高赖氨酸蛋白基因在转基因生菜中的表达和遗传转化

以生菜(LactucasativaL.)为受体材料,将含有高赖氨酸蛋白基因的植物表达载体pLBI用农杆菌介导法进行遗传转化,获得47株转基因生菜植株.PCR和Southern-blotting检测证实目的片段在T0代的整合,RT-PCR检测表明目的基因的转录.T0代植株赖氨酸(Lys)含量的测定分析表明,赖氨酸有不同程度的提高,比对照提高最高的可达9.46%.T1代植株PCR检测证明,目的基因能够遗传.图5表1参25     

施用氮、磷对油松幼苗叶形态与解剖结构的影响

施用氮和磷素对油松生长有一定的促进作用,为进一步了解其对针叶形态与解剖结构的影响,采用田间小区实验,设置4种处理,即对照(不施氮和磷)、施氮(+N,每次20 kg/hm~2,连续施用7次,下同)、施磷(+P,每次10 kg/hm~2)及氮磷共施(+NP,每次N 20 kg/hm~2+P 10 kg/hm~2),对油松当年生针叶开展研究.结果显示:施氮、施磷或氮磷共施增加了油松的地径、冠幅和地上部生物量,施氮或磷后叶氮或磷含量显著增加.对叶长、宽、厚和表面积施氮影响不大但施磷显著增加.施氮显著增加了叶中柱和转输组织的面积及所占比例,而施磷显著增加了表皮层、树脂道腔、中柱、转输组织、木质部和韧皮部的面积,同时树脂道腔、中柱和转输组织面积所占横截面积比例也有显著增加.施氮影响22个形态和解剖结构参数中的4个,而施磷对14个参数都有显著影响,表明施磷对油松针叶形态和解剖结构的影响大于施氮.主成分分析表明施磷主要影响光合和树脂生产能力,而施氮或施磷均增加了转输组织功能.本研究表明磷是油松幼苗生长的主要限制因子,因此生产实践中要重视磷肥的使用.     

拟南芥SHI基因在烟草中的表达和矮化、延迟开花的烟草植株的获得

利用聚合酶链式反应(PCR)从拟南芥基因组DNA中克隆了拟南芥SHOTINTERNODS(SHI)基因,并进行了序列测定.以载体pBI121为基本骨架构建了SHI的植物表达载体,并导入根癌农杆菌EHA105.通过农杆菌介导的叶盘法将拟南芥SHI基因转入烟草.PCR和RT-PCR检测证明,拟南芥SHI基因已整合到烟草基因组中并得以表达,获得了矮化、延迟开花的烟草植株.图5参12     

改变碳输入对太岳山油松林土壤酶活性的影响

土壤酶是土壤微生物作用于土壤环境的媒介,其活性对土壤环境的变化十分敏感,因此测定土壤酶活性对了解土壤微生物群落功能与环境因子的关系具有重要意义.为了解碳输入变化对土壤酶活性的影响,本文以山西太岳山油松(Pinus tabulaeformis)天然林和人工林为研究对象,自2009年7月开始进行对照(无人为干扰,CK)、去凋(去除凋落物,LR)、切根去凋(切断根系并去除凋落物,LRNR)3种处理,于2012年7、9月和2013年5月采集表层0-20 cm的土样,测定了多酚氧化酶(Polyphenol oxidase)、过氧化物酶(Peroxidase)、纤维素酶(Cellulase)、蔗糖酶(Invertase)、脲酶(Urease)和中性磷酸酶(Neutral phosphatase)的活性,另于2012年10月采集表层0-20 cm的土样测定土壤化学性质.结果显示:碳输入的改变对土壤酶活性影响显著,去凋处理和切根去凋处理均显著降低了土壤碳、氮含量(P0.05),并且显著抑制了土壤纤维素酶、蔗糖酶、脲酶、中性磷酸酶等水解酶的活性(P0.05),但对多酚氧化酶和过氧化物酶等氧化酶的影响不显著(P0.05).切根去凋处理对水解酶的抑制作用大于去凋处理,并略微提高了土壤过氧化物酶活性.另外在天然林中蔗糖酶活性的下降比人工林更为显著,而人工林中纤维素酶活性的下降比天然林更显著.研究表明:油松天然林中的土壤微生物群落功能倾向于分泌蔗糖酶,而油松人工林中的土壤微生物群落功能倾向于分泌纤维素酶.土壤酶活性的变化说明,在山西油松林,碳输入的减少会降低参与有机质降解的土壤酶活性,而对参与腐殖质合成的土壤酶活性没有显著影响.图3表3参31     

