生产植物次生产物的新途径

若干年来,人们对利用植物细胞培养物生产精细化学药品就很感兴趣。但是,除少数情况外,商品化生产的进展缓慢,主要原因是许多未分化培养物的生产能力很低并且(或者)不稳定。新近用发根土壤杆菌(Agrobacterium rhizogenes)遗传转化植物以后,产生了生长迅速、器官化的“毛状”根(hairy root)培养物。这对于植物细胞生物技术的某些领域可能是一场革命。本文将讨论应用毛状根技术生产植物次生代谢产物的问题。植物至今仍然是药物和其它精细化学药品的主要来源。目前使用的许多化合物在结构上是复杂的,使得它们的化学合成在经济上是难以实行的。很久以来,人们就认识到利用培养的植物细胞生产这些称为“次生”的代谢产物,以取代从植物组织中抽提是合算的。但是,     

流行性出血热病毒R22株cDNA克隆及其特异性鉴定

用家鼠型流行性出血热病毒R22株RNA,经polyA接尾,以Oligo-dT做引物,合成cDNA。用pUC18为载体转染E.coli Mc1061,建立cDNA克隆。再经菌落杂交,选择病毒特异性的5个阳性克隆制成缺口翻译探针,与病毒RNA3个片段进行反杂交,确定RNA片段的特异性。结果表明,3个克隆为中(M)片段的cDNA,另两个分别为大(L)和小(S)片段cDNA。核苷酸序列分析证明,克隆的DNA中含病毒特异的核苷酸序列。     

Biochemical Plant Responses to Ozone : II. Induction of Stilbene Biosynthesis in Scots Pine (Pinus sylvestris L.) Seedlings

Formation of the stilbenes pinosylvin and pinosylvin 3-methyl ether, as well as the activity of the biosynthetic enzyme stilbene synthase (pinosylvin-forming), were induced several hundred- to thousandfold in primary needles of 6-week-old pine (Pinus sylvestris L.) seedlings upon exposure to a single pulse of ozone of at least 0.15 microliters per liter. The seedlings required 4 hours of exposure as a minimum for the induction of stilbene biosynthesis when exposed to 0.2 microliters per liter ozone. Both stilbene synthase activity and stilbene accumulation increased with the duration of ozone treatment. The activity of phenylalanine ammonia-lyase and the activity of chalcone synthase, a key enzyme of the flavonoid pathway that uses the same substrates as stilbene synthase, were also stimulated about twofold by ozone. Stilbene biosynthesis appears to represent the first example of a dose-dependent biochemical response to ozone in a conifer species and may serve as a useful biomarker to study stress impacts on pine trees.     

Hydroperoxide Lyase and Other Hydroperoxide-Metabolizing Activity in Tissues of Soybean, Glycine max

Hydroperoxide lyase (HPLS) activity in soybean (Glycine max) seed/seedlings, leaves, and chloroplasts of leaves required detergent solubilization for maximum in vitro activity. On a per milligram of protein basis, more HPLS activity was found in leaves, especially chloroplasts, than in seeds or seedlings. The total yield of hexanal from 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13S-HPOD) from leaf or chloroplast preparations was 58 and 66 to 85%, respectively. Because of significant competing hydroperoxide-metabolizing activities from other enzymes in seed/seedling preparations, the hexanal yields from this source were lower (36-56%). Some of the products identified from the seed or seedling preparations indicated that the competing activity was mainly due to both a hydroperoxide peroxygenase and reactions catalyzed by lipoxygenase. Different HPLS isozyme compositions in the seed/seedling versus the leaf/chloroplast preparations were indicated by differences in the activity as a function of pH, the K_m values, relative V_max with 13S-HPOD and 13(S)-hydroperoxy-cis-9,trans-11,cis-15-octadecatrienoic acid (13S-HPOT), and the specificity with different substrates. With regard to the latter, both seed/seedling and chloroplast HPLS utilized the 13S-HPOD and 13S-HPOT substrates, but only seeds/seedlings were capable of metabolizing 9(S)-hydroperoxy-trans-10,cis-12-octadecadienoic acid into 9-oxononanoic acid, isomeric nonenals, and 4-hydroxynonenal. From 13S-HPOD and 13S-HPOT, the products were identified as 12-oxo-cis-9-dodecenoic acid, as well as hexanal from 13S-HPOD and cis-3-hexenal from 13S-HPOT. In seed preparations, there was partial isomerization of the cis-3 or cis-9 into trans-2 or trans-10 double bonds, respectively.     

