淡紫拟青霉对辛硫磷的降解效应

采用菌、药混合培养的方式研究淡紫拟青霉对降解辛硫磷的降解作用,并探讨不同培养基、不同加药量及继代培养对降解效能的影响.结果表明:菌、药混合培养5d,辛硫磷的降解率可达96%以上.降解率随加药浓度的升高而下降,摇瓶培养液中的加药量达到8000μg/mL时,辛硫磷的降解率明显下降为25.51%.察氏、孟加拉红、PDA培养液降解力最强,在辛硫磷添加量达400μg/mL的摇瓶培养液中,菌、药混合培养5d,降解率可达98%以上.短期内继代培养对淡紫拟青霉菌的降解能力没有明显影响,所有继代培养处理对辛硫磷的校正降解率均在94%左右,且无显著差异.以上结果初步证实,淡紫拟青霉对有机磷农药具有显著的生物降解活性.图2表3参19     

假单胞菌表达载体pYMB03的构建与性能分析

恶臭假单胞菌(Pseudomonas putida)是具有强抗逆性能的环境优势菌,构建其质粒表达载体有着明显的应用潜力.将恶臭假单胞菌AB92019菌株中肽聚糖相关脂蛋白编码基因的启动子PoprL和质粒载体pTrcHis-B的多克隆位点片段插入到质粒载体pUCPl8的EcoRL/HInIII位点,获得了重组载体pYMB03.用绿色荧光基因gfp作为标记进行外源蛋白表达的结果表明,该载体能分别在恶臭假单胞菌AB92019菌株和大肠杆菌DH5a菌株中,由启动子PoprL启动组成型表达GFP蛋白并使细胞产生可见荧光.经SDS-PAGE验证,所产生的GFP蛋白分别占细胞总蛋白的12.5%和5.O%.重组菌株YMB001中GFP表达量与菌体培养时间有关,在稳定期后期其相对荧光强度达到最大值(D600nm=l.0),但与培养温度未见相父性.对携带该载体的2株重组恶臭假单胞菌7次168 h继代培养测定,载体pYMB03的稳定性均为100%.     

黄孢原毛平革菌酵母双杂交cDNA文库的构建及应用

作为研究木质素降解系统的模式微生物黄孢原毛平革菌,迄今还没有蛋白-蛋白相互作用方面的报道.本研究利用酵母双杂交技术构建了低氮培养基中培养2d和3d的酵母双杂交cDNA文库,分别得到2.5×105和3.0×105个重组子.以14-3-3蛋白为钓饵筛选3d cDNA文库,在SD/-Ade/-His/-Leu/-Trp缺陷培养基平板上共获得了约600个单克隆,进一步分析发现,有30个克隆可激活3个报告基因.分离自这些克隆中的质粒DNA能转化大肠杆菌.利用HaeⅢ和Sau3AⅠ限制酶切分析可将它们分为4类.序列测定和同源分析结果表明,其中一类为14-3-3蛋白基因,提示14-3-3蛋白可形成二聚体,另外3类在GenBank没有明显同源的基因.通过SBASE网站预测,编号为Y2H333的克隆含有一个OB-fold核酸结合结构域(OB-fold nucleic acid binding domain).这些研究结果表明,所构建的黄孢原毛平革菌cDNA酵母杂交文库是成功的,14-3-3蛋白在黄孢原毛平革菌中能以蛋白-蛋白相互作用的形式发挥功能.图4表1参28     

质粒pACYC184电转化松材线虫携带的荧光假单胞菌GcM5－1A

以质粒pACYC184为材料,研究了松材线虫携带的荧光假单胞菌的最适电转化条件.结果表明,荧光假单胞菌GcM5-1A培养至D600nm为0.5时制备感受态细胞,以300 mmol/L蔗糖溶液为电击缓冲液,质粒DNA终浓度为0.01 ng/μL,在25 μF电容、9 kV/cm电场强度、200 Ω电阻的电击条件下电击一次,可得最大的电转化效率,即5.5×106/μg(DNA).SDS-PAGE分析显示,转化质粒pACYC184的氯霉素抗性基因在受体细胞内得到了表达.本方法的建立为松材线虫携带的荧光假单胞菌的基因导入提供技术支持.为进一步通过基因敲除的方法,选择性地失活特定功能基因,进而确定这些基因在松材线虫病病理进程中的作用奠定基础.图2参16     