一种改进的SON-PCR基因扩增方法

单侧寡聚核苷酸嵌套PCR(single oligonucleotide nested PCR,SON-PCR)是一种简便易行的由已知序列克隆其侧翼序列的方法,但该法的第二轮PCR反应引发效率低,而且得到的产物两端含相同的引物序列,不可直接测序.针对该问题,将第二轮PCR改进为两段式扩增,即先以单侧嵌套引物引发5′端做选择性线性扩增,然后再加入第一轮引物特异引发3′端,使特异性和效率都得到很大提高,并可用PCR产物直接测序.分别用已报道的和改进的SON-PCR法克隆Botrytis cinerea羟甲基戊二酰CoA还原酶基因侧翼序列,后者获得期望结果,证明改进的SON-PCR方法行之有效.国内外尚未见同类报道.图4参7     

低浓度SO2对油松苗生长的影响

油松(PinustabulaeformisCarr.)苗暴露在SO2浓度分别为0(CK)、0.1mgm?3(T1)、0.2mgm-3(T2)和0.4mgm?3(T3)的4个人工气候箱内60昼夜.TI、T2组油松生长情况与CK无显著性差异.T3组油松针叶颜色发现异常,针叶中类胡萝卜素含量下降29%,含硫量增加242%,从针叶渗出的钾离子量比CK增加198%,光合速率下降7%,蒸腾速率增加17%,单株干重下降19%,相对生长速率下降3μgmg-1d-1.图7表3参8     

黄土丘陵区不同土地利用类型下土壤酶活性和养分特征

研究了土地利用类型对土壤酶活性和土壤养分的影响.首先通过最小显著性差异(LSD)评价各样地之间和同一样地土壤剖面四个不同层次的土壤酶活性和养分的差异,其次分析四种土壤酶(转化酶、碱性磷酸酶、脲酶和过氧化氢酶)活性和土壤养分之间相关性;最后以农地为对照,应用土壤恢复指数研究不同土地利用类型对土壤的改良效果.研究结果表明,同农地相比,不同土地利用类型的表层(0～5 cm)土壤四种酶活性、全氮、有机质和碱解氮含量均有所提高,且在同一土地利用类型下表层土壤酶活性和养分均显著大于下部各土层.相关性分析表明,土壤四种酶活性与有机质、全氮、碱解氮呈极显著正相关关系.同农地相比,其余土地利用类型表层土壤质量显著提高,而以杨树、侧柏、油松、仁用杏和刺槐5种人工林地的土壤质量提高最为明显,同龄树种中,杨树对土壤的恢复效果大于刺槐和油松.     

温度胁迫对草坪草生理指标影响

冷季型草坪草营养器官适宜的生长温度为10～24℃.在不同温度胁迫下,各种草坪草的各项生理指标都会发生变化,如细胞膜的热稳定性、蛋白质含量和酶活性、脯氩酸质量分数的变化等.这些变化可以反应出不同草种抗逆性的强弱.由于涉足冷季型草坪草研究领域的人很少,因此本实验利用草地早熟禾(Poa pralensis)、多年生黑麦草(Lolium perenne)、高羊茅(Festucaarudinacea)3个冷季型品种为材料进行研究测定.处理温度为4℃、35℃、45℃.对照25℃(室温);测定指标为过氧化物酶(POD)、脯氨酸质量分数、过氧化物酶活性和质膜相对透性.干热处理在人工气候箱内进行;4℃条件下是将供试材料放入温度设置为4℃的LRH-250-GS人工气温箱中进行处理,共处理4 d,每隔24 h取一次实验材料,重复3次测定.经实验测定与盆栽材料观察发现:早熟禾属材料具有叶色浓郁,质感适中等特点,在抗热性方而综合性状较好,是营建高质景草坪的前选材料.黑麦草生长迅速,5～6 d就可出苗,初期耐干热较强,抵抗低温的能力较强.实验结果为北方地区建造好的草坪提供选择草种的理论依据参考.     

