focused on the mechanisms of its biocontrol and the isolation of secondary metabolites. In this study, screening antifungal C. spp., mutation breeding of C. cupreum and interspecies protoplast fusion between C. cupreum and C. globosum were carried out, respectively. The corresponding results are as follows: Firstly, among more than 40 C. spp., the strains produced anti-fungal antibiotics were screened by agar diffusion experiments. Results showed that both CH08 and CH23 had inhibition against Colletotrichum gloeosporioides, Cladosporium fulvum, and Phytophthora infestans. Both CH16 and CH17 had inhibition against Colletotrichum gloeosporioides and Cladosporium fulvum. In addition, CH21 exhibited anti-fungal activity against Fusarium oxysporum f. sp niveum and Colletotrichum capsici. Furthermore, CH08, CH16, CH17 and CH23 were identified as C. globosum, CH21 was proved to be C. cupreum based on morphology. The comparison of the anti-fungal spectrum between C. cupreum and C. globosum, showed they could produce different antibiotics. Secondly, specified protocols for preparing and regenerating protoplasts from mycelia of C. cupreum CH21 and C. globosum CH08 were studied. The effects of the age mycelia, the concentration of enzyme, digestion temperature and time, kinds of osmotic stabilizer and regeneration medium on protoplasts preparation and regeneration were all optimized, respectively. In one protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1.5 h at 30 ºC, 2.02×107 protoplasts from each gram mycelia were obtained from cultures of C. cupreum CH21 grown in potato dextrose broth (PDB) medium for 54 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA (potato dextrose agar with osmotic stabilize), the regeneration efficiency of protoplasts was 51.45%. In another protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1 h at 30 ºC, 1.57×108 protoplasts from each gram mycelia were obtained from cultures of C. globosum CH08 grown in PDB for 48 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA, the regeneration efficiency of protoplasts was 41.48%. Thirdly, the mutagenesis conditions and secondary screening model of C. cupreum CH21 were explored. An 80% to 85% death rate could be achieved when the protoplasts of C. cupreum CH21 were irradiated by 15 w UV lamp from 30 cm distance for 90 s. In addition, the doublelayer plate’s method for the primary screening of high-producing antibiotics strains was established. A high yielding antibiotic mutant CH21-I-402 was obtained through the primary screening on plate and the secondary selection in Erlenmeyer flask, compared to the original CH21 strain, the antifungal activity of the mutant CH21-I-402 was increased by 18.3%. Fourth, the sensitivity to antibiotics of both C. cupreum CH21-I-402 and C. globusm CH08 was detected. Results showed C. cupreum CH21-I-402 was sensitive to G418 (Geneticin) (Gs) and resistant to Hygromycin B(Hr), and C. globusm CH08 was sensitive to both G418 (Geneticin) (Gs) and Hygromycin B(Hs). At the same time, neomycin phosphotransferase II (npt II) gene was transformed into C. globusm CH08(Gs, Hs) mediated by Agrobacterium tumefaciens EHA105, and the npt II gene was verified by polymerase chain reaction in resistance to G418 strains CH08-GR70 and CH08-GR120. The transformants still showed sensitive to Hygromycin B(Hs). Finally, a selection system for hybrids was set up by interspecies protoplast fusion between C. cupreum and C. globusm using dominant selective drug resistance markers. At first, protoplasts of C. cupreum CH21-I-402 (Hr, Gs) and C. globusm CH08-GR70 (Hs, Gr) were prepared, then the protoplasts were fused in the presence of 35% polyethylene glycol 6000 and regenerated on OPDA medium with 65 μg/ml Hygromycin B and 60μg/ml G418, at last 46 colonies with Hr and Gr were obtained. Even after 5 generations’ subculture, most of the colonies displayed significant difference in taxonomic characteristics with their parental strains. Regenerated strains PF1 and PF26 were confirmed as fusants by amplified fragment length polymorphisms analysis with the genomic DNA as the model. PF26 showed higher inhibitory activity against Colletotrichum gloeosporioides and Macrophoma kuwatsukai than that of the parental strain C. globusm.

Cross sections for transfer ionization in ion-helium collisions

A simple model has been developed within the independent-particle model (IPM) based on the Bohr-Lindhard model and classical statistical model. Cross sections for transfer ionization of helium by ions A(q+) (q = 1-3) are calculated for impact energies between 10 and 6000 keV/u. The calculated cross sections are in good agreement with the experimental data of helium by He(1-2)+ and Li(1-3)+.

