荧光假单胞菌Tn5转座诱变及对青枯病生防特性

目的：利用Tn5转座诱变荧光假单胞菌PF20001,研究所获得的突变株对青枯病的生防效果。方法：利用三亲本杂交方式,将带有转座子Tn5的Tn5-102（含luxAB）的质粒pTR102成功地转入PF20001,利用平板相互拮抗法分析突变株对青枯病致病菌的拮抗作用。结果：通过诱导Tn5转座,得到荧光假单胞菌PF20001的Tn5插入突变库。经平板相互拮抗实验发现,菌株PF20001-lux-48拮抗圈明显大于野生型（半径达0.35cm）。用Tn5-lux特异引物进行PCR扩增,结果显示只有以该突变株的DNA为模板才能得到300bp的扩增产物,证实该菌株基因组中有Tn5插入。结论：Tn5的插入使菌株PF20001对青枯病生物防治能力增强。     

N_2和NH_4~ 培养下粪产碱菌固氮酶铁蛋白的生物合成及特性比较

应用DEAE-纤维素和凝胶柱层析,分别将N_2和NU_1~ (30 mmol/L)培养的粪产碱菌固氮酶铁蛋白(Af2和Af~ 2)分离并提纯52倍,在SDS-PAGE上呈均一状态。Af2的比活性达1540 nmol C_2H_4mg~(-1)protein min~(-1),Af~*2无活性。Af2和Af~*2理化性质基本相同,分子量为64.5 kD;均由2个亚基组成,每个亚基分子量为32.5kD;氨基酸种类相同,总残基数分别为537和553,不含色氨酸;每分子Af2和Af~ 2均含有4个Fe原子和4个酸不稳定S~(2-)原子;UV-vis光谱吸收特征相同;荧光探剂测定结果为:每分于Af2和Af~*2均络合2个分子MgATP或2个分子MgADP。     

固氮粪产碱菌谷氨酰胺合成酶的分离纯化及其特性

联合固氮细菌粪产碱菌Ａ１５０１菌体经超声破碎后,无细胞粗提液以ＰＥＧ－６０００分级沉淀,丙酮沉淀,再经蓝球脂糖亲和层析分离、纯化。获得的纯谷氨酰胺合成酶（ＧＳ）在ＳＤＳ－ＰＡＧＥ和４－３０％梯度ＰＡＧＥ上均呈均一的一条带。ＧＳ亚基及整酶分子量分别为５５ＫＤ和６４５ｋＤ,亚基由４５６个氨基酸残基组成。ＧＳ的Ｋｍ值。在以Ｇｌｕ为源的介质中培养时分别为２０ｍｍｏｌ／Ｌ（Ｇｌｕ）,５０ｍｍｏｌ／Ｌ（ＡＴＰ     

固氮粪产碱菌谷氨酰胺合成酶的构象变化及调节作用

应用园二色谱测定了粪产碱菌谷氨酰胺合成酶（ＧＳ）各构象,结果表明在Ｇｌｕ培养下ａ螺旋为２８％,β折叠为２２％,无规则卷曲占５０％;而在ＮＨ４＾＋培养下,三者相应为２０％,２０％,６０％。荧光光谱及付立叶红外光谱也证明,两种培养条件下ＧＳ的构象存在着差异。不同氮源对粪产碱菌ＧＳ的形成有显著的影响。高浓度ＮＨ４＾＋培养下ＧＳ合成受到阻遇,而Ｇｌｕ或低浓度ＮＨ４＾＋则对ＧＳ合成无明显的影响。ＮＨ４＾＋培     

维生素C生产用菌系2980产酸菌基因转移的筛选模型及转座子Tn5诱变

维生素C的生产目前国内广泛采用由产酸菌(Gluconobacter oxydans)与伴生菌(Bacillus megaterium)组成的2980菌系,在2980中G.oxydans单独生长传代困难,其生长和产酸需要B.megaterium参与。以Bacillus subtilis Ki-2-132(pUB110)作为伴生菌与原2980的G.oxydans组合,获得稳定产酸的新菌系。在此基础上建立了适合我国混菌发酵产酸菌外源基因(Kanr)转移的筛选模型。同时报道了以携带有自杀性载体P1：：Tn5的大肠杆菌E.coli W3110为供体菌对G.oxydans进行Tn5诱变的条件和结果。     