放线菌产纤维素酶固定化研究

选择琼脂、明胶、琼脂-明胶3类固定化载体对具有降解纤维素能力的放线菌2235产生的纤维素酶进行固定化,在分别以羧甲基纤维素钠( CMC - Na)和滤纸为底物条件下,通过测定不同方法固定化后的纤维素酶活性,确定用于固定化纤维素酶的优良载体.结果表明,明胶法固定化效果最差;琼脂-明胶协同法固定化效果最优,采用该方法测得的滤纸酶和CMC酶活性分别可达3 317和8 868 nkat·mL-1.     

三种木本植物对酸雨的敏感性和抗性

酸雨污染对生态系统和人类生存环境造成严重影响,研究植物抗性及其对环境的适应能力是大气污染胁迫退化生态系统恢复中植物种类选择的基础和前提。作者选择陶瓷工业污染区南海西樵山和相对清洁区肇庆鼎湖山自然生长的木本植物鸭脚木(Schefflera octophylla)、荷木(Schima superba)当年生叶和一年生叶以及马尾松(Pinus massoniana)一年生针叶进行生理指标的测定和分析。结果表明:在酸雨污染环境下,3 种植物叶片的细胞膜透性受到损害,光合色素降解,PSII 最大光化学效率下降;对酸雨污染的敏感性次序为:马尾松>鸭脚木≥荷木,马尾松为酸雨敏感种。初步讨论了树木对酸雨污染的部分抗性生理机制,为酸雨污染地区退化生态系统恢复之植被选择提供理论依据。     

金鸡菊(Coreopsis drummondii)的抗TMV活性物质

采用活性跟踪法从金鸡菊根中分离获得抗病毒活性物质,经质谱和核磁共振分析,鉴定该物质为1-苯基-1,3,5-三庚炔.采用半叶枯斑法、叶圆盘法测定了该物质对烟草花叶病毒的抑制效果,结果表明,0.2 mg/mL的该化合物对TMV表现出较好的体外抑制侵染和增殖活性,其对TMV侵染和复制的抑制率分别为73.5%和84.3%.实时荧光定量PCR测定结果表明,该化合物对TMV外壳蛋白基因的表达有明显的抑制作用,0.2 mg/mL的该化合物对TMV外壳蛋白基因表达的抑制率为79.8%.图6表1参19     

Oviposition of carrot fly (Psila rosae) in response to foliage and leaf surface extracts of host plants

Summary. Foliage of twelve host and two non-host species and surrogate leaves treated with the respective leaf extracts were presented to laboratory populations of the carrot fly (Psila rosae) in oviposition choice assays. The stimulatory activity of dichloromethane surface extracts and the diethyl ether fraction of hot water extracts did not reflect accurately the differences in acceptability observed among intact leaves. A better correlation was found using hexane extracts prepared in a microwave oven. Two out of five fractions of this crude hexane extract obtained by silica gel column chromatography stimulated oviposition. The diethyl ether fraction, which contained the previously identified oviposition stimulants (propenylbenzenes, furanocoumarins, polyacetylenes), could account for only a minor part of the variation in the acceptability of host leaves. The preference hierarchy for intact leaves corresponded better to the ranking of species according to activity of the methanolic fraction, which apparently contains unknown stimulatory compounds.? The water fractions of the hot water extracts were shown to reduce egg-laying underneath surrogate leaves treated with a stimulatory extract. This oviposition-deterring effect was particularly strong with the non-preferred species Pimpinella major, which is also highly resistant in the field. Hence, unidentified inhibitory compounds may also contribute to differential accept ability of host plants. It is concluded that antixenotic (non-preference) resistance of host plants to carrot fly attack depends on complex mixtures of semiochemicals. Received 11 June 1997; accepted 26 November 1997.     