不同水分胁迫方式对沙地樟子松幼苗光合特性的影响

沙地樟子松引种栽培的成功已使该树种成为中国北方沙区人工造林的首选树种,但由于早期引种的沙地樟子松人工林出现了衰退现象,使得人们对在干旱、半干旱沙地进行大面积樟子松造林产生疑问.为进一步研究沙地樟子松人工林衰退问题,该文以2年生沙地樟子松幼苗为材料,采用盆栽控水和聚乙二醇(PEG)处理法对苗木进行水分胁迫试验,比较两种胁迫处理苗木的光合特性.结果表明,土壤水分胁迫与PEG模拟水分胁迫(处理1 h)对2年生樟子松幼苗光合生理特征及其水分利用效率影响基本一致;当土壤含水量为40%田间持水量时,沙地樟子松已表现出干旱胁迫,土壤含水量为20%田间持水量时胁迫达到最大.10%PEG处理对2年生樟子松幼苗光合生理指标影响与对照具有相同趋势,表明该处理未对苗木造成严重干旱胁迫.20%与30%PEG处理对樟子松幼苗光合指标影响的趋势相同,胁迫超过2 h后樟子松幼苗光合速率、气孔导度、蒸腾速率都降到较低值且相对稳定.土壤含水量为20%田间持水量的胁迫对樟子松幼苗的水分利用效率几乎没有影响,轻度(40%田间持水量)胁迫甚至增高了水分利用效率;PEG胁迫的前期(4 h之前),苗木的水分利用效率低于对照;在胁迫处理4 h后,20%与30%PEG处理的樟子松的水分利用效率均超过了对照.这表明樟子松在较低的土壤含水量下,具有忍耐、适应干旱胁迫的能力.另外,不同形式的强度胁迫处理(30%PEG和20%田间持水量)的各光合特征指标相对值之间没有差异,表明樟子松苗木在强度胁迫条件下各指标相对值已降至相当低的程度.3种PEG浓度(10%、20%、30%)干旱胁迫处理在2 h以内的各指标的相对值与3种土壤水分胁迫处理(40%、30%、20%田间持水量)基本一致,因此,可以认为2 h的PEG胁迫处理与土壤水分胁迫处理(7~10 d)具有相同的效果.

非营养缺陷型原生质体融合选育角蛋白酶生产菌

以两性霉素B抗性作为标记 ,通过单株灭活 ,进行栖土曲霉种内非营养缺陷型原生质体融合 .在 40 %的PEG(Mr=6 0 0 0 )促融下 ,融合频率为 1.6 2× 10 -5.连续传接 10代后 ,获得稳定的融合子 .对孢子体积、核DNA含量、生长速度进行了测定 ,并利用RAPD技术分析比较了亲本及融合子的基因组DNA指纹多态性 ,证明融合子为杂合二倍体 ,产角蛋白酶活力较亲本显著提高 .图 3表 4参 17

干旱胁迫对杨树光合生理指标的影响

采用PEG模拟干旱胁迫的方法,利用气体交换法和叶绿素荧光技术,研究了干旱胁迫下小青杨(Populuspseudo-simonii)的光合生理变化.结果表明,干旱胁迫初期,小青杨的净光合速率(Pn)、蒸腾速率(Tr)、气孔导度(gs)和胞间CO2浓度(Ci)值均随干旱胁迫增强而下降,杨树Pn的下降主要是由于gs下降引起的;干旱胁迫后期,Ci值逐渐升高,非气孔限制成为光合作用的主要限制因子.干旱胁迫后期,PSⅡ原初光能转化效率(Fv/Fm)和PSⅡ潜在活性(Fv/Fo)明显下降,光抑制增强,光合电子传递受阻.POD酶的活性在胁迫初期升高,后期降低,说明干旱胁迫初期对保护系统酶活性升高有诱导作用,随着胁迫时间的延长,Fv/Fm和Fv/Fo降低,活性氧清除酶活性下降,活性氧代谢的平衡被打破,导致光合器官的伤害.由此表明,干旱胁迫后期Pn的降低与PSⅡ荧光参数及POD酶活性下降有关.

