Isolation and characterization of a hemocyte aggregation inhibitor from hemolymph of Manduca sexta larvae

A protein that inhibits hemocyte aggregation has been isolated from hemolymph of Manduca sexta larvae and named hemocyte aggregation inhibitor protein (HAIP). HAIP has a M_r = 50,000, pI = 8.5, and contains 7% carbohydrate. It is present at 230 ± 20 μg/ml in hemolymph of day 3 fifth instar larvae. Antibodies to HAIP do not cross-react with M. sexta hemolin, which is similar in size and charge and also inhibits hemocyte aggregation. HAIP and hemolin have some similarity in amino acid composition and NH₂-terminal sequence, but are different in overall secondary structure, as determined by CD spectroscopy. The concentration of HAIP in hemolymph is not affected by injection of larvae with bacteria. A protein of approximately 50,000 daltons that reacts with antibody to M. sexta HAIP is present in hemolymph of Bombyx mori, Heliothis zea, and Galleria mellonella. Although the function of HAIP in vivo is not yet clear, it may have a role in modulating adhesion of hemocytes during defensive responses. © 1994 Wiley-Liss, Inc.     

Interactions of polyamines and nitrogen nutrition in plants

Biogenic amines occupy an important position among the many nitrogenous plant compounds. Polyamines are part of the overall metabolism of nitrogenous compounds, yet they do not seem to function in the 'normal' nitrogen nutrition. Rather, these widespread polycations (e. g. putrescine, spermidine and spermine) are involved in the regulation of growth and stress, probably by binding to negatively charged macromolecules. In addition, some diamines and polyamines are metabolized to yield 'secondary 'metabolites such as nicotine and other alkaloids. Previous studies have indicated that the ratio of nitrate to ammonium nutrition affects polyamine biosynthesis and content in intact plants. Thus, an increase in putrescine accumulation was found under conditions of excess ammonium ions, relative to nitrate. Modifications of nitrogen sources in the culture medium of tobacco cell suspensions (depletion of ammonium nitrate, or potassium nitrate, or both) resulted in marked changes in the content of cellular free polyamines. Considerable changes in the content of specific polyamines were also found with exposure to specific inhibitors of polyamine biosynthesis (difluoromethyl ornithine, difluoromethyl arginine, cyclohexylamine, methylglyoxal-bis-guanylhydrazone). However, a combination of nitrogen depletion of the medium and some inhibitors resulted in a very marked over-production of spermidine and spermine. The significance of these findings is discussed in relation to the assumption that polyamines act as a metabolic buffer, and maintain cellular pH under conditions where ammonium assimilation produces an excess of protons.     

Development of a microtargeting device for particle bombardment of plant meristems

A gene transfer system for meristem cells was developed on the basis of a ballistic approach. In order to meet some important prerequisites for an efficient transfer system, such as for example aiming at small tissues and control of penetration of the microprojectiles, we developed an acceleration system fundamentally different from the usual macroprojectile driven approach. Instead of a macroprojectile, microtargeting uses the law of Bernoulli for accleration of highly uniform-sized gold particles. The system is able to deliver 80% of the particles to an area as small as 150 micron in diameter, which corresponds to the size of a meristem. Microtargeting yields gene delivery (measured as number of transiently GUS expressing cells to up to 3% of the cells exposed in the target area or up to 35 × 10³ cells per cm². Stable transformation of tobacco microcolonies with the microtargeting device was shown to have an efficiency up to one stable transformant per 1000 cells exposed to the shot, or up to one transformant per shot. We perform 4 or 5 shots per min. After 30 to 40 shots, reloading can take up to 2 min. Microtargeting is very flexible and allows for the adjustment of the important parameters to fit the requirements of the respective tissue.     

Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.     