Cytotoxic and apoptotic activity of Scrophularia oxysepala in MCF-7 human breast cancer cells

Breast cancer is the most common cancer in women. Therefore, there is an urgent need to identify and develop therapeutic strategies against this deadly disease. This study is the first to investigate the cytotoxic and apoptotic effects of Scrophularia oxysepala in MCF-7 human breast cancer cells. Dichloromethane, methanol and n-hexane extracts of Scrophularia oxysepala were examined. The 3-(4,5-dimethylethiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction and trypan-blue exclusion assays were performed in MCF-7 cells as well as control cell line MCF10A to analyze the cytotoxic activity of the plant. Furthermore, the apoptosis-inducing action of extracts of the plant was evaluated using cell death detection ELISA, the terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay, Western blotting and real-time polymerase chain reaction (PCR). The results showed that dichloromethane and methanol extracts significantly inhibited cell growth and viability without inducing damage to MCF10A cells. Cell death detection ELISA, morphological changes of cells in TUNEL assay, Western blot detection of poly (ADP-ribose) polymerase (PARP) cleavage and increase in the mRNA expression levels of p-53, caspase-3 and c-myc showed that the induction of apoptosis was the main mechanism of cell death. Our results strongly suggest that this plant may contain potential bioactive compound(s) for the treatment of breast cancer.     

抗除草剂转基因作物实时荧光定量PC检测

建立了一种以SYBR Green Ⅰ为结合染料、快速准确检测转抗除草剂基因成分的实时荧光定量PCR方法.以转基因大豆与转基因玉米标准品为材料,通过使用特异性引物和SYBR Green Ⅰ结合染料实时荧光定量PCR技术,对转基因农作物中外源抗除草剂基因进行了定量检测,绘制了两种基因扩增的标准曲线图,根据标准曲线方程计算外源基因含量;并作了溶解曲线、检测方法检测灵敏度和精密度的分析.研究发现,两者标准曲线方程线性关系良好.R~2 值分别达到0.993 9与0.992 4.通过已知标准品进行验证,实测值与真值接近,与实际含量的相埘偏差是6.52%和7.90%.结果表明,SYBR Green Ⅰ结合染料法完全可以用于转基因农作物定量PCR检测.图5表2参11     

Antimicrobial activities of extracts from tropical Atlantic marine plants against marine pathogens and saprophytes

Studies investigating disease resistance in marine plants have indicated that secondary metabolites may have important defensive functions against harmful marine microorganisms. The goal of this study was to systematically screen extracts from marine plants for antimicrobial effects against marine pathogens and saprophytes. Lipophilic and hydrophilic extracts from species of 49 marine algae and 3 seagrasses collected in the tropical Atlantic were screened for antimicrobial activity against five ecologically relevant marine microorganisms from three separate kingdoms. These assay microbes consisted of the pathogenic fungus Lindra thalassiae, the saprophytic fungus Dendryphiella salina, the saprophytic stramenopiles, Halophytophthora spinosa and Schizochytrium aggregatum, and the pathogenic bacterium Pseudoaltermonas bacteriolytica. Overall, 90% of all species surveyed yielded extracts that were active against one or more, and 77% yielded extracts that were active against two or more assay microorganisms. Broad-spectrum activity against three or four assay microorganisms was observed in the extracts from 48 and 27% of all species, respectively. The green algae Halimeda copiosa and Penicillus capitatus (Chlorophyta) were the only species to yield extracts active against all assay microorganisms. Among all assay microorganisms, both fungi were the most resistant to the extracts tested, with less than 21% of all extracts inhibiting the growth of either L. thalassiae or D. salina. In contrast, over half of all lipophylic extracts were active against the stramenopiles H. spinosa and S. aggregatum, and the bacterium P. bacteriolytica. Growth sensitivity to hydrophilic extracts varied considerably between individual assay microorganisms. While 48% of all hydrophilic extracts were active against H. spinosa, 27% were active against P. bacteriolytica, and only 14% were active against S. aggregatum. Overall, more lipophilic extracts inhibited microbial growth than hydrophilic extracts. The variability observed in the antimicrobial effects of individual extracts against each assay microorganism reflects the importance of choosing appropriate test microbes in assays from which ecologically relevant information is sought. Results from this survey demonstrate that antimicrobial activities are prevalent among extracts from marine algae and seagrasses, suggesting that antimicrobial chemical defenses are widespread among marine plants.     