一组高效木质素降解复合菌的筛选

木质素的有效降解是秸秆等农业废物减量化及资源化利用的难点.采用连续驯化培养的方法,从农业废物堆肥过程升温、降温和腐熟3个阶段的微生物菌群中分别筛选驯化出3组具有木质素降解能力的复合菌MC1、MC2和MC3.通过初筛和复筛实验,筛选出一组性能稳定并具有高效木质素降解能力的复合菌,并对其继代培养的稳定性进行了验证.结果表明,从堆肥升温阶段筛选出的复合菌MC1的木质素降解能力最强.在37℃静置条件下液态发酵培养14d,d6时复合菌MC1各酶活值均达到最大,其中木质素过氧化物酶酶活为258.37UL-1,锰过氧化物酶酶活486.39UL-1,漆酶酶活为49.25UL-1;d14时木质素降解率达到36.25%.继代培养实验结果表明复合菌MC1具有较好的稳定性.图2表1参19     

发根农杆菌转化海边香豌豆及转化体的体细胞胚胎发生

将海边香豌豆无功苗的子叶和下胚轴切段在附加ρ（2,4-D)=1mgL^-1,ρ（6BA)=0.5mgL^-1的MS培养基上预培养3d，然后与发根农杆菌A4菌液共培养30min。洗涤之后，在附加羧苄青霉素的无激素的MS培养基上培养。7－10d后，外植体切面处长出许多毛状根。继代培养时生长旺盛，且产生许多侧根和分枝。志状根的诱导频率与无菌苗培养天数和外植体来源部位有关。子叶切块的诱导率明显高于下胚轴，16d龄无菌苗的子叶切块诱导毛状根的频率最高。乙酰丁香酮处理菌液可以有效提高子叶外植体的毛状根诱导频率。将毛状根切段培养在含有ρ（2，4－D）＝1.0mgL^-1的MS培养基上可诱导出愈伤组织。该种愈伤组织转移到含有ρ（2，4－D）＝0.3mgL^-1的MS培养基上培养时，诱导出了许多早期体细胞胚。检测的转化组织均含有农杆碱和甘露醇。图2表1参19     

铜绿假单胞菌D1产鼠李糖脂及降解原油效果

铜绿假单胞菌是生物表面活性剂鼠李糖脂最主要的生产菌株,对石油增溶、降解有很好的效果.从轻质原油样品中富集分离获得菌株D1,利用CTAB-亚甲基蓝平板法初步确定该菌株可以合成鼠李糖脂,分析其16S rRNA基因序列确定菌株D1属于铜绿假单胞菌(Pseudomonas aeruginosa).以菜籽油和甘油为碳源,菌株D1能够分别合成18.4 g/L和11.4 g/L鼠李糖脂.分析菌株D1可利用碳氢化合物种类,发现在以原油和柴油的培养基中,D1细胞数量分别增加了39.2倍和33.9倍.在10 d培养过程内,菌株D1能够分解基本培养基中33.3%的原油.相同时间内,补加0.5 g/L的甘油,菌株D1能够分解培养基中71.8%的原油.上述表明菌株D1能够合成鼠李糖脂和有效地降解原油,在石油污染的生物修复方面具有较好的应用潜力.     