固氮粪产碱菌谷氨酰胺合成酶的分离纯化及其特性

联合固氮细菌粪产碱菌（Ａｌｃａｌｉｇｅｎｅｓｆａｅｃａｌｉｓ）Ａ１５０１菌体经超声破碎后,无细胞粗提液以ＰＥＧ－６０００分级沉淀,丙酮沉淀,再经蓝琼脂糖（ＢｌｕｅＳｅｐｈａｒｏｓｅＣＬ－６８）亲和层析分离、纯化。获得的纯谷氨酰胺合成酶（ＧＳ）在ＳＤＳ－ＰＡＧＥ和４－３０％梯度ＰＡＧＥ上均呈均一的一条带。ＧＳ亚基及整酶分子量分别为５５ｋＤ和６４５ｋＤ,亚基由４５６个氨基酸残基组成。ＧＳ的Ｋｍ值,在以Ｇｌｕ为氮源的介质中培养时分别为２０ｍｍｏｌ／Ｌ（Ｇｌｕ）,５０ｍｍｏｌ／Ｌ（ＡＴＰ）和４５ｍｍｏｌ／Ｌ（ＮＨ~＋_４）;在以ＮＨ~＋_４为氮源的介质中培养时则分别为７０ｍｍｏｌ／Ｌ（Ｇｌｕ）,４９ｍｍｏｌ／Ｌ（ＡＴＰ）和８０ｍｍｏｌ／Ｌ（ＮＨ~＋_４）,表明ＮＨ~＋_４培养下形成高度腺苷化的ＧＳ对Ｇｌｕ及ＮＨ~＋_４的亲和力有所下降。     

固氮粪产碱菌ntrC-lacZ融合基因的构建及其在水稻根部的表达

将粪产碱菌（Alcaligenesfaecalis）A1501ntrC基因和lacZ基因正向克隆于广泛性转移载体pLA2917上,获得多拷贝ntrC质粒pLAC1和含ntrC－lacZ融合基因的重组质粒pLAC2,采用双亲结合方法,将上述两个重组质粒导入A1501中,获得含ntrC－lacZ融合基因的结合子A15C2和ntrC多拷贝结合子A15C1。采用X－gal原位显色技术、显微切片、扫描电镜观察及ntrC部分缺失突变株研究粪产碱菌ntrC在根部的表达及功能,结果表明粪产碱菌A1501在水稻根部有较强的定殖能力,并能侵进入水稻根内定殖。在水稻主侧根伸长区及根内皮层薄壁细胞及侧根分生区ntrC－lacZ融合基因表达活性明显高于别的部位。高铵条件下多拷贝ntrC结合子根表定殖能力大于野生型,而ntrC突变株则低于野生型。表明ntrC基因参与固氮菌根表结合的过程。     

EFFECT OF INDOLE-3-ACETIC ACID ON GROWTH OF CODIUM FRAGILE SUBSP. TOMENTOSOIDES (CHLOROPHYCEAE) IN CULTURE1

Growth of Codium fragile subsp. tomentosoides (van Goor) Silva in culture depends upon the season of seawater collection. One factor responsible for this variation in growth may be indole-3-acetic acid (IAA). When 10^?9 to 10^?4 M IAA is added to cultures of Codium fragile, optimum growth is at 10^?6 M. The response to exogenous IAA depends upon the time of year when the sea-water is collected. The growth in a range of known IAA concentrations allows the prediction of a seasonal cycle of IAA, or its physiological equivalent, in Rhode Island coastal waters. Such a compound may be an important ecological factor for some algal species.     