Antimicrobial activities of extracts from Indo-Pacific marine plants against marine pathogens and saprophytes

This study is the second of two surveys designed to systematically screen extracts from marine plants for antimicrobial effects against ecologically relevant marine microorganisms, and to compare results on a geographical basis. In the preceding survey, extracts from tropical Atlantic marine algae and seagrasses were screened in growth inhibition assays against the pathogenic fungus Lindra thalassiae, the saprophytic fungus Dendryphiella salina, the saprophytic stramenopiles, Halophytophthora spinosa and Schizochytrium aggregatum, and the pathogenic bacterium Pseudoaltermonas bacteriolytica. In this study, the same assay microorganisms were used to examine the antimicrobial effects of lipophilic and hydrophilic extracts from 54 species of marine algae and two species of seagrasses collected from Indo-Pacific reef habitats. Overall, 95% of all species surveyed in this study yielded extracts that were active against one or more, and 77% yielded extracts that were active against two or more assay microorganisms. Broad-spectrum activity against three or four assay microbes was observed in the extracts from 50 to 21% of all species, respectively. Extracts from the green alga Bryopsis pennata and the red alga Portieria hornemannii inhibited the growth of all assay microorganisms. Given that antimicrobial activity was prevalent among extracts of Indo-Pacific marine plants, it is interesting to note that the inhibitory effects of each extract varied considerably between the assay microorganisms. Overall, H. spinosa and D. salina were the most susceptible while L. thalassiae, S. aggregatum, and P. bacteriolytica were the most resistant to the extracts tested. These results provide good evidence that antimicrobial chemical defenses are widespread among Indo-Pacific marine plants. Further, the activity profiles of plant extracts suggest that antimicrobial secondary metabolites can have pathogen-selective or broad-spectrum effects. To confirm these results, chemical studies will be needed to isolate and characterize the compounds responsible for the observed antimicrobial activities.     

固相萃取/气相色谱质谱法测定灰尘中的氯代多环芳烃

建立了固相萃取(SPE)/气相色谱质谱(GC/MS)联用检测灰尘中氯代多环芳烃(ClPAHs)的方法.以正己烷和二氯甲烷混合液为提取溶剂,索氏提取灰尘样品中的20种ClPAHs,活性硅胶层析柱与活性炭混合硅胶SPE柱协同净化.净化后的提取液采用GC/MS测定,SIM模式扫描,并用质谱特征离子定量分析.结果表明,填充量为0.2 g(W(活性炭)∶W(硅胶)=1∶40)的活性炭混合硅胶SPE小柱能有效地将ClPAHs分离出来,载样后采用反向溶剂洗脱,既提高了回收率又减少了洗脱剂甲苯的用量,净化效果好.处理灰尘样品后检测分析,20种ClPAHs的平均回收率稳定在60.4%—120.1%,相关系数>0.99,检出限为0.04—0.17 ng.g-1,相对标准偏差为1.6%—10.2%.本方法前处理简单,定性、定量准确可靠,可广泛应用于环境介质中氯代多环芳烃的检测.     