荧光假单胞菌高效广谱载体的构建及分析

为促进高GC含量基因在荧光假单胞菌(Pseudomonas fluorescens)中表达效果更加理想、操作更加简便,本研究首先采用不依赖基因序列和连接反应的克隆(Sequence and ligation independent cloning,SLIC)方法将载体pCIBhis上与复制相关的序列和标记基因片段构建成克隆载体pCIBS1.然后优化荧光假单胞菌转化方法,用电转化法将pCIBS1导入荧光假单胞菌BL915中,随后又将T7和tac基因启动子分别插入pCIBS1中,成功构建了表达载体pCIBS3和pCIBS2.研究发现载体pCIBS1在大肠杆菌和荧光假单胞菌中均较为稳定,并且将绿色荧光蛋白基因插入表达载体中,在大肠杆菌BL21(DE3)中获得表达,验证了表达载体功能.本研究构建的表达载体和建立的荧光假单胞菌BL915电转化方法,为高GC含量基因在荧光假单胞菌中的表达奠定了基础.     

parDE基因对重组质粒pLM2在Enterobacter gergoviae 57-7中稳定性的影响

将 parDE基因克隆到载有组成型表达nifA基因的质粒 pST10 2 1上 ,得到重组质粒pLM2 .将 pLM2 ,pST10 2 1分别转到玉米联合固氮菌Enteorbactergergoviae 5 7 7(E5 7 7) ,获得转化子E5 7 7(pLM2 ) ,E5 7 7(pST10 2 1) ,二者在c(NH+ 4 ) =5 0mmolL-1的Kp培养基中表达的固氮活性一致 .将pLM2 ,pST10 2 1分别转到E .coliDH5α(pMGFP2 .1) ,培养物用 4 88nm光激发后在 5 10nm均有相同的的荧光特征发射峰 .上述实验表明 ,重组质粒pLM2仍保留了原质粒pST10 2 1中nifA的功能 .但质粒 pLM2和 pST10 2 1在E5 7 7中的稳定性试验表明 ,E5 7 7(pST10 2 1)在无抗生素的LB中继代培养 70代后 ,质粒几乎全部丢失 (仅存 1% ) ;而E5 7 7(pLM2 )继代培养 10 0代后 ,质粒全部存在 (10 0 % ) .图 4表 1参 12     

龙眼胚性细胞悬浮培养再生植株

以龙眼胚性愈伤组织为起始材料 ,研究了胚性悬浮细胞培养的影响因素、继代保持方法及其体胚发生与植株再生 .结果表明 ,建立龙眼优良胚性悬浮细胞系的技术关键在于 :由松散型胚性愈伤组织作为起始材料 ;以去除琼脂的保持松散型胚性愈伤组织的培养基作为启动培养基 ;采用分别附加 5mgL-1AgNO3 、10 0mgL-1肌醇的培养基交替继代保持 .胚性悬浮细胞在附加 2 %蔗糖、0 .7%琼脂、4 0 0mgL-1LH、10 0mgL-1肌醇的MS固体培养基上形成的体胚数量可达 7× 10 4～ 14× 10 4g-1.经过成熟培养后 ,体胚转换植株的频率在 6 5 %以上 .图版 1图 1表 3参 16     

Effects of different endogenous fungi on the mass loss and nutrient content of leaf litter of Cunninghamia lanceolata北大核心CSCD