云南山楂中吲哚乙酸、脱落酸、玉米素和玉米素核苷的内源水平及其生根率

以云南山楂(Crataegus scabrifolia (Franch.)Rchd.)几个株系的成年树和实生苗的离体培养芽条为材料,用酶联免疫吸附测定法(ELISA)测定了它们的叶片中吲哚乙酸(IAA),脱落酸(ABA),玉米素和玉米素核苷(Z+ZR)的内源水平,探讨了这三种激素及其之间的平衡与生根率的相关性。结果发现:1.IAA内源水平之差异存在于株系间。2.内源(Z+ZR)在株系间无显著差异,而幼树发育至成年树的过程中,与IAA的平衡是多样的,同成年树的生根率似有以下相关性,当IAA水平降低,同时(Z+ZR)水平升高,有利于生根,反之则抑制生根。3.ABA内源水平无论成年或幼年树都呈低水平且无显著差异。     

萘乙酸、激动素和伤害对绿豆子叶愈伤组织形成的作用以及和色氨酸、吲(口朶)乙酸生物合成的关系

本文以绿豆子叶为材料研究了切伤、外源萘乙酸及激动素诱导形成愈伤组织的作用及其与内源色氨酸和吲哚乙酸生物合成的关系。实验结果表明,切伤对于愈伤组织的形成具有重要作用,切伤面积的大小与愈伤组织的增殖成正比。在绿豆子叶愈伤组织形成的初期,游离色氨酸和内源吲哚乙酸的水平均降低,而在后期,组织内部游离色氨酸和吲哚乙酸的含量都有增加。在培养基中加入外源色氨酸可以部分代替萘乙酸促进愈伤组织的形成。可以认为,外源激素诱导愈伤组织的形成是通过促进内源色氨酸和内源吲哚乙酸的生物合成而实现的。受伤对愈伤组织的形成也起了重要的协同作用。     

GC-SIM-MS DETECTION AND QUANTIFICATION OF FREE INDOLE-3-ACETIC ACID IN BACTERIAL GALLS ON THE MARINE ALGA PRIONITIS LANCEOLATA (RHODOPHYTA)

Indole-3-acetic acid (IAA), a plant hormone necessary for terrestrial plant growth and development, was detected and quantified in the marine red alga Prionitis lanceolata Harvey (Halymeniaceae, Gigartinales, Rhodophyta) using gas chromatography–selective ion-monitoring mass spectrometry (GC-SIM-MS). This allowed comparison of free IAA levels between the algal thallus and eubacterially induced galls on this alga characterized by abnormal algal growth and cell division and extensive, intercellular microbial proliferation. The levels of free IAA in the P. lanceolata thallus averaged 2.5 (±1.1) ng·g⁻¹ fresh wt. Free IAA levels in galls were more variable, ranging from ca. 4 to 39 (8.3 ± 10.9) ng·g⁻¹ fresh wt, but were significantly higher overall ( P = 0.0022). The identity of the IAA in this marine florideophycean alga was confirmed by full scan GC-MS analysis of both galls and thalli. The levels of free IAA in P. lanceolata were two to three orders of magnitude higher than those observed previously in the Rhodophyta. The origin of elevated IAA levels in P. lanceolata galls is unknown because it is possible that this compound is produced by either the gall-inducing bacterial symbiont or the host alga.     

EFFECT OF INDOLE-3-ACETIC ACID,KINETIN, AND ETHYLENEDIAMINETETRAACETIC ACID ON PLANT GROWTH AND UPTAKE AND TRANSLOCATION OF LEAD,MICRONUTRIENTS, AND MACRONUTRIENTS IN ALFALFA PLANTS

Alfalfa plants germinated and grown for 15 d in soil containing 80 mg Pb kg^?1 were treated with ethylenediaminetetraacetic acid (EDTA) at 0.8 mM and indole-3-acetic acid-kinetin (IAA-KN) at 100 μM. Fifteen days after the treatment application, the concentration of lead (Pb), macronutrients, and micronutrients was determined using inductively coupled plasma/optical emission spectroscopy. The chlorophyll content and plant growth were also measured. Roots of plants exposed to Pb alone, Pb–EDTA, and Pb–EDTA-IAA-KN had 160, 140, and 150 mg Pb kg^?1 DW, respectively. Pb was not detected in the stems of plants exposed to Pb alone; however, stems of plants treated with EDTA and EDTA–IAA-KN had 78 and 142 mg Pb kg^?1 DW, respectively. While the Pb concentration in leaves of plants treated with EDTA and EDTA–IAA-KN was 92 and 127 mg kg^?1 DW, respectively. In addition, EDTA and EDTA–IAA-KN significantly increased the translocation of zinc and manganese to leaves. The x-ray absorption spectroscopic studies demonstrated that Pb(II) was transported from roots to leaves without a change in the oxidation state.     