Secondary metabolites of the leaf surface affected by sulphur fertilisation and perceived by the diamondback moth

Summary. Summary. Oilseed rape, Brassica napus L. (cv Express), plants were grown under three different sulphur regimes: sulphur-free (S₀), normal sulphur (S_n, normal field concentration) and a sulphur-rich (S₊, 2 × concentration of S_n). We performed dual choice oviposition assays with the diamondback moth, Plutella xylostella, using real plants and, for the first time with this insect, artificial leaves sprayed with methanolic leaf-surface extracts. The results mirrored those of a separate study of preferences for whole plants. Females laid more eggs on surrogate leaves that were treated with S_n extracts than on S₀ plants, while only a slight, not significant, difference was observed between extracts of normal and sulphur-rich plants. This shows that chemical compounds on the leaf surface mediate the oviposition preference and that the female insect can perceive the quality of the host-plants in terms of their fertilisation status.Since leaf volatiles are known to be oviposition stimulants, we investigated the effects of leaf-surface extracts on insect olfactory responses using electroantennograms (EAGs). In agreement with the behavioural data, we found that extracts of sulphur-treated plants yielded higher EAG amplitudes than the S₀ extracts. Since the leaf content of the volatiles isothiocyanates is influenced by sulphur nutrition, we analysed the extracts for these compounds. Above the detection threshold of our GC-MS system, no isothiocyanates were found. Thus, other compounds present in the surface extracts must be perceived by the antenna.However, the HPLC analysis revealed 11 different glucosinolates. Progoitrin (2-Hydroxy-3-butenyl) and gluconapoleiferin (2-Hydroxy-4-pentenyl), which belong to the hydroxy-alkene class of glucosinolates, were the most abundant compounds. The total glucosinolate content sharply increased from S₀ to S_n plants, whereas it was slightly lower in _n versus S₊ plants. Since it is known that glucosinolates can stimulate oviposition, it seems likely that the increased content we observed was influencing the insect preference in this study too.     

一株生物素依赖型菌株bio-5:分离,初步鉴定及其用于生物素浓度测定

从西红柿根圈土壤中分离纯化到一株生物素依赖型菌株bio - 5 ,经测定其内源抗生素抗性和脂肪酸特征 ,初步定名为假单胞菌bio - 5 .这一菌株对生物素非常敏感 .建立了以此菌株用于测定生物素浓度的标准方法 .利用这一方法 ,测定了不同作物叶子中生物素的含量 ,并将这一方法成功用于筛选转stv基因烟草植物 .与传统生物素测定菌株LactobacillusplantarumATCC80 14比较 ,我们所分离的菌株具有易于获得 ,易于培养 ,快速用于生物素浓度测定的特点 .图 7表 2参 2 2     

In vitro antibacterial and anti-inflammatory properties of seaweed extracts against acne inducing bacteria, Propionibacterium acnes

This study was conducted to evaluate the antimicrobial activities of common seaweeds from the coast of South Korea against the etiologic agents of acne vulgaris. Fifty-seven species of seaweed were screened for potential antimicrobial activity. Methanol extracts of 13 species (22.8%) showed inhibitory effects against Propionibacterium acnes. The aqueous extracts of only two species (3.5%) showed antimicrobial activity. When tested with the agar disk diffusion method, Ecklonia cava, E. kurome, Ishige sinicola, and Symphyocladia latiuscula had the strongest inhibitory effects. However, these four seaweed extracts showed no antibacterial activity against Staphylococcus epidermidis at 5 mg disk-1. The minimum inhibitory concentration (MIC) values of E. cava and E. kurome were both 0.31 mg ml-1 and the MIC values of l. sinicola and S. latiuscula were 0.26 and 0.21 mg ml-1, respectively. Among whole plants of E. cava and E. kurome, extracts of the pinnate blade had the highest inhibitory activity on bacterial growth. In cytotoxicity assays, methanol extracts of E. cava, E. kurome, and I. sinicola showed no effect on cell viability at concentrations of 200 microg ml-1. However, the methanol extracts of S. latiuscula reduced cell viability rates to 50% at the same concentration. Additionally, methanol extracts of E. cava, E. kurome, and I. sinicola potently inhibited the in vitro production of nitric oxide. These results suggest that the methanol extracts from these three species may be useful in the development of therapeutic agents for acne vulgaris. Further investigations to determine the bioactive compound are in progress.