聚乙二醇模拟水分胁迫对沙地樟子松种子萌发影响研究

以引种区沙地樟子松种子为材料,观测了聚乙二醇(PEG)模拟水分胁迫对沙地樟子松种子萌发的影响.结果表明,不同浓度PEG处理胁迫对种子的萌发均有一定的延缓作用;种子的发芽率、发芽指数和发芽势随胁迫强度的增加呈现明显下降趋势.30%PEG处理的种子在试验结束后仍未能萌发,表明樟子松种子的萌发的临界PEG水分胁迫值小于30%,相当于-1.20MPa的水势.种子发芽后胚根和胚轴的生长亦受到PEG模拟水分胁迫,当PEG浓度为10%时(相当于-0.2MPa水势),胚根、胚轴的长度都较短,说明樟子松种子的胚根、胚轴的生长对PEG模拟干旱胁迫更敏感;但胚根/胚芽的比值随PEG模拟水分胁迫的强度增加而增加,表明樟子松种子萌发后对水分胁迫具有较强的适应性.由此可见,干旱胁迫影响引种区沙地樟子松种子的萌发可能是导致沙地樟子松人工林不能天然更新的因素之一.

科尔沁沙地南缘樟子松人工林主要外生菌根真菌的最适培养条件(英文)

在实验室条件下模拟环境因子进行沙地樟子松林下常见3种外生菌根真菌的纯培养试验,探讨培养基、pH值、水分和温度对外生菌根真菌生长的影响,以期得到3种外生菌根真菌的最适生长条件。结果表明:外生菌根真菌在含有松针汁、马铃薯汁、酵母粉和维生素的培养基里生长较好;pH值对外生菌根真菌的生长影响较小,L.deliciosus和B.sp.的最适生长pH分别为6.0和5.0,L.sp.在较广的pH范围内均可生长得良好,且在中性和弱碱性的培养基中长势均好于另外两种;水分对实验菌株的影响较大,在较低的PEG浓度(100gPEG·kg-1H2O)下,三种菌株生长良好,最适得生长PEG浓度为10%;高温容易导致外生菌根真菌死亡,3种外生菌根真菌的最适生长温度范围是25-28℃。

黄土高原不同密度刺槐(Robinia pseudoacia)林地水分生产力与土壤干燥化效应模拟

应用WinEPIC模型模拟研究了1957~2001年期间黄土高原半湿润区长武和半干旱区延安不同密度刺槐(Robinia pseudoacia)林地水分生产力演变规律和深层土壤干燥化效应。结果:(1)长武和延安高密度(6000株/hm2)、中高密度(4500株/hm2)、中低密度(3000株/hm2)和低密度(1500株/hm2)等4种处理刺槐林地逐年生物量模拟值均呈现快速增加、达到最大值后又逐年波动性降低的变化趋势,林地密度越高早期逐年生物量越高,后期逐年生物量差异缩小,两地均以低密度处理逐年生物量平均值和累积生物量模拟值最高;(2)4种密度处理45年生刺槐林地年均耗水量值基本相等,长武和延安分别为603mm和566mm,但生长前期年耗水量明显高于后期,并高于同期年降水量,林地密度越高前期耗水量越高,中期以后各密度处理耗水量基本接近且波动趋势基本一致,林地密度越高干旱胁迫程度越重;(3)模拟生长初期,4种密度处理刺槐林地0~10m土层逐月土壤有效含水量均呈现强烈的波动性降低趋势,长武各密度处理刺槐林地分别在7~23年生、延安分别在7~17年生之后逐月土壤有效含水量均在0~200mm较低水平上随降水量变化而波动..

膜荚黄芪氮磷钾优化施肥模式研究

采用氮、磷、钾三因素二次D-饱和最优设计,通过田间试验建立了氮、磷、钾的施肥量编码值与膜荚黄芪根产量、多糖含量的效应函数。结果表明,氮、磷、钾肥对膜荚黄芪根产量的增产作用大小依次为氮肥>钾肥>磷肥;氮、磷、钾肥对黄芪多糖含量的作用大小依次为钾肥>磷肥>氮肥,其中钾肥为负效应,氮肥、磷肥为正效应。寻优结果表明,膜荚黄芪目标产量在6000～7000 kg/hm~2之间,95%置信区间的优化施肥量为N 66.85～102.92 kg/hm~2、P_2O_5 64.64～94.95 kg/hm~2、K_2O 119.78～166.48kg/hm~2;膜荚黄芪多糖含量在13%～14%之间,95%置信区间的优化施肥量为N 66.85～102.92kg/hm~2、P_2O_5 64.64～94.95kg/hm~2、K_2O 119.78～166.48 kg/hm~2;膜荚黄芪高产优质高效栽培优化施肥量为N 99.52～102.92kg/hm~2、P_2O_5 94.20～94.95kg/hm~2、K_2O 119.78～166.48kg/hm~2,N、P_2O_5、K_2O的最佳比例为1:0.92～0.95:1.16～1....