Photoacoustic Study of Changes in the Energy Storage of Photosystems I and II during State 1-State 2 Transitions

Using photoacoustic spectroscopy, state 1-state 2 transitions were demonstrated in vivo in intact sugar maple leaves (Acer saccharum Marsh.) by following the changes in energy storage of photosystems (PS) I and II. Energy storage measured with 650 nm modulated light (light 2) in the presence of background white light indicated the total energy stored by both photosystems (ES_t), and in the presence of background far-red light showed the energy stored by PSI (ES_psi). The difference between ES_t and ES_psi gave the energy stored by PSII (ES_psii). While ES_t remained nearly constant during state transitions, both ES_psi and ES_psii changed considerably. The ratio of ES_psii to ES_psi, an indicator of the energy distribution between the two photosystems, decreased or increased during transition to state 2 or state 1, respectively. State transitions were completed in about 20 min and were fully reversible. During transition from state 1 to state 2, the fraction of excitation energy gained by PSI was nearly equal to that lost by PSII. This fraction of excitation energy transferred from PSII to PSI accounted for about 5% of the absorbed light (fluorescence is not considered), 19% of ES_t, 34% of ES_psii, and 43% of ES_psi in state 2. NaF treatment inhibited the transition to state 1. Data in the present study confirm the concept of changes in absorption cross-section of photosystems during state transitions.     

N-Cyclo-N'-(4-Dimethylamino-alpha-Naphthyl)Carbodiimide Inhibits Membrane-Bound and Partially Purified Tonoplast ATPase from Maize Roots

Certain carboxylic acid groups within the primary structure of proton translocating proteins are thought to be involved in the proton pathway. In this report, the effects of a lipophilic carboxylic acid reactive reagent, N-cyclo-N′(4-dimethylamino-α-naphthyl)carbodiimide (NCD-4), on the two types of proton pumps in maize (Zea mays L.) root microsomes were investigated. NCD-4 was found to inhibit the vacuolar-type H⁺-ATPase in microsomal preparations; however, the plasma membrane-type H⁺-ATPase was unaffected. The H⁺-ATPase in highly purified tonoplast vesicles was also inhibited by NCD-4. Inhibition was dependent on the concentration and length of exposure to the reagent. However, there was little, if any, increase in the fluorescence of treated vesicles, indicating few carboxylic acid residues were reacting. Inhibition of the tonoplast H⁺-ATPase by NCD-4 was examined further with a partially purified preparation. The partially purified H⁺-ATPase also showed sensitivity to the NCD-4, supporting the hypothesis that this carboxylic acid reagent is an inhibitor of the tonoplast ATPase from maize roots.     

Chloroplast Structure and Function Is Altered in the NCS2 Maize Mitochondrial Mutant

The nonchromosomal stripe 2 (NCS2) mutant of maize (Zea mays L.) has a DNA rearrangement in the mitochondrial genome that segregates with the abnormal growth phenotype. Yet, the NCS2 characteristic phenotype includes striped sectors of pale-green tissue on the leaves. This suggests a chloroplast abnormality. To characterize the chloroplasts present in the mutant sectors, we examined the chloroplast structure by electron microscopy, chloroplast function by radiolabeled carbon dioxide fixation and fluorescence induction kinetics, and thylakoid protein composition by polyacrylamide gel electrophoresis. The data from these analyses suggest abnormal or prematurely arrested chloroplast development. Deleterious effects of the NCS2 mutant mitochondria upon the cells of the leaf include structural and functional alterations in the both the bundle sheath and mesophyll chloroplasts.     

Are antibodies responsible for a decreased superovulatory response in goats which have been treated repeatedly with porcine follicle-stimulating hormone?

Repeated administration of xenogenic gonadotropins in human or animal species may be responsible for antibody production and refractoriness. An experiment was conducted in which goats were treated with porcine FSH (p-FSH) at 6-week intervals for a period of 7 months. A sensitive radioimmunoassay (RIA) was used to detect antibodies to p-FSH in plasma samples taken at short-term intervals during a 7-month period. Antibodies appeared after the first injection, and levels increased following booster injections. A high correlation rate existed between antibody level and superovulatory response. Refractoriness in goats was associated with a high level of antibodies.     

Ovarian function and pregnancy rates in reindeer calves (Rangifer tarandus ) in Southern Norway

Reindeer calves (n = 632) were slaughtered in November/December (n = 476) or in January (n = 156). Dressed weights and amount of perirenal fat were recorded, and the reproductive organs were collected. A separate group of 130 reindeer calves were weighed at 7 months of age and were followed up with repeated weighings and pregnancy examinations up to 21 months. The onset of puberty and the pregnancy rate were significantly influenced by body weight and the amount of perirenal fat. Approximately 60 g of perirenal fat and 22 kg dressed weight were found at the lower limits for pregnancy. A total of 222 (35%) animals had reached puberty and 126 (20%) were pregnant when examined after slaughter. Animals which conceived during their first autumn showed only a moderate weight gain the following year, and the calf mortality rate in these animals was 47.4%. It was concluded that calf pregnancies are common among the reindeer of Southern Norway and that measures need to be taken to prevent them.