Developmental regulation of an acyl carrier protein gene promoter in vegetative and reproductive tissues

The expression of an Arabidopsis acyl carrier protein (ACP) gene promoter has been examined in transgenic tobacco plants by linking it to the reporter gene -glucuronidase (GUS). Fluorometric analysis showed that the ACP gene promoter was most active in developing seeds. Expression was also high in roots, but significantly lower in young leaves and downregulated upon their maturation. Etiolated and light-grown seedlings showed the same level of GUS activity, indicating that this promoter is not tightly regulated by light. Histochemical studies revealed that expression was usually highest in apical/ meristematic zones of vegetative tissues. Young flowers (ca. 1 cm in length) showed GUS staining in nearly all cell types, however, cell-specific patterns emerged in more mature flowers. The ACP gene promoter was active in the stigma and transmitting tissue of the style, as well as in the tapetum of the anther, developing pollen, and ovules. The results provide evidence that this ACP gene is regulated in a complex manner and is responsive to the array of signals which accompany cell differentiation, and a demand for fatty acids and lipids, during organogenesis.     

Inhibition of tobacco NADH-hydroxypyruvate reductase by expression of a heterologous antisense RNA derived from a cucumber cDNA: Implications for the mechanism of action of antisense RNAs

Tobacco plants were genetically transformed to generate antisense RNA from a gene construct comprised of a full-length cucumber NADH-dependent hydroxypyruvate reductase (HPR) cDNA placed in reverse orientation between the cauliflower mosaic virus 35S promoter and a nopaline synthase termination/polyadenylation signal sequence. In vivo accumulation of antisense HPR RNA within eight independent transgenic tobacco plants resulted in reductions of up to 50% in both native HPR activity and protein accumulation relative to untransformed tobacco plants (mean transgenote HPR activity=67% wild type, mean transgenote HPR protein=63% wild type). However, in contrast to previous reports describing antisense RNA effects in plants, production of the heterologous HPR antisense RNA did not systematically reduce levels of native tobacco HPR mRNA (mean transgenote HPR mRNA level=135% wild type). Simple regression comparison of the steady-state levels of tobacco HPR mRNA to those of HPR antisense RNA showed a weak positive correlation (r value of 0.548, n=9 ; n is wild type control plus eight independent transformants; significant at 85% confidence level), supporting the conclusion that native mRNA levels were not reduced within antisense plants. Although all transgenic antisense plants examined displayed an apparent reduction in both tobacco HPR protein and enzyme activity, there is no clear correlation between HPR activity and the amount of either sense (r=0.267, n=9) or antisense RNA (r=0.175, n=9). This compares to a weak positive correlation between HPR mRNA levels and the amount of HPR activity observed in wild-type SRI tobacco plants (r=0.603, n=5). The results suggest that in vivo production of this heterologous HPR antisense RNA is inhibitory at the level of HPR-specific translation and produces its effect in a manner not dependent upon, nor resulting in, a reduction in steady-state native HPR mRNA levels. In this context, the observed antisense effect appears to differ mechanistically from most antisense systems described to date.     

土壤理化性质驱动烤烟根际细菌群落的组配及其共现性网络互作

【目的】解析土壤微生物在植物根际的组配机制对于认识和维护农田生态系统的稳定性至关重要。【方法】通过Illumina高通量测序和生物信息学分析方法明确了我国主要种植烟草生态区烤烟根际土壤细菌群落与土壤理化性质的互作关系。【结果】烤烟根际细菌类群主要为放线菌纲(Actinobacteria)、α-变形菌纲(Alphaproteobacteria)、γ-变形菌纲(Gammaproteobacteria)和嗜热油菌纲(Thermoleophilia)。细菌群落组成按生态区聚类,且样本空间距离和细菌群落相似度显著负相关。共现性网络分析表明,烤烟根际细菌群落间协同作用大于拮抗作用,武陵秦巴生态区、黄淮平原生态区、南岭丘陵生态区和沂蒙丘陵生态区细菌群落高度模块化,小单胞菌属(Micromonospora)为南岭丘陵生态区和黄淮生态区细菌共现性网络的网络中心,Bryobacter和气单胞菌属(Arenimonas)为南岭丘陵生态区细菌网络的模块核心,其菌群特性而非相对丰度决定了其在稳定细菌网络中的重要作用。冗余分析结果证实pH、有效铁、交换性镁和有效锰能显著影响烤烟根际细菌群落结构。【结论】烤烟根际细...