To explore the role of endophytic fungi in the decomposition of litter, the endophytic fungi Penicillium sp. strain CG2 (A), Fusarium flavum strain AY13 (B), and Talaromyces strain AJ14 (C) of Cunninghamia lanceolata were added to experimental pots in different forms (mycelium, sterilized fermentation broth, single fungus, and mixed fungi), and a control treatment (CK) was set up (no fungi added). At 10, 30, 60, 90, and 120 days after litter decomposition, a study on the decomposition dynamics of C. lanceolata litter under different treatments was performed. The results showed that the rate of leaf mass loss was the highest in the sterilized fermentation broth treatment A after 120 days, and that there was a significant difference (P < 0.05) between the mycelium treatment AC and the control treatment after 60 days (23.97% higher than the control group). On day 60, the litter carbon content from the mycelium treatment A was significantly different from that of the control (P < 0.05), showing a 16.74% lower value, whereas the litter carbon content of the mycelium treatment B was 21.13% lower than that of the control after 90 days. The nitrogen content of the litters of most mycelium and sterilized fermentation broth treatments was increased compared to that of the control group; there was significant difference (P < 0.05) between the sterilized fermentation broth treatment A and the control (P < 0.05), with a 17.05% higher value than that of the control. Similar to nitrogen, the litter phosphorus content also increased; there was a statistically significant difference between the mycelium treatment A and the control group, with treatment A showing a 46.67% higher value than the control group. The potassium content was 28% lower than that of the control group under the sterilized fermentation broth treatment C, a result that was significantly different from that of the control group (P < 0.05). After treatment for 90 days, the ratio of carbon to nitrogen was the lowest under the treatments with the mycelium A and the mycelium B, with values 25.54% and 25.11% lower than that of the control group, respectively, and a statistically significant difference from that of the control group (P < 0.05). The ratio of carbon to phosphorus was the lowest under the treatment with mycelium A after 60 days, and the result was significantly different from that of the control (P < 0.05), with a 43.05% lower value than the control. Thus, the three endophytic fungi had different effects on the mass loss rate and nutrient content of the litter. The Penicillium sp. strain CG2 (A) had statistically significant effects on the mass loss and nutrient content of leaf litter, which was within the range of fungi fertilizer reference values for the breeding of C. lanceolata. © 2018 Science Press. All rights reserved.     

溶藻菌发酵液及其溶藻产物的生物急性毒性试验

应用发光细菌（Photobacteriumphosphoreum）急性毒性试验,研究了溶藻菌（Streptomycessp．HJC-D1）发酵液及其对铜绿微囊藻（Microcystisaemgmosa）抑制产物的生物急性毒性。结果表明,溶藻菌发酵液本身对发光细菌具有一定的低毒性,发酵3-5d时其相对发光度为（67．59％1：3．11％）-（72．35％±2．76％）;体积分数5％的溶藻菌发酵液可有效抑制初始质量浓度高达（O．1483±0．0032）mg-L-1的铜绿微囊藻生长,其抑藻率达85％以上,且藻液毒性明显低于对照组;以微囊藻毒素为主要溶藻产物进行毒性试验发现,其半抑制质量浓度为1096．92μg·L-1,水体藻毒素质量浓度低于20μg·L-1时其生物毒性较低。     

Examination of hydroxamate-siderophore production by neritic eukaryotic marine phytoplankton

Species of neritic eukaryotic marine phytoplankton were investigated during 1982 for hydroxamate-type siderophore production under iron-sufficient and iron-deficient culture conditions. Three of the 5 Prorocentrum species examined produced siderophores. Prorocentrin, the extracellular hydroxamate-type siderophore isolated from P. minimum, was also produced by P. mariae-lebouriae and P. gracile. P. maximum and P. micans grew poorly in iron-deficient medium and did not produce intracellular or extracellular hydroxamate-type siderophores. Thalassiosira pseudonana and Dunaliella tertiolecta produced extracellular siderophores under iron-deficient conditions, but siderophore production was not detected in the other two species, Skeletonema costatum and Olisthodiscus luteus. Each species which produced extracellular Csaky-positive hydroxamate showed a similar pattern of production. Under iron-sufficient conditions there was no measurable siderophore found either intracellularly or extracellularly. Under iron-deficient culture conditions hydroxamate-type siderophore was produced 1 to 2 d after the cessation of growth in the stationary phase. Production was over a short period of time (1 to 2 d) and the siderophore did not remain in the medium. The rate of siderophore disappearance from the medium was similar to the rate of production. Each species which produced siderophores showed an increase in in vivo fluorescence coincidental with the disappearance of the extracellular siderophore from the culture medium. There was no corresponding increase in in vivo fluorescence in iron-sufficient cultures. It is suggested that in vivo fluorescence may be used as a screening procedure for determining hydroxamate-type siderophore production in eukaryotic phytoplankton. An hypothesis on the iron uptake mechanism is proposed.     

