Demonstration of putative membrane and cytosol steroid receptors for 17α, 20β-dihydroxy-4-pregnen-3-one in brook troutSalvelinus fontinalis oocytes by photoaffinity labelling using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione (R5020)

Cytosol from brook trout ovarian follicles (stages 1–3) was photoaffinity (PA) labelled using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione ([³H]R5020). The covalently bound cytosol protein had a relative mass of 501,000 M_r following Sephacryl S-300 column chromatography. The zona radiata membrane fraction from brook trout oocytes which had gone through the first phase of meiotic maturation (stages 6–7) was isolated by ultracentrifugation of the whole oocytes. The zona radiata solubilized protein presumably from the oocyte membrane was also PA labelled and found to give a peak at 355,000 M_r. The SDS PAGE of the cytosol and zona radiata PA labelled protein gave very similar subunits indicating that the membrane protein and the cytosol protein, both of which bind the maturation inducing steroid (MIS) 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP), have similar subunit structures. The isolated zona radiata protein showed cooperativity of binding to [³H]17,20-DHP and PA labelling to [³H]R5020. The association constant (K_a) was 2.0×10⁷M^–1 and maximum binding capacity (N_max) 427 fmoles/mg protein with MIS [³H]17,20-DHP.No evidence for nuclear binding of MIS [³H]17,20-DHP or PA labelling of [³H]R5020 to nuclei was observed. The nuclei were isolated from stages 1 and 3 fresh ovarian follicles of brook trout. The experimental evidence presented demonstrates the presence of MIS 17,20-DHP receptor-like protein from the zona radiata membranes by PA labelling in brook trout oocytes during final stages of maturation.     

(三唑基-~(14)C-)粉锈宁的标记合成

本文报道了(三唑基-14C)-粉锈宁的制备。由14C-甲酸和重碳酸氨基胍形成(5-14C)-3-氨基-1,2,4-三唑,再经重氮化脱氨得到(5-14C)-1,2,4-三唑,最后再与对氯酚和二氯片呐酮反应得到(三唑基-14C)-粉锈宁。放化收率为26％(从甲酸-14C计),放化纯度大于95％。     

Localization of potato leafroll virus in leaves of secondarily-infected potato plants

Potato leafroll virus (PLRV) antigen was localized by immunogold labelling in semi-thin leaf sections of secondarily-infected potato plants cv. Bintje. Viral antigen was present in all cell types of the phloem tissue. but occurred most abundantly in the companion cells. Detectable amounts of PLRV antigen were found only in the sieve elements in veins with a large number of infected companion cells. Occasionally, parenchyma cells were also found to be infected. PLRV was not exclusively limited to the phloem tissue in the infected potato plants, but was also found in mesophyll cells neighbouring minor phloem vessles. Spread of virus from cell to cell in the mesophyll was not observed. The distribution of PLRV in the potato leaf tissue has implication on its availability, for acquisition by aphids.     

AM真菌共生系统中硝态氮吸收转运途径及对寄主生长的作用

研究了AM真菌共生系统中硝态氮NO3-吸收转运、铵和硝态氮吸收合成精氨酸及对寄主生长的影响。利用AM真菌（Glomus intraradices）与毛根农杆菌质粒DNA转化的胡萝卜根（Ri T-DNA transformed carrotroots）建立的双重培养系统,以及同位素示踪技术研究了AM真菌共生系统中硝态氮NO3-转运吸收途径,研究了铵和硝态氮吸收合成精氨酸和其转运动态;并用农田试验研究铵和硝态氮吸收转运对寄主生长的影响。研究发现AM真菌菌丝在NH4+和NO3-共存时,优先吸收NH4+。当AM真菌的根外菌丝在NH415NO3培养1周时,虽然根外菌丝的自由氨基酸没有被15N标记,包括精氨酸,但是菌根组织中的自由氨基酸是被15N标记的,揭示了15NO3-沿着菌丝直接扩散或转运至菌根组织而不是来自于精氨酸转运的新模式;而根外菌丝在15NH4NO3培养时菌根组织中只有精氨酸被15N标记的结果,而其它氨基酸合成的氮素主要来自从菌丝室运转来的14NO3-,所以没有标记。AM真菌根外菌丝施加13C6-葡萄糖后,培养6周后,发现菌根组织的精氨酸和蛋白质中都没有13C标记,说明了其根外菌丝不能利用葡萄糖。当在菌丝室施加13C1,2-乙酸钠时,发现菌根组织的精氨酸和蛋白质中都有13C标记,分别为8.5?2.3%和7.6?0.7%,说明了其根外菌丝能吸收利用乙酸盐中的碳素,当在菌丝室施加13C1,2-乙酸钠+15NO3时,随着氮源的增加,提高了其自由精氨酸浓度为54.2?19.3%,菌根蛋白质中精氨酸浓度变化不大;同时大大提高了菌根组织的精氨酸和蛋白质中C/N同位素标记丰度分别为57.4?4.8%和50.3?2.8%。说明了菌丝室加碳源乙酸和氮源,可以提高精氨酸的合成。大田试验中,在低磷条件下,接种AM真菌之后,添加硝酸钾可以明显地提高菌根化甜玉米茎叶重,相比对照的甜玉米提升了12.28%;硫酸铵则不如硝酸钾对AM真菌菌根化甜玉米株重的促进作用,反而是降低了其生物量8.19%,尿素则降低了13.02%,但是尿素再加有机肥则可以缓解对生物量的降低作用。AM真菌对铵和硝态氮的吸收和转运是有两种不同模式,对于铵态氮（NH4+和尿素）,AM真菌通过根外菌丝内谷氨酰氨合成酶-谷氨酸合成酶（GS-GOGAT）途径被吸收利用的,而吸收的氮大都是整合入精氨酸（Arg）分子,合成的精氨酸可以被AM真菌根外菌丝完整地运转至根内菌丝,而对于NO3-,用同位素示踪技术揭示了AM真菌共生系统中硝态氮NO3-通过菌丝吸收转运至根内菌丝的途径;硝态氮对寄主甜玉米生长有促进作用,而铵则相反有抑制作用。     