CARBON SOURCE DEPENDENCE OF THE RATIO OF δ‐AMINOLEVULINIC ACID BIOSYNTHESIS VIA THE C5 AND SHEMIN PATHWAYS IN EUGLENA GRACILIS (EUGLENOPHYCEAE)1

The ratio of two biosynthetic pathways was estimated, the C5 and Shemin pathways, to δ‐aminolevulinic acid (ALA, a biosynthetic intermediate of tetrapyrrole) from the ¹³C‐enrichment ratios (¹³C‐ER) at the carbon atoms of chl a (after conversion to methyl pheophorbide a) biosynthesized by Euglena gracilis G. A. Klebs when l ‐[3‐¹³C]alanine was used as a carbon source. On the basis of these estimations, we confirmed that ALA was efficiently biosynthesized via both the C5 and Shemin pathways in the plastids of E. gracilis, and we determined that the ratio of ALA biosynthesis via the Shemin pathway was increased in the ratio of 14%–67%, compared with that in our previous d ‐[1‐¹³C]glucose feeding experiment ( Iida et al. 2002 ). This carbon source dependence of the contributions of the two biosynthetic pathways might be related to activation of gluconeogenesis by the amino acid substrate. The methoxy carbon of the methoxycarbonyl group at C‐13² of chl a was labeled with the ¹³C‐carbon of l ‐[methyl‐¹³C]methionine derived from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl coenzyme A (CoA), through the atypical tricarboxylic acid (TCA) cycle, gluconeogenesis, and l‐ [3‐¹³C]serine. The phytyl moiety of chl a was also labeled on C‐P2, C‐P3¹, C‐P4, C‐P6, C‐P7¹, C‐P8, C‐P10, C‐P11¹, C‐P12, C‐P14, C‐P15¹, and C‐P16 from ¹³C‐isoprene (2‐[1,2‐methyl,3‐¹³C₃]methyl‐1,3‐butadiene) generated from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl CoA.     

真菌中脱落酸生物合成代谢关键酶的初步研究*

应用逆转录PCR(RT-PCR)技术测定脱落酸产生菌Botrytis cinerea 3-羟-3-甲基戊二酰CoA(HMG-CoA)还原酶mRNA含量,结果表明经诱变筛选得到的脱落酸高产菌HMG-CoA还原酶含量显著高于野生菌,提示HMG-CoA还原酶可能为真菌ABA生物合成的关键酶。     

细胞因子和视黄酸调节肺癌细胞补体的生物合成

目的探讨近年用来治疗恶性肿瘤的视黄酸与细胞因子(TNF-α、IL-1β、IL-4、IL-6、IFN-γ)对人肺癌细胞株A549分泌C3及Β因子的影响。方法分别用ELISA、Western blot和RT-PCR检测培养上清中C3及B因子的水平及细胞C3及B因子mRNA表达。结果TNF-α、IL-1β、IL-4、IL-6和IFN-γ诱导A549细胞分泌C3及B因子是未处理组的6.84、11.0,7.27、8.04,6.16、1.08,4.75、1.43和2.10、1.59倍。视黄酸对Α549细胞分泌C3及B因子没有影响,但它能增强TNF-α、IL-1β、IL-4、IL-6和IFN-γ诱导的A549分泌C3及B因子分别是TNF-α、IL-1β、IL-4、IL-6和IFN-γ组的1.46、1.90,1.44、1.90,1.18、1.69,0.94、2.52,和1.09、2.39倍。RT-PCR半定量分析C3及B因子mRNA的结果与蛋白水平相似。结论视黄酸能调节诸细胞因子增强诱导人肺癌细胞株A549细胞分泌C3及B因子,这可提高细胞因子对肿瘤细胞的杀伤作用。