Enzyme activities of marine bacteria involved in Laminaria-thallus decomposition and the resulting sugar release

The extracellular decomposing enzyme activities of marine bacteria, including 60 and 16 strains of Laminaria-thallus decomposing bacteria (LDB) and non-LDB, respectively, were determined against several algal polysaccharides. A number of LDB decomposed alginate, fucoidan, and cellulose, but not laminarin. Clear decomposing activity was not observed in the culture supernatant from nutrient broth, but was detected in that from a Laminaria-thallus medium, suggesting that decomposing enzymes were induced. During the incubation of LDB in Laminaria-thallus medium, depolymerized sugar materials with a wide range of molecular size were released into the culture fluid. Total sugars released into the culture fluid after 4 d of incubation amounted to a maximum of 95% of the initial content in the thallus tissues. These findings suggest that microbial decomposition of Laminaria thallus in sea water could be a good source of carbohydrates which may support the heterotrophic growth of marine organisms.     

稳健统计在快速生物降解性试验质量分析中的应用

快速生物降解性试验在新化学物质的生态毒理测试中占据重要地位,其测试数据的可靠性直接影响后续的测试策略和物质的申报登记、以及新化学物质进入环境后的风险管理。为研究OECD 301系列试验(301B、301D和301F)的质量,选用本机构近5年来积累的标准化物质苯甲酸钠14 d快速生物降解率数据,首次采用稳健统计技术,以稳健总计统计量的Z比分数作为评价指标,对各试验数据和试验系统的质量开展评估。结果显示,当301B、301D和301F试验的苯甲酸钠降解率分别在63.8%~88.0%、63.4%~89.2%和66.0%~99.8%范围内时,表明测试结果满意,该批次试验数据质量良好;若苯甲酸钠降解率不在此范围,则表明测试结果可能存在问题,甚至得到的可能是错误数据,需要进一步验证。据此提出了对实际测试工作具有指导意义的质量分析对照表,以期为测试机构开展新化学物质快速生物降解性试验时的质量分析提供参考。     

Effects of photoperiod on photosynthetic characteristics and quality of Tetragonia tetragonoides (Pall.) Kuntze in a plant factory北大核心CSCD

为探究光周期对番杏[Tetragonia tetragonoides(Pall.)Kuntze]生长及品质的影响,利用植物工厂中光源高度可控的优势对番杏进行不同光照时间(8、10、12、14、16 h)处理,测定分析番杏的生长、光合特性及营养品质.结果表明:(1)番杏各农艺性状随光照时间延长而增长,在光照时间14 h与16 h处理之间未呈显著性差异(P<0.05);(2)适宜光周期有利于光合色素积累,在16 h处理下叶绿素a、总叶绿素和类胡萝卜素含量最大,叶绿素b含量则在14 h光照时最大;净光合速率(P_(n))、蒸腾速率(T_(r))和气孔导度(G_(s))随光照时间延长上升,其中净光合速率在14 h与16 h光照之间变化不显著,胞间CO_(2)(C_(i))则呈先降后升的趋势;光周期变化对最大光化学效率F_(v)/F_(m)与光化学淬灭系数q_(P)未造成显著影响,非光化学淬灭系数NPQ随光照时间延长呈先降后升的趋势,14-16 h光照处理之间变化不显著,电子传递效率ETR则随光周期延长而降低;(3)不同光周期对番杏各营养成分含量的影响有所差异,可溶性蛋白变化差异较小,硝酸盐随光照时间延长而降低,14 h光照下可溶性糖含量最大.本研究表明14-16 h光照处理有利于番杏生长和营养物质积累.(图5表2参37)     

Quantitative genetic analysis of growth in larval scallops (Placopecten magellanicus)