Towards a better understanding of carbon flow in the rhizosphere: a time-dependent approach using carbon-14

 Controversies exist in interpreting rhizosphere C flow obtained by different ¹⁴CO₂ labelling methods. However, there is a need for the standardisation of methods in order to be able to compare values obtained for different plants, different stages of development and different habitats. Perennial bromegrass (Bromus erectus Huds) grown in soils of different fertility was exposed to a ¹⁴CO₂ atmosphere for different periods of time: 1 h, 298 h and 78 days. The evolution of ¹⁴CO₂ in the soil was measured during and after labelling. The ¹⁴C contents of plant and rhizosphere compartments were then estimated. The time-sequence of the rate of ¹⁴CO₂ evolution after 1 h of labelling, indicated a maximum after around 20 h, followed by an exponential decrease. When expressed as a percentage of net ¹⁴C assimilation, root-soil respiration accounted for 14% and 18% in the nutrient-poor and nutrient-rich soils, respectively. Integration of the hourly values over several days showed that the dynamics of the evolution rate were similar for the 298-h and 78-day experiments, thus indicating that rhizosphere C flow was dominated by newly assimilated C. This was confirmed by the proportions of below-ground ¹⁴C, measured for roots, respiration and soil, which were not significantly affected by the labelling regime. The differences were, however, found to be significant between the two types of soils. The conclusion was that the conditions for plant growth during labelling were more important than the length of time of labelling, and that this explained the discrepancies in the literature-cited values. A succession of short-term ¹⁴C labelling of plants at different development stages followed by an allocation period of about 1 week is proposed to give a reliable estimation of the dynamics of C flow in the rhizosphere. Received: 7 June 1999     

Extractability of microbial N as influenced by C : N ratio in the flush after drying or fumigation

 Extractability of microbial N was estimated using in situ labelling of the microbial population with ¹⁵N. Four arable soils (one grey forest soil and three chernozems with different long-term fertilization) were amended with (NH₄)₂SO₄ (unlabelled or labelled with ¹⁵N) and d-glucose with a C : N ratio of 10 : 1 or 20 : 1 for the grey forest soil and 50 : 1 for the chernozems. d-glucose and labelled N with a C : N ratio of 20 : 1 did not cause microbial immobilization of unlabelled N. The use of substrates with a C : N ratio of 50 : 1 led to a pronounced priming action on soil N and decreased the extractability of immobilized ¹⁵N. Values of the extractable biomass N fraction (k _EN ) assessed for the fumigation-extraction and rehydration procedures were similar and varied in inverse proportion to the C : N ratio of the flush. The k _EN factor was calculated using values of the C : N ratio in flushes and the fixed C : N ratio of structural cell components, with the assumption that the C : N ratio of the extractable cytoplasmic cell fraction is variable. The ratio between the extractable and non-extractable biomass N fraction (k _EC ) and the C : N ratio of non-extractable cell components were assessed as equation parameters optimized for the measured k _EN and C : N ratio of flush data. Received: 31 October 1997     

植物病毒标记胶体金探针的制备

对用于植物病毒标记的几类胶体金探针的制备及其标记技术进行了研究。比较了不同情况下胶体金探针的选择、制备与保存的关键技术环节,比较了提高标记强度与特异性的方法,并且对五种植物病毒进行了标记。     

兔病毒性出血症Dot-IGSS检测方法的建立

[目的]建立一种检测兔病毒性出血症IgG抗体的斑点免疫金银染色法（Dot—IGSS）。[方法]选择抗原包被浓度20μg/ml,被检血清稀释度1：160;鼠抗兔IgG稀释度1：200、金标羊抗鼠IgG稀释度1：20的工作条件,用所建立的Dot—IGSS与血凝抑制试验（HI）对96头份被检血清进行平行检测。[结果]Dot—IGSS和HI的检测阳性率分别为94％和31％。[结论]所建立的Dot—IGSS是一种灵敏、特异和稳定的血清学捡测方法,适用于兔病毒性出血症的诊断和抗体检测。     

甘蔗花叶病毒VPg蛋白的原核表达及其在玉米中的免疫定位

 利用RT-PCR方法从感染甘蔗花叶病毒北京分离株(SCMV-BJ)的玉米叶片中扩增得到VPg基因,将VPg基因连接到原核表达载体pET-28a上。获得的重组子转化大肠杆菌BL21(DE3)后,用IPTG进行诱导表达。SDS-PAGE分析表明,VPg在大肠杆菌中获得了高效表达,产生的融合蛋白分子量为30 kD。将融合蛋白纯化后免疫兔子获得了特异性较高的抗血清。ACP-ELISA测定抗血清的效价为1/4096。利用该抗血清对健康玉米和病毒侵染的玉米茎、叶脉和叶片进行免疫金标记,结果表明在茎和叶脉的韧皮部筛管的细胞壁处有金颗粒。