抗氧化剂对渗透胁迫下沙棘叶片膜脂过氧化的保护作用

以沙棘叶片为材料 ,研究了外源抗氧化物质 Vc、VE、β-胡萝卜素等对 PEG渗透胁迫下细胞膜的保护作用。结果表明 :加入外源 Vc、β-胡萝卜素能有效保护膜系统 ,使膜透性和丙二醛在胁迫条件下增加小于对照 ,尤其在胁迫前期作用明显 ,而对叶绿素的保护作用在后期表现更突出 ,使叶绿素含量维持在较高水平 ;外源 VE 对防止膜透性增加作用不大 ,对减低丙二醛含量与保护叶绿素方面有所贡献。同时也证明沙棘叶片内 Vc在渗透胁迫下含量下降 ,而 Pro含量显著升高。证明这些物质是构成沙棘抗旱性的重要基础。

温度、pH及干旱胁迫对沙地樟子松外生菌根菌生长影响

通过对采集于沙地樟子松人工林内的3种外生菌根真菌(牛肝菌Boletussp.、乳菇Lactariussp.、高环柄菇Macrolepiota procera)进行纯培养,在不同温度、pH值和PEG模拟干旱胁迫条件下,观测了外生菌根菌的生长发育状况。结果表明,3种外生菌根菌生物量在不同pH范围(3~7)内存在着显著差异(P<0.05);在低于5℃和高于37℃时,3种外生菌根菌均不能生长,其最适生长温度分别为:牛肝菌,25℃高环柄菇,25~28℃,乳菇,25~30℃;在低浓度PEG胁迫处理条件下(10%,-0.20MPa),3种外生菌根菌的生长均有所增加,当PEG胁迫达到30%(-1.53 MPa)时,3种外生菌根菌的生长均受到抑制。与上述结果比较,沙地樟子松人工林地土壤的实际pH值在外生菌根菌的最适生长范围内;林地最高气温值远远超过3种外生菌根菌的最适生长值的时间在每年的生长季都会出现;在极端干旱年份(如1996年),林地土壤的实际含水量在生长季节(5~9月)远远低于抑制外生菌根菌生长的土壤含水量,可能影响到外生菌根菌的生存。因此,可以推断,沙地樟子松人工林地的外生菌根菌的生长与发育,在干旱胁迫及高温作用下受到很大程度的影响,如果仅从微生物角度分析,外生菌根菌在干旱和高温条件下不能生长或已经死亡是导致沙地樟子松人工林衰退的一个原因。

On the chemical synthesis and drug delivery response of folate receptor-activated, polyethylene glycol-functionalized magnetite nanoparticles

We describe here the chemical synthesis and in vitro drug delivery response of polyethylene glycol (PEG)-functionalized magnetite (Fe3O4) nanoparticles, which were activated with a stable ligand, folic acid, and conjugated with an anticancer drug, doxorubicin. The functionalization and conjugation steps in the chemical synthesis were confirmed using Fourier transform infrared spectroscopy. The drug-release behavior of PEG-functionalized and folic acid-doxorubicin-conjugated magnetic nanoparticles was characterized by two stages involving an initial rapid release, followed by a controlled release. (C) 2007 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Square lamellar structure having phase-separated microdomain in H-shaped block copolymer thin film

The morphology of a H-shaped block copolymer (poly(ethylene glycol) backbone and polystyrene branches (PS)(2)PEG(PS)(2)) in a thin film has been investigated. A peculiar square lamella that has a phase-separated microdomain at its surface is obtained after spin coating. The experimental temperature plays a critical role in the lamellar formation. The copolymer first self-assembles into square lamellar micelles with an incomplete crystalline core due to the crystallizability of PEG.

Ring-shaped morphology in H-shaped block copolymer thin films

The formation of ring-shaped structures in an H-shaped block copolymer [a poly(ethylene glycol) backbone with polystyrene branches, i.e., (PS)(2)PEG(PS)(2)] thin film was investigated when it was annealed in saturated PEG-selective acetonitrile vapor. Our results clearly indicate that ring formation is determined by the initial morphology of the spin-coated film, the solvent vapor selectivity and the environmental temperature of the solvent-annealing process. Only the films with the initial core-shell cylindrical structure in strongly PEG-selective acetonitrile vapor could form the ring-shaped structures.