We estimated the broad-sense heritability of larval size in 20 full-sib families of the giant scallop, Placopecten magellanicus (Gmelin, 1791) grown in laboratory culture in August and September 1991. The goal was to compare scallops with other bivalves which have been shown to have significant heritabilities for larval growth. Secondly, we estimated the lipid content of occytes from female parents, since this is hypothesized to affect larval growth and survival. Finally, we estimated the among-family variation in mortality from 4 to 21 d as a test of possible genetic variation for viability among larval scallops. Significant genetic variation (h²=1.10 to 1.24) was estimated for larval shell length at 4, 14, and 21 d. There was a significant correlation (r=0.66) between larval size at 4 d and lipid content of oocytes, but only when two females with high levels of lipid oocyte^–1 were excluded as outliers. There was no correlation between larval size at 14 and 21 d and lipid content of oocytes. Mortality among families from 4 to 21 d was high (69 to 97%), and was significantly different among families. These results indicate that there is significant heritability for larval growth which is largely independent of the lipid content of the oocytes. A high heritability for larval growth may indicate that this trait is only weakly correlated with fitness.     

Laboratory observations on spawning,brood size and egg hatchability of the Antarctic krill Euphausia superba from Prydz Bay,Antarctica

Forty-three female Euphausia superba were collected from three sites in Prydz Bay, Antarctica in January 1985. Most gravid females took 3 to 14 d to spawn after capture and some less mature females released eggs after 21 d. Most females spawned only once; overall mean brood size was 2 809 eggs and mean hatchability for eggs from the premoult females was 69.7%. For seven females spawning a second time, overall mean brood size was 597 eggs. Mean hatchability for second-spawn eggs from five premoult females was 67.1%. Possible origins of the second-spawned eggs were small numbers of matured eggs left in the ovaries after the first spawning or eggs developed from those of peripheral nucleolus and vitellogenic stages. Throughout the 8 wk of observations, there was no apparent development of eggs in females which had released all their eggs at one spawning.     

Cytogenetic analysis of Epinephelus marginatus (Pisces: Serranidae), with the chromosome localization of the 18S and 5S rRNA genes and of the (TTAGGG)n telomeric sequence

 A cytogenetic analysis was carried out on specimens of Epinephelus marginatus (Lowe, 1834) from three localities in the Mediterranean Sea, to deepen knowledge of the chromosome complement of the species and identify any possible population-specific cytogenetic markers. All specimens had a 2n = 48 acrocentric karyotype with two Ag-, chromomycin A₃- and C-positive NORs (nucleolar organizer regions) in the subcentromeric region of the smallest chromosome pair. The constitutive heterochromatin was distributed centromerically. Except for NORs, neither eu- nor heterochromatin show fluorescence after fluorochrome staining, i.e. there is no localized increase of AT- or GC-rich DNA. In all populations, the (TTAGGG) _n telomeric sequences are restricted to the telomeres, and the 18S and the 5S rDNA clusters are located on different chromosome pairs. In specimens from Sardinia, additional signals after C-banding, Ag-staining and FISH (fluorescence in situ hybridization) with 18S rDNA can be observed in the telomeric region of one or two large-sized chromosomes, classified as No. 2. This suggests that additional and variable NORs could be detected in the species in addition to those on the genus-specific, NOR-bearing Chromosome Pair 24. Received: 20 October 1999 / Accepted: 14 April 2000     

丙烯腈对小鼠精液运动参数的影响(Effects of Acrylonitrile on Sperm Motility in Male Mice)

为探讨丙烯腈(Acrylonitrile,AN)对雄性小鼠的生殖毒性作用机制,将250只SPF级昆明种雄性小鼠按体重随机分为5组:3个AN染毒组(1.25、2.50、5.00mg·kg-1)、1个阴性对照组(生理盐水0.01mL·g-1)和1个阳性对照组(环磷酰胺40mg·kg-1),腹腔注射染毒5d,每天1次.于初次染毒后第7、14、21、28、35d分五批处死小鼠,检测分析小鼠精子运动参数的变化.结果表明,5个观察终点各剂量组精液参数(精子密度、活动度、运动速度、运动方式参数、空间位移程度)变化与阴性对照均无显著差异(p>0.05).提示,在试验浓度范围内,AN对小鼠精子运动